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UNIVERSIDADE FEDERAL DO CEARÁ
REDE NORDESTE DE BIOTECNOLOGIA - RENORBIO
PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA
JOSÉ JACKSON DO NASCIMENTO COSTA
EXPRESSÃO DE MARCADORES DE CÉLULAS GERMINATIVAS E DE OÓCITOS
EM FIBROBLASTOS BOVINOS TRATADOS COM 5-AZA-CITIDINA E EM
CÉLULAS-TRONCO ADULTAS CULTIVADAS IN VITRO NA PRESENÇA DE BMP-2,
BMP-4 OU FLUIDO FOLICULAR
SOBRAL - CE
2016
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JOSÉ JACKSON DO NASCIMENTO COSTA
EXPRESSÃO DE MARCADORES DE CÉLULAS GERMINATIVAS E DE OÓCITOS
EM FIBROBLASTOS BOVINOS TRATADOS COM 5-AZA-CITIDINA E EM
CÉLULAS-TRONCO ADULTAS CULTIVADAS IN VITRO NA PRESENÇA DE BMP-2,
BMP-4 OU FLUIDO FOLICULAR
Tese apresentada ao Curso de Doutorado em
Biotecnologia da Rede Nordeste de Biotecnologia
– RENORBIO da Universidade Federal do Ceará,
como parte dos requisitos para obtenção do título
de Doutor em Biotecnologia. Área de
concentração: Biotecnologia em Agropecuária.
Orientador: Prof. Dr. José Roberto Viana Silva
Co-orientadora: Profa. Dra. Márcia Viviane Alves
Saraiva
SOBRAL - CE
2016
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A Deus, pela minha existência, pela força,
coragem e determinação que me foi dada para
alcançar mais esse objetivo, porque nada nos é
possível se não for de Sua vontade,
Dedico
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AGRADECIMENTOS
A Deus, pelo seu amor e pela sua infinita misericórdia manifestados a cada dia em
minha vida. Pela proteção, força e coragem para enfrentar todas as dificuldades da vida pessoal e
profissional. Senhor, a minha confiança descansa em tuas mãos. Sempre espero e confio em ti.
Obrigado por mais essa vitória.
À minha esposa Amélia Araújo, agradeço pelo amor, amizade, companheirismo,
apoio nos momentos mais difíceis e pela compreensão que me deram suporte na trajetória final
deste trabalho. “Entre as coisas mais lindas que eu conheci, só reconheci suas cores belas quando
eu te vi. Entre as coisas bem-vindas que já recebi, eu reconheci minhas cores nela então eu me
vi.” Sou muito mais feliz com você e com nossa filha Val. Que o Senhor continue nos
abençoando cada dia mais.
Aos meus pais José Ponte Costa e Maria de Fátima do Nascimento, por terem me
dado a vida e por todo amor e dedicação fundamentais em todos os momentos da minha vida.
Aos meus irmãos Janilson Costa e sua esposa Cristiane Santos Costa, Jailson Costa e Jamile
Costa, com os quais dividi momentos de alegrias e tristezas, e que sempre estarão me
incentivando e torcendo pelo meu sucesso. De forma especial, agradeço à minha sobrinha, Ana
Clara Costa, uma das maiores alegrias da minha vida, você é muito especial.
Aos meus avós Pedro do Nascimento e Maria da Conceição do Nascimento, por todo
o amor e carinho dedicados a mim, vocês são essenciais para a minha vida. E ainda agradeço a
todos os meus tios(as) e primos(as), que longe ou perto sempre me ajudaram, torcem por mim e
sempre estarão no meu coração. Em especial ao meu tio José Maria Costa e José Astélio Costa,
por cuidarem de mim desde a infância, educando e sempre orientando os caminhos retos a seguir.
À família Araújo, em especial à dona Amélia Araújo, sr. João Elmiro Soares,
Marcigleide Araújo, Elenilton Roratto, Júlia Roratto, Ana Sofia Roratto, Olga Araújo e Samira
Araújo. Por fazerem me sentir parte da família, dividindo comigo momentos de tristezas, mas
principalmente momentos de muitas alegrias. Vocês moram no meu coração.
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Ao orientador Prof. Dr. José Roberto Viana Silva, pela orientação deste trabalho,
paciência, pela dedicação dispensada, confiança e profissionalismo demonstrados no decorrer de
nossa convivência. Agradeço pela contribuição decisiva na minha formação e pelo muito que
aprendi durante os anos de mestrado e doutorado.
À co-orientadora e amiga Profa. Dra. Márcia Viviane Alves Saraiva pela dedicação
constante e por sempre estar disposta a ajudar em todos os momentos.
Aos queridos amigos integrantes do grupo de pesquisa de Reprodução e Cultura de
Células: Ellen de Vasconcelos da Cunha, Francisco Taiã Gomes Bezerra, Laís Feitosa, Ana Kelry
Carneiro Lopes, Éverton Pimentel, Adriel Pereira, Edilcarlos Max, Hozana Braga, Pedro Alves,
Miguel Fernandes, Laryssa Barrozo, Bruno Matos Brito e Bianca Silva.
À querida amiga Juliane Passos e a toda família Passos, Cleuton Monteiro, tia Mazé e
Neyla, agradeço pela amizade, companheirismo, apoio e incentivo desde o início e por me
tratarem sempre com muito carinho, como se eu fosse um irmão. Obrigado pelo apoio e pelas
palavras de carinho nos momentos mais difíceis.
Aos amigos e vizinhos Jordânia Marques, Clayrtiano Freire, João Arthur Freire e
Maria das Graças Oliveira, agradeço de forma especial, pelo convívio, confiança, amizade e
companheirismo durante todos esses anos. Que Deus continue abençoando nossa amizade.
Aos amigos Anderson Weiny Silva, Regislane Pinto Ribeiro, José Renato de Souza
Passos e Glaucinete Borges, agradeço pela amizade, pelos conselhos, pelos momentos de trabalho
e pelos momentos de descontração. Admiro a competência de vocês.
À amiga Joyla Maria Pires Bernardo agradeço por ter tornado o dia a dia muito mais
divertido e agradável, sempre trazendo alegria e descontração durante a execução de todos os
experimentos.
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Aos amigos Tânia de Azevedo Lopes, Antônia Moemia Lucia Rodrigues Portela,
Gisvani Lopes de Vasconcelos, Katianne dos Santos Freitas e Danielle Val, obrigado por me
aguentarem todos esses anos, obrigado pela amizade, carinho e momentos de diversão e de
trabalho também. Sem as risadas de vocês tudo teria sido mais difícil. É bom ter o privilégio da
presença de vocês na minha vida. Sou grato a Deus por nossa amizade, vocês são especiais.
Ao prof. Dr. Rodrigo Maranguape pelo incentivo dado aos meus primeiros passos na
pesquisa durante minha iniciação científica e a toda equipe do Laboratório de Genética Molecular
do NUBIS, de forma especial a Auxiliadora Oliveira, João Garcia, Tatiana Farias, Nayane Hardy,
Aurilene Cajado, Jedson Aragão, pela convivência durante todos esses anos.
Aos integrantes da banca examinadora, Profa. Dra. Ana Paula Ribeiro Rodrigues;
Profa. Dra. Maria Helena Tavares de Matos; Profa. Dra. Valdevane Rocha Araújo; Prof. Dr. Igor
Iuco Castro da Silva; Prof. Dr. Antonio Silvio do Egito; Profa. Dra. Juliana Jales De Hollanda
Celestino; por terem gentilmente aceito o convite para participar da banca de defesa desta tese e
pela solicitude em contribuir no engrandecimento deste trabalho.
Aos funcionários da Faculdade de Medicina de Sobral pela convivência, atenção e
disponibilidade durante todos esses 10 anos de convívio.
A todos que de alguma forma me deram força e incentivo na realização do meu
doutoramento, seja profissionalmente ou sentimentalmente e por participarem da minha vida.
Por fim, agradeço a todos que contribuíram, de alguma forma, ou torceram para que
eu chegasse até aqui, compartilhando comigo um momento tão importante. A todos vocês, de
coração, o meu MUITO OBRIGADO!!!
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“Porquanto, ainda que a figueira não floresça,
nem haja fruto na vide; o produto da oliveira
minta, e os campos não produzam mantimento;
Todavia eu me alegrarei no Senhor, exultarei no
Deus da minha salvação. O Senhor é minha força
e me fará andar sobre as minhas alturas.”
(HABACUQUE, 3:17-19)
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RESUMO
Este estudo teve como objetivo investigar o efeito da 5-Aza-citidina durante a indução da
pluripotência, e avaliar os efeitos do cultivo em meio contendo BMP-2, BMP-4 ou fluido
folicular na diferenciação de fibroblastos em células germinativas primordias (CGP) e oócitos
(fase 1). Além disso, isolar células-tronco do epitélio ovariano de bovinos e avaliar os efeitos da
BMP-2, BMP-4 ou fluido folicular sobre a diferenciação destas células-tronco em estruturas
semelhantes a oócitos (fase 2). Na fase 1, os fibroblastos foram tratados com 0.5, 1,0 ou 2.0 μM
de 5-Aza por 18, 36 ou 72 h. Foi avaliada a morfologia, viabilidade celular e a expressão gênica
(OCT-4, NANOG, REX e SOX2), para a seleção da concentração/tempo mais eficientes. Os
fibroblastos foram então cultivados em meio suplementado com 10 ng/mL de BMP-2, ou 10
ng/mL de BMP-4 ou 5% de fluido folicular bovino, por 7 ou 14 dias. Posteriormente, foi feita a
avaliação da morfologia e viabilidade celular, e expressão gênica (VASA, DAZL, C-KIT, SCP3,
ZPA e GDF-9). Para a fase 2, as células-tronco da superfície ovariana foram isoladas,
expandidas, cultivadas em meio de diferenciação contendo as 50 ng/mL de BMP-2, ou 50 ng/mL
de BMP-4, ou BMP-2+BMP-4 ou 5% de fluido folicular bovino por 14 dias. Foram avaliadas as
características morfológicas, viabilidade celular, e expressão da fosfatase alcalina e expressão
gênica (VASA, DAZL, C-KIT, SCP3, ZPA e GDF-9). Os resultados de expressão gênica foram
analisados usando ANOVA seguido pelo Teste de Kruskal Wallis (P<0,05). Na fase 1, o cultivo
com 2.0 μM de 5-Aza por 72 h provocou mudanças na morfologia e na taxa de proliferação
celular, e aumentou significativamente a expressão de fatores de pluripotência. Já o cultivo em
meio contendo BMP-2, BMP-4 ou fluido folicular, durante 7 ou 14 dias, alterou a morfologia
celular, e a expressão de genes específicos para células germinativas e oócitos. Na fase 2, as
células-tronco do ovário expressaram genes de pluripotência e após o cultivo, as células
apresentaram características morfológicas que se assemelhavam a CGP e a células semelhantes a
oócitos, incluindo a expressão da fosfatase alcalina e a expressão de genes específicos de células
germinativas e oócitos. Em conclusão, o presente estudo descreve a possibilidade de converter os
fibroblastos da pele de bovinos e células-tronco da superfície ovariana, em células semelhantes a
oócitos, através do processo de reprogramação celular, associado à suplementação do meio com
BMP-2, BMP-4 e fluido folicular.
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Palavras-chave: Expressão gênica. Fibroblastos bovinos. Células-tronco adultas. Cultivo in
vitro.
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ABSTRACT
This study aimed to investigate the effect of 5-Aza-cytidine during induction of pluripotency, and
evaluate the effects of culture in medium containing BMP-2, BMP-4 or follicular fluid in the
differentiation of fibroblasts in primordias germ cells (CGP) and oocytes (stage 1). Furthermore,
isolation of stem cells from the bovine ovarian epithelium and evaluate the effects of BMP-2,
BMP-4 or follicular fluid on the differentiation of these stem cells into structures similar to
oocytes (stage 2). In phase 1, fibroblasts were treated with 0.5, 1.0 or 2.0 µM of 5-Aza for 18, 36
or 72 h. Morphology cell viability and gene expression (OCT-4, NANOG, SOX2 and REX), were
assessed, for the selection of the concentration/time more efficient. The fibroblasts were then
cultured in medium supplemented with 10 ng/mL BMP-2 or 10 ng/mL BMP-4 or 5% bovine
follicular fluid by 7 or 14 days. Subsequently, evaluation of the morphology and cell viability
was taken, and gene expression (VASA, DAZL, c-Kit, SCP3, ZPA and GDF-9). For phase 2, the
stem cells of ovarian surface were isolated, expanded, grown in differentiation medium
containing 50 ng/mL BMP-2 or 50 ng/mL BMP-4, or BMP-2+BMP-4 or 5% bovine follicular
fluid for 14 days. Morphological characteristics, cell viability and expression of alkaline
phosphatase and gene expression (VASA, DAZL, C-KIT, SCP3, ZPA and GDF-9), were evaluated.
The gene expression results were analyzed using ANOVA followed by Kruskal-Wallis test (P
<0.05). In stage 1, the culture with 2.0 µM of 5-Aza for 72 h caused changes in morphology and
cell proliferation rate, and significantly increased the expression of pluripotency factors. The
culture in medium containing BMP-2, BMP-4 or follicular fluid for 7 or 14 days, altered cellular
morphology, and expression of specific genes for stem cells and oocytes. In stage 2, the ovarian
stem cells expressed pluripotent genes, and after culture, this cells showed morphologic
characteristics similar to PGC and oocytes, including the expression of alkaline phosphatase, and
the expression of specific genes for PCG and oocytes. In conclusion, the present study describes
the possibility of conversion of the skin fibroblasts and stem cells from ovarian surface of the
bovine, in oocyte-like cells, similar oocyte cells, through the cell reprogramming process
associated with the supplementation of BMP-2, BMP- 4 and follicular fluid.
Keywords: Gene expression. Bovine fibroblasts. Adult stem cells. In vitro culture.
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LISTA DE ILUSTRAÇÕES
Figura 1 - Desenho esquemático do ovário de mamíferos com suas diversas
estruturas........................................................................................................................
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Figura 2 - Representação esquemática do desenvolvimento das CGP e dos sinais
envolvidos na regulação da migração e colonização CGP em camundongos............... 31
Figura 3 - Especificação da linhagem germinativa e eventos moleculares.................. 34
Figura 4 – Representação esquemática do papel da sinalização das BMPs e WNT
durante a formação das células germinativas primordiais (CGPs) em embriões..........
36
Figura 5 – Regulação da transcrição da especificação das CGPs................................. 37
Figura 6 – Isolamento e diferenciação das células-tronco embrionárias...................... 43
Figura 7 – Isolamento e diferenciação de células-tronco mesenquimais..................... 45
Figura 8 – Reprogramação de células adultas e formação das células iPS.................. 50
CAPÍTULO 1
Figure 1 - Schematic presentation of the development of stem cells into oocytes-like
cells, and the stimulating substances and markers for each stage of differentiation…. 92
Figure 2 - Procedure for restoring fertility by differentiating iPS into oocytes............ 93
CAPÍTULO 2
Figure 1 - Representative pictures of the morphological changes in bovine skin
fibroblasts exposed to 5-Aza. (A) The fibroblast cells isolated from bovine fetal ear
skin (untreated cells). (B) Fibroblasts exposed to 2.0 µM of 5-Aza for 72 h. (C)
Fluorescence staining of viable cells for Calcein AM. (D) Fluorescence staining of
apoptotic cells for ethidium homodimer-1. Scale bar = 100
µm………………………………………….………………………...……………….. 133
Figure 2 - Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in
fibroblasts cultured for 18 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0
µM)………………………………………………………………………………….. 134
Figure 3 - Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in
fibroblasts cultured for 36 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0
13
µM)…………………………………………………………………………………... 135
Figure 4. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in
fibroblasts cultured for 72 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0
µM)…………………………………………………………………………………… 136
Figure 5. Levels of mRNA for of pluripotency genes, (A) SOX2, (B) NANOG, (C)
OCT4 and (D) REX, in fibroblasts cultured for 18 h, 36 h or 72 h in with different
concentrations of 5-Aza (0.5, 1.0 or 2.0 µM)………………………………………… 137
Figure 6. Representative pictures of the morphological characterization in bovine
skin fibroblasts exposed to 5-Aza and cultured in differentiation medium for 14
days. Fibroblast cultured for 14 days in differentiation medium supplemented with
10 ng/mL of BMP-2 (line 1), 10 ng/mL of BMP-4 (line 2), 5% follicular fluid (line
3), (A, D, G) cell analyzed by light microscopy, (B, E, H) Fluorescence staining of
viable cells for Calcein AM; (C, F, I) Fluorescence staining of apoptotic cells for
ethidium homodimer-1. Scale bar = 100 µm…………………………………………. 138
Figure 7. Levels of mRNA for markers of germ cells [VASA (A, B), DAZL (C, D),
C-KIT (E, F)] in cells cultured for 7 (A, C, E) or 14 (B, D, F) days in control
medium or supplemented with BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5%
follicular fluid………………………………………………………………………… 139
Figure 8. Levels of mRNA for markers of oocytes [ZPA (A, B), GDF-9 (C, D),
SCP3 (E, F)] in cells cultured for 7 (A, C, E) or 14 (B, D, F) days in control medium
or supplemented with BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular
fluid…………………………………………………………………………………… 140
Figure 9. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-
KIT (C)] and oocytes [ZPA (D), GDF-9(E), C-KIT (F)] after culture cells for 0 h (5-
Aza), 7 or 14 days in control medium……………………………………………....... 141
Figure 10. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-
KIT (C)] and oocytes [ZPA (D), GDF-9(E), C-KIT(F)] after culture cells for 0 h (5-
Aza), 7 or 14 days in medium supplemented with 10 ng/mL of BMP-2……………... 142
Figure 11. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-
KIT (C)] and oocytes [ZPA (D), GDF-9(E), C-KIT(F)] after culture cells for 0 h (5-
Aza), 7 or 14 days in medium supplemented with 10 ng/mL of BMP-4…………… 143
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Figure 12. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-
KIT (C)] and oocytes [ZPA (D), GDF-9(E), C-KIT(F)] after culture cells for 0 h, 7 or
14 days in medium supplemented with 5% follicular fluid…………………………... 144
CAPÍTULO 3
Figure 1. Fluorescence staining of ovarian stem cells before (A-B) and after 14 days
culture in minimum essential medium (α-MEM) (C-D) or supplemented with BMP2
(E-F), BMP4 (G-H), both BMP2 and BMP4 (I-J) and follicular fluid (K-L). Scale
bar = 100 µm. Green (calcein) staining cells are viable and red (ethidium
homodimer) staining cells are not…………………………………………………….. 167
Figure 2. Alkaline phosphatase activity in ovarian stem cells at day 0. Scale bar =
100 µm………………………………………………………………………………... 168
Figure 3. Cycle threshold (CT) after amplification of mRNA for pluripotency stem
cell markers (OCT4 and SOX2) by real time qRT-PCR……………………………… 168
Figure 4. Levels of mRNA for specific germline cell [VASA (A), DAZL (B)] and
oocyte [SCP3 (C), GDF9 (D) and ZPA (E)] markers in ovarian stem cells cultured
for 14 days in MEM supplemented with BMP2, BMP4, BMP2 and BMP4 and
follicular fluid. Significant differences at P<0.05……………………………………. 169
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LISTA DE TABELAS
CAPÍTULO 1
Table 1. Molecular markers for stem cells, PCGs, oocytes and their functions............ 89
CAPÍTULO 2
Table 1. Primer pairs used in real-time PCR for quantification of markers of
pluripotency, germ cells and oocytes genes expressed in cells cultured……………...
132
CAPÍTULO 3
Table 1. Primer pairs used in real-time PCR…………………………………………. 166
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LISTA DE ABREVIATURAS E SIGLAS
Português Inglês
Alk2 Receptor de ativina semelhante à
quinase tipo 2
Activin receptor-like kinase-2
AP Fosfatase alcalina Alkaline phosphatase
AP2g Proteína de ligação e ativação do
regulador - 2 gama
Activating enhancer binding protein 2
gamma
ASC Células-tronco adultas Adult stem cells
AVE Endoderma visceral anterior Anterior visceral endoderm
Blimp1 Proteína de maturação induzida por
linfócitos B - 1
B lymphocyte-induced maturation
protein-1
BMP Proteína morfogenética óssea Bone morphogenetic protein
BMP-15 Proteína morfogenética óssea - 15 Bone morphogenetic protein – 15
BMP-2 Proteína morfogenética óssea - 2 Bone morphogenetic protein – 2
BMP-4 Proteína morfogenética óssea - 4 Bone morphogenetic protein – 4
BMP-8b Proteína morfogenética óssea - 8b Bone morphogenetic protein - 8b
BOULE Proteína Boule Boule protein
BRG1 Brahma-Related Gene - 1
BSA Albumina sérica bovina Bovine serum albumin
CD133 Antígeno também conhecido por
prominina-1
Antigen also known as prominin-1
CDH1 Caderina-1 Cadherin-1
CDX2 Caudal-related homeobox
CGP Célula Germinativa Primordial Primordial germ cell
c-Kit Receptor para kit ligante Kit ligand receptor
c-MYC Myc proto-oncogene protein
COLA4 Colagenase microbiana Microbial collagenase
Cx43 Conexina 43 Connexin 43
DA Aorta dorsal Dorsal aorta
DAZL Suprimido na azoospermia Deleted in azoospermia like
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DDX-4 Polipeptídeo caixa DEAD 41 DEAD box polypeptide 4
Dmc1 Proteina de recombinação meiótica 1 DNA meiotic recombinase 1
DMEM/F12 Dulbecco's Modified Eagle Medium:
Nutrient Mixture F-12
DNA Ácido desoxirribonucleico Deoxyribonucleic acid
DNMT DNA metiltransferase Deoxyribonucleic acid
methyltransferase
DNMT1 DNA metiltransferase 1 Deoxyribonucleic acid
methyltransferase 1
Dpc Dias pós-concepção Days post coitum
Dppa3 Gene de pluripotência associada ao
desenvolvimento – 3
Developmental pluripotency
associated 3 pseudogene 2
DVE Endoderma visceral distal Distal visceral endoderm
EBs Corpos embrióides Embryoid bodies
EM Mesoderma embrionária Embryonic mesoderm
Epi Epiblasto Epiblast
ESC Células-tronco embrionárias Embryonic stem cells
Evx1 Even-Skipped Homeobox 1
ExE Ectoderma extra-embrionário Extra-embryonic ectoderm
ExM Mesoderma extra-embrionário Extra-embryonic mesoderm
FGSCs Células-tronco de linha germinativa
feminina
Female germline stem cell
FIGα Factor in the germline alpha
FOP Falha ovariana precoce Premature ovarian failure
FOXH1 Forkhead box protein H1
Fragilis Mouse interferon-induced protein like
gene-1
FSH Hormônio Folículo Estimulante Follicle-stimulating hormone
FSHR Receptor do Hormônio Folículo
Estimulante
Follicle-stimulating hormone receptor
GATA3 Proteína 3 de ligação a GATA GATA binding protein 3
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GDF3 Fator de diferenciação de crescimento
tipo 3
Growth differentiation factor type 3
GDF-9 Fator de diferenciação de crescimento
tipo 9
Growth differentiation factor type 9
GFP Proteína fluorescente verde Green fluorescent protein
giPS Células-tronco de pluripotência
induzidas caprinas
Goats induced pluripotent stem cells
GV Vesícula germinativa Germinal vesicle
H2AX Histona membro da família X H2A histone family member X
HDAC Histona deacetilase Histone deacetylase
HDM Histona demetilase Histone demethylase
hESC Células-tronco embrionárias de
humanos
Human embryonic stem cells
Hg Intestino primitivo Hindgut
HGF Fator de crescimento de hepatócitos Hepatocyte Growth Factor
HMT Histona metiltransferase Histone methyltransferase
Hoxa1 Homeobox protein Hox-A1
Hoxb1 Homeobox protein Hox-B1
ICM Massa celular interna Inner cell mass
Ifitm3 Proteína transmembranar induzida 3 Interferon-induced transmembrane
protein 3
IGF2 Fator de crescimento semelhante à
insulina-2
Insulin like growth factor-2
IM Mesoderma intermediário Intermediate mesoderm
iPS Células-tronco de pluripotência
induzidas
Induced pluripotent stem cells
IVF Fertilização in vitro In vitro fertilization
KLF4 Fator semelhante a Kruppel – 4 Kruppel-like factor 4
KRT7 Queratina 7 Keratin 7
KRT8 Queratina 8 Keratin 8
LIF Fator inibidor de leucemia Leukemia inhibitory fator
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LIN28 Lin-28 homolog A
MAPK Proteína cinase ativada por mitógenos Mitogen-activated protein kinase
MEM Meio essencial mínimo Minimal essential medium
mESCs Células-tronco embrionárias de ratos Mouse embryonic stem cells
mRNA Ácido Ribonucléico mensageiro Messenger Ribonucleic Acid
MSC Células-tronco mesenquimais Mesenchymal stem cells
mTS Células-tronco do trofoblasto murinho Stem cells from murine trophoblast
Mvh Proteína de rato homóloga a proteína
VASA
Mouse vasa homolog
NaB Butirato de sódio Sodium butyrate
Nanog Nanog Homeobox
NANOS3 Nanos Homolog 3
NeC Cordão nefrogênico Nephrogenic cord
NoC Notocorda Notochord
NODAL Fator de diferenciação de crescimento
nodal
Nodal growth differentiation factor
NT Tubo neural Neural tube
Oct4 Fator de transcrição ligado ao
octâmero 4
Octamer-binding transcription factor 4
OCT-4A Fator de transcrição ligado ao
octâmero 4A
Octamer-binding transcription factor
4A
OGSCs Células-tronco germinativas do ovário Ovarian germ stem cells
OLC Células semelhantes a oócitos Oocyte-like cells
OMS Organização Mundial de Saúde World Health Organization
ORF Quadro aberto de leitura Open reading frame
OSC Células-tronco ovarianas Ovarian stem cells
OSE Epitélio de superficie do ovário Ovarian surface epithelium
OSKM OCT4, SOX2, KLF4 e MYC OCT4, SOX2, KLF4 and MYC
PGC Célula germinativa primordial Primordial germ cell
Pgc7 Proteína 7 em células germinativas
primordiais
Primordial germ cell protein 7
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PGCLC Células semelhantes a células
germinativas primordiais
Primordial germ cell-like cells
pH Potencial hidrogeniônico Potential hydrogen
PMSG Gonadotrofina de égua prenhe Pregnant mare serum gonadotropin
Pou5f1 Domínio POU, classe 5, fator de
transcrição 1
POU domain, class 5, transcription
factor 1
Prdm1 Proteína dedo de zinco de domínio
PR1
PR domain zinc finger protein 1
Prdm14 Proteína dedo de zinco de domínio PR
14
PR domain zinc finger protein 14
REC8 Proteína de recombinação meiótica 8 Meiotic recombination protein 8
RG108 N - Ftalil – L – Triptofano N-Phthalyl-L-Tryptophan
RNA Ácido Ribonucléico Ribonucleic acid
RNAm Ácido Ribonucléico mensageiro Messenger ribonucleic acid
R-Smads Receptor regulado dos mensageiros
intracelulares
Receptor-regulated mothers against
decapentaplegic homolog
SCP1 Proteína 1 do complexosinaptonêmico Synaptonemal complex protein 1
SCP2 Proteína 2 do complexosinaptonêmico Synaptonemal complex protein 2
SCP3 Proteína 3 do complexosinaptonêmico Synaptonemal complex protein 3
Smad1 Mensageiro intracelular do tipo 1 Mothers against decapentaplegic
homolog 1
SMAD2 Mensageiro intracelular do tipo 2 Mothers against decapentaplegic
homolog 2
Smad4 Mensageiro intracelular do tipo 4 Mothers against decapentaplegic
homolog 4
Smad5 Mensageiro intracelular do tipo 5 Mothers against decapentaplegic
homolog 5
Sox2 Região determinante do sexo no
cromossomo Y - Caixa 2
Sex determining region Y-box 2
SSEA Antígeno embrionárioestágio-
específico
Stage specific embryonic antigen
21
SSEA-1 Antígeno embrionárioestágio-
específico 1
Stage specific embryonic antigen 1
SSEA-3 Antígeno embrionárioestágio-
específico – 3
Stage specific embryonic antigen – 3
SSEA-4 Antígeno embrionárioestágio-
específico – 4
Stage specific embryonic antigen – 4
STAT3 Transdutor de sinal e ativador da
transcrição - 3
Signal transducer and activator of
transcription 3
Stella Gene de pluripotência associado ao
desenvolvimento - 3
Developmental pluripotency-associated
3
STELLAR Pseudogene de pluripotência 2
associado ao desenvolvimento - 3
Developmental pluripotency
associated 3 pseudogene 2
Stra8 Gene 8 estimulado pelo ácido
retinóico
Stimulated by retinoic acid gene 8
SYCP3 Proteína 3 do complexosinaptonêmico Synaptonemal complex protein 3
Tcfap2c Fator de transcrição AP-2 gama Transcription factor AP-2, gamma
TEKT1 Tektina-1 Tektin-1
TERT Transcriptase inversa da telomerase Telomerase reverse transcriptase
TGF-β Superfamília de fatores de
crescimento transformante beta
Transforming growth factor beta
Tnap Fosfatase alcalina não especifica a
tecidos
Tissue non-specific alkaline
phosphatase
TRA1-60 Gene semelhante a podocalixina 60 Podocalyxin like 60
TRA1-81 Gene semelhante a podocalixina 81 Podocalyxin like gene 80
TS Células-tronco do trofoblasto Trophoblast stem cells
TSA Tricostatina A Trichostatin A
UCB Sangue de cordão umbilical Umbilical cord blood
Vasa Proteína Vasa Vasa protein
VE Endoderma visceral Visceral endoderm
VEGF Fator de crescimento endotelial
vascular
Vascular endothelial growth fator
22
VPA Ácido valpróico Valproic acid
VSEL Células-tronco semelhante à células
embrionárias pequenas
Small embryonic-like stem cells
WNT3 Wingless-Type MMTV Integration
Site Family, Member 3
XEN Células-tronco do endoderma extra-
embrionário
Extraembryonic endoderm stem cells
ZP2 Glicoproteína da zona pelúcida 2 Zona pellucida glycoprotein 2
ZPA Glicoproteína da zona pelúcida A Zona pellucida glycoprotein A
ZPC Glicoproteína da zona pelúcida C Zona pellucida glycoprotein C
β-hCG Gonadotrofina coriônica humana beta Beta human chorionic gonadotropin
23
LISTA DE SÍMBOLOS
Português Inglês
% Percentagem Percentage
~ Aproximadamente Aproximately
± SEM Erro padrão da média Standard error of the mean
°C Graus Celsius Degrees Celsius
µg Micrograma Microgram
µL Microlitro Microliter
µm Micrômetro Micrometer
µM Micromolar Micromolar
CO2 Dióxido de carbono Carbon dioxide
h Hora Hour
IU/mL Unidades internacionais por mL International units per mL
min Minuto Minute
mg Miligrama Milligram
mL Mililitro Milliliter
mM Milimolar Millimolar
mm Milímetro Millimeter
ng Nanograma Nanogram
nm Nanômetro Nanometer
P < 0,05 Probabilidade de erro menor do que
5%
Error probabilities is less than 5%
P > 0,05 Probabilidade de erro maior do que
5%
Error probabilities is more than 5%
24
SUMÁRIO
1 INTRODUÇÃO............................................................................................. 25
2 REVISÃO DE LITERATURA..................................................................... 28
2.1 Ovário mamífero........................................................................................... 28
2.2 Formação e desenvolvimento das células germinativas e de oócitos........ 29
2.2.1 Mecanismos de especificação das CGPs........................................................ 32
2.2.1.1 Sinalização da especificação das CGPs.......................................................... 35
2.2.2 Migração de células germinativas primordiais............................................. 37
2.3 Células-tronco: definição, classificação e aplicações.................................. 39
2.3.1 Células-tronco embrionárias.......................................................................... 42
2.3.2 Células-tronco adultas.................................................................................... 44
2.3.2.1 Células-tronco ovarianas................................................................................ 47
2.3.3 Células-tronco de pluripotência e reprogramação celular.......................... 49
2.4 Proteínas morfogenéticas ósseas e seu papel durante a formação das
CGPs............................................................................................................... 53
2.5 Fluido folicular e seu papel durante a formação das CGPs e
oócitos............................................................................................................. 56
3 CAPÍTULO 1
ARTIGO I: In vitro differentiation of primordial germ cells and oocytes-
likes cells from stem cells................................................................................
58
4 PROBLEMA.................................................................................................. 94
5 JUSTIFICATIVA.......................................................................................... 95
6 HIPÓTESES CIENTÍFICAS....................................................................... 97
7 OBJETIVOS.................................................................................................. 98
7.1 Objetivos gerais............................................................................................. 98
25
7.2 Objetivos específicos...................................................................................... 98
8 CAPÍTULO 2
ARTIGO II: Expression of markers for germ cells and oocytes in cow
dermal fibroblast treated with 5-aza-cytidine and cultured in presence of
BMP-2, BMP-4 and follicular fluid.................................................................
99
9 CAPÍTULO 3
ARTIGO III: Bovine ovarian stem cells differentiate into germ cells and
oocyte-like structures after culture in vitro.....................................................
145
10 CONCLUSÕES............................................................................................. 170
11 PERSPECTIVAS........................................................................................... 171
REFERÊNCIAS............................................................................................ 172
25
1 INTRODUÇÃO
A infertilidade tem sido reconhecida pela Organização Mundial de Saúde (OMS),
como um problema de saúde pública em todo o mundo, emergindo como um dos principais
desafios do novo milênio para aqueles envolvidos no tratamento de infertilidade e da reprodução
assistida (VAYENA et al., 2001). No mundo, estima-se que a infertilidade afete
aproximadamente 14% dos casais (BOIVIN et al., 2007). Assim, nos últimos anos, novas
tecnologias reprodutivas tem sido desenvolvidas na tentativa de ajudar a reduzir este problema.
Entretanto, nos países em desenvolvimento, as tecnologias reprodutivas mais recentes não estão
disponíveis ou tem elevado custo financeiro (OMBELET et al., 2008). Além disso, a elucidação
de mecanismos e fatores envolvidos na biologia reprodutiva possibilitará o desenvolvimento de
protocolos de preservação de gametas de animais de elevado patrimônio genético ou em risco de
extinção. As biotécnicas reprodutivas devem garantir a manutenção da morfologia folicular e a
obtenção de oócitos viáveis a serem utilizados em programas de criopreservação, cultivo e
fecundação in vitro, melhorando a produtividade de animais de alto valor zootécnico ou em via
de extinção.
A infertilidade feminina, com a exaustão prematura do pool de oócitos, pode ocorrer
em decorrência de diferentes patologias e devido a uma série de perturbações como doenças auto-
imunes, doenças genéticas ligadas ao cromossomo X, ooforectomia por neoplasias ovarianas
benignas ou malignas, endometriose e cistos ovarianos, por exemplo. Além disso, os tratamentos
de quimioterapia convencionais são as causas mais comuns de infertilidade em pacientes com
câncer. A quimioterapia exerce efeito anti-mitótico inibindo a divisão celular e a replicação do
DNA, e a exposição do ovário a estas drogas induz danos irreversíveis aos folículos e aos oócitos,
resultando em infertilidade (SILVESTRIS et al., 2015). Há várias técnicas de reestabelecimento
da fertilidade, como a criopreservação de tecido ovariano seguido por transplante oocitário
autólogo, congelamento de embriões, cultivo in vitro do tecido ovariano, de folículos e de
oócitos, os quais previnem danos ao conteúdo nuclear (SILVESTRIS et al., 2015). No entanto,
estas técnicas não são totalmente seguras, pois, com o efeito da estimulação hormonal para a
maturação do pool de oócitos, tumores sensíveis ao estrogênio como os cânceres de mama e de
endométrio podem crescer rapidamente antes do tratamento de quimioterapia. Por outro lado, as
técnicas baseadas na criopreservação de tecido ovariano, após a estimulação hormonal e
subsequente transplante autólogo, implica no risco de reintroduzir as células malignas,
26
especialmente em pacientes com leucemia que podem abrigar células malignas na corrente
sanguínea.
Além dos tratamentos de reprodução assistida estabelecidos acima, os cientistas estão
desenvolvendo novas abordagens baseadas em células-tronco para o tratamento da infertilidade
(CHIRPUTKAR; VAIDYA, 2015), visto que as células-tronco têm a capacidade de reconstituir
os diversos tecidos corporais. As células-tronco embrionárias (ESC) são isoladas a partir da
massa celular interna, permanecendo em estado indiferenciado e capazes de diferenciar-se em
células das três camadas germinativas in vitro e in vivo. Já as células-tronco adultas (ASC) são
conhecidas por estar presentes em vários órgãos do corpo, como a pele, medula óssea, cérebro,
coração, tecido adiposo, etc. Estas células embora presentes na fase quiescente, podem ser
estimuladas pela secreção de fatores solúveis para a reconstituição tecidual (CHIRPUTKAR;
VAIDYA, 2015). As células de pluripotência induzida (iPS) foram mencionadas pela primeira
vez em 2006, por Takahashi e Yamanaka, sendo então intensamente estudadas. Através desta
técnica, células somáticas adultas, são modificadas e adquirem comportamento muito semelhante
ao das células-tronco embrionárias, inclusive molecularmente. O uso das iPS reduz questões
éticas relacionadas ao uso de células-tronco de origem embrionária. No entanto, os mecanismos
biológicos relacionados à formação e diferenciação das iPS bovinas ainda não foram
completamente elucidados (TAKAHASHI; YAMANAKA, 2006; YAMANAKA, 2008). Além
disso, durante muitos anos, acreditou-se que não haveria a possibilidade de renovação da a
reserva oocitária. Porém, com o avanço dos estudos da oogênese, a teoria se tornou alvo de
grande controvérsia (TILLY; JOHNSON, 2007). Diante do exposto, as pesquisas que visam
solucionar os problemas de infertilidade em humanos, bem como aumentar a eficiência
reprodutiva de animais de alto valor genético ou em via de extinção, são de grande importância.
Assim, a possibilidade de formação de novos oócitos após o nascimento, utilizando-se células-
tronco de diferentes origens, pode ter uma importante aplicação terapêutica reprodutiva, mas
ainda não se sabe quais os mecanismos ideiais para a produção de gametas.
Para um melhor entendimento da importância deste trabalho, a seguir serão
abordados aspectos relacionados ao ovário mamífero, formação das células germinativas e
diferenciação em oogônias, formação e diferenciação de células-tronco embrionárias, células-
tronco adultas, células-tronco de pluripotência induzida, as funções das proteínas morfogenéticas
27
ósseas (BMP) e do fluido folicular no processo de formação de células germinativas e
diferenciação em oócitos.
28
2 REVISÃO DE LITERATURA
2.1 Ovário mamífero
A palavra ovário é derivada do latim ovum que significa ovo. Este órgão, além de ser
a gônada feminina que contém o suprimento de células germinativas para a produção da próxima
geração, funciona também como uma glândula reprodutiva que controla diversos aspectos
fisiológicos e do desenvolvimento das fêmeas (EDSON; NAGARAJA; MATZUK, 2009). Dessa
forma, o ovário possui dois papéis primários: (i) a liberação de um oócito inteiramente
competente para fertilização e desenvolvimento embrionário, caracterizando a função
gametogênica e (ii) a preparação de órgãos reprodutivos acessórios para a gestação e o
nascimento através da produção de hormônios esteroides, que retrata a função endócrina deste
órgão (GOUGEON, 2004).
Figura 1 - Desenho esquemático do ovário de mamíferos com suas diversas estruturas.
O ovário mamífero apresenta folículos em diferentes estágios de desenvolvimento (primordiais, primários,
secundários, terciários e pré-ovulatórios), corpo lúteo e oogônias, na superfície ovariana (TILLY; JOHNSON, 2007).
Fonte: Adaptado de http://academic.rcc.edu/moore/docs/Bio30/Lecture%208%20-%20Oogenesis.pdf.
29
Nos mamíferos, o tamanho e o formato do ovário variam de acordo com a espécie e
com a fase do ciclo estral/menstrual (HAFEZ; HAFEZ, 2004). Em geral, os ovários dos
mamíferos estão organizados em córtex e medula (Figura 1) (ARAKI, 2003; GARTNER;
HIATT, 2007). O epitélio superficial, pode variar o formato de pavimento à cuboide (mais
comum), que cobre o ovário é denominado epitélio germinativo. Imediatamente abaixo deste
epitélio, fica a túnica albugínea, uma cápsula de tecido conjuntivo denso não modelado, que é
pouco vascularizada (GARTNER; HIATT, 2007). O córtex do ovário, com uma alta densidade
celular, é constituído por uma estrutura de tecido conjuntivo, o estroma, que contém células
semelhantes a fibroblastos (GARTNER; HIATT, 2007), assim como folículos ovarianos e/ou
corpos lúteos em vários estágios de desenvolvimento e regressão (HAFEZ; HAFEZ, 2004). A
medula, por sua vez, é constituída principalmente por tecido conjuntivo frouxo, altamente
vascularizado (vasos sanguíneos e linfáticos), e fibras nervosas (GARTNER; HIATT, 2007).
Histologicamente, não há um limite bem definido entre essas duas regiões (GARTNER; HIATT,
2007).
Os eventos que marcam a morfogênese do ovário fetal incluem a colonização por
células germinativas primordiais (CGP), interação das células germinativas primordiais com
células somáticas, formação dos cordões ovígeros e, finalmente, o desaparecimento dos cordões
ovígeros com concomitante estabelecimento de uma população de folículos primordiais e uma
complexa rede vascular (JUENGEL et al., 2002). A cronologia no desenvolvimento desses
eventos que culminam na formação dos folículos primordiais parece ser semelhante em todos os
mamíferos (SAWYER et al., 2002).
Durante muitos anos, acreditou-se que a reserva folicular ovariana fosse formada na
embriogênese e que não haveria a possibilidade de renovação. Porém, com o avanço da ciência e
consequente aumento de metodologias para o estudo da foliculogênese, a teoria se tornou alvo de
grande controvérsia, pois demonstrou-se, então, a possibilidade de renovação, todavia, ainda não
se sabe se ela realmente ocorre naturalmente e em quais condições poderia ocorrer (TILLY;
JOHNSON, 2007).
2.2 Formação e desenvolvimento das células germinativas e de oócitos
30
As CGP foram identificadas pela primeira vez em mamíferos por Chiquoine em
1954, que encontrou uma população de células capaz de gerar oócitos e espermatozoides
(CHIQUOINE, 1954). As CGP, que se originam do epiblasto proximal, são os precursores
embrionários dos gametas e a especificação destas células é uma das primeiras decisões do
destino celular feita pelo embrião (YOUNG; DIAS; LOVELAND, 2010). Em camundongos,
após cerca de 4,0-4,5 dias pós-concepção (dpc), o blastocisto é composto por três tipos de células,
o trofectoderma, o ectoderma primitivo e a endoderme primitivo (ROSSANT; TAM, 2009). Após
a implantação, em torno de 6,0 dpc, um pequeno grupo de células do epiblasto proximal,
originado do ectoderma primitivo, é direcionado para entrar na linhagem germinativa e formar as
CGPs (DE SOUSA LOPES et al., 2004; 2007) (Figura 2).
As CGP tornam-se identificáveis durante o início da gastrulação pela a atividade
positiva da fosfatase alcalina (AP). Além disso, formam um grupo de ~40 células na base do
alantóide incipiente no mesoderma extra-embrionário (ExM), por volta do 7,25 dia embrionário
(GINSBURG; SNOW; MCLAREN, 1990; LAWSON; HAGE, 1994). Posteriormente, e
concomitante com um aumento em seu número, elas começam a migrar em direção ao
endoderma do intestino primitivo em desenvolvimento e se movem através dele. Através de
movimentos ameboides, as CGP, então, saem do endoderma e seguem para o mesentério, em
torno do dia E10,5 para colonizar gônadas embrionárias, onde elas proliferam (elas estarão
sofrendo mitoses, e o número dessas células aumenta significativamente) (SOTO-SUAZO;
ZORN, 2005), e iniciam mais uma diferenciação em oócitos ou espermatozóides dependendo do
sexo (BOWLES; KOOPMAN, 2007). Nem todas as CGP irão obter sucesso nesta migração, e
aquelas que não alcançarem a crista genital irão se degenerar (ADAMS et al., 2008). Um evento
chave que ocorre nas CGP durante esta fase proliferativa em ambos os sexos (machos e fêmeas) é
uma reprogramação epigenética, mais notavelmente, uma desmetilação do DNA de todo o
genoma, que inclui a inativação do imprinting genômico (SAITOU; KAGIWADA; KURIMOTO,
2012). No embrião fêmea (XX), as CGP continuam a proliferar até ~E13,5 (quando atingem
cerca de 25.000 células) e, posteriormente, entram na prófase I das divisões meióticas
(HILSCHER et al.,1974; SPEED, 1982). Em ovinos e bovinos, a diferenciação das CGP em
oogônias ocorre no 31º e 42º dia de gestação, respectivamente (RÜSSE, 1983). A fase que
antecede este processo meiótico é marcada pela replicação do DNA (GORDON, 1994). Nesta
fase, as oogônias se diferenciam e passam então a ser denominadas de oócitos (FAIR, 2003).
31
Figura 2 - Representação esquemática do desenvolvimento das CGP e dos sinais envolvidos na
regulação da migração e colonização CGP em camundongos.
Breve resumo da especificação e migração das CGP, bem como colonização das gônadas em camundongos. Epi:
epiblasto; (AVE: endoderma visceral anterior; ExE: ectoderma extra-embrionário; CGP: Células germinativas
primordiais; EM: mesoderma embrionário; ExM: mesoderma extra-embrionário; Hg: intestino primitivo; DA: aorta
dorsal; NT: tubo neural; NoC: notocorda; IM: mesoderma intermediário; NeC: cordão nefrogênico. Fonte: Adaptado
de SAITOU; YAMAJI, (2010); NIKOLIC et al., (2016).
32
As oogônias são células-tronco germinativas que expandem sua população por meio
de uma alta frequência de divisões mitóticas (PICTON; BRIGGS; GOSDEN, 1998). Devido às
rápidas e consecutivas divisões mitóticas, as oogônias são muitas vezes unidas em aglomerados
por pontes citoplasmáticas intercelulares, formando um sincício ou ninhos de oogônias,
organizados em cordões ovígeros (PICTON, 2001; TINGEN; KIM; WOODRUFF, 2009).
Durante a replicação do DNA, o ácido retinoico atua ativando proteínas responsáveis por
desencadear o início da meiose, como por exemplo, STRA-8, REC-8, DMC-1 e SCP-3
(MARQUES-MARI et al., 2009; BOWLES et al., 2006; KOUBOVA et al., 2006). As oogônias
apresentam um maior número de organelas intracelulares, antes de se diferenciarem em oócitos
primários (SUH; SONNTAG; ERICKSON, 2002; ABIR et al., 2006). Os oócitos primários
continuam a divisão meiótica passando pelos estágios de leptóteno, zigóteno e paquíteno da
prófase I, até atingirem o estágio de diplóteno (ARAKI, 2003). Os oócitos permanecem nesse
estágio até pouco antes da ovulação, em que após estimulação hormonal os oócitos completam a
primeira divisão meiótica com a extrusão concomitante do primeiro corpo polar e param
novamente na segunda divisão meiótica (oócito secundário), a qual será somente é concluída
após a fertilização (BOWLES; KOOPMAN, 2007). Durante a fertilização pelo espermatozoide, o
oócito completa a segunda divisão meiótica e extrusa o segundo corpo polar.
2.2.1 Mecanismos de especificação das CGPs
A origem exata da linhagem de células germinativas e o mecanismo de especificação
das CGP são ainda indefinidos, especialmente devido à ausência de marcadores específicos que
retratem os primeiros processos de especificação destas células (SAITOU; YAMAJI, 2010).
Entretanto, sabe-se que as CGP precursoras são formadas sob o controle de sinais oriundos das
células vizinhas, tais como as proteínas morfogenéticas ósseas (BMPs), -2, -4, e -8 (Figura 3)
(HUMMITZSCH et al., 2015). A análise da expressão gênica da população de CGP fundadoras
com E7,25 identificou dois genes, Fragilis e Stella, que são altamente e especificamente
expressos em CGP, respectivamente (SAITOU; BARTON; SURANI, 2002). Fragilis (Mouse
interferon-induced protein like gene-1, mil-1 ou ainda conhecido como interferon-induced
transmembrane protein 3, Ifitm3) (TANAKA; MATSUI, 2002) é um membro das proteínas
transmembranares induzíveis pelo interferon. Enquanto que Stella (também conhecido como
33
primordial germ cell 7, Pgc7, ou ainda conhecido como Developmental pluripotency-associated
3, Dppa3) (SATO et al., 2002) é uma pequena proteína que se transloca do citoplasma ao núcleo
e ainda na direção oposta. Fragilis começa a ser expresso nas células no epiblasto proximal, com
~E6,25-E6,5, e sua expressão se intensifica no mesoderma extra-embrionário posterior. Já a
expressão de Stella começa especificamente nas células que já estão expressando Fragilis no
mesoderma extra-embrionário (~E7,0-E7,25), e continua a ser expressa na migração das CGP.
Em aproximadamente E7, pequenos grupos de CGP, que são estabilizados por E-caderina, foram
localizados posterior à linha primitiva no mesoderma extra-embrionário (HUMMITZSCH et al.,
2015).
As células Stella-positivas mostram uma elevada expressão de fosfatase alcalina
tecido não específica (Tnap), um gene para a atividade AP em CGP (MACGREGOR;
ZAMBROWICZ; SORIANO,1995). As células com expressão positiva para Stella e altos níveis
de RNAm para Fragilis reprimem a expressão de genes Homeobox tais como Hoxb1, Hoxa1,
Evx1 e VL1, enquanto que as células Fragilis-positivas, mas Stella-negativas mantém a expressão
de genes Hox (SAITOU; BARTON; SURANI, 2002). Saitou et al. (2002) propuseram que as
células Stella-positivas e Homeobox-negativas são importantes para o estabelecimento das CGP.
Além disso, estudos revelaram que nem Fragilis e nem Stella são genes essenciais para a
especificação das CGP (PAYER et al., 2003; LANGE et al., 2008).
Essa variação na expressão dos genes Homeobox é importante, uma vez que o papel
desses genes é especificar a identidade das células ao longo do eixo do corpo ou induzir a
diferenciação das células para linhagens celulares somáticas específicas. Isto sugere que as
células germinativas fundadoras adquirem a capacidade de evitar a especificação somática,
prevenindo ou suprimindo a expressão dos genes Homeobox. Esta poderia ser uma das principais
características que as células germinativas de mamíferos possuem e lhes permite manter ou
recuperar a totipotência (NIKOLIC et al., 2016). Este conceito é apoiado pela expressão
continuada de Oct4 (Octamer-binding transcription factor 4, também conhecido por Pou5f1), e
outros genes de pluripotência em células germinativas (YEOM et al., 1996). O gene Oct4 é
considerado um gene chave para pluripotência (NIWA, 2007), além de ser usado como um
marcador específico para a linhagem de células germinativas, apenas após ~E7,75 (YEOM et al.,
1996).
34
A análise do transcriptoma das CGP levou à identificação de dois genes reguladores
chave para a especificação das CGP, Blimp1 (B lymphocyte-induced maturation protein-1,
também conhecido como PR domain zinc finger protein 1 ou ainda PR domain containing 1,
[Prdm1]) e Prdm14 (PR domain zinc finger protein 14 ou ainda PR domain containing 14)
(OHINATA et al., 2005; VINCENT et al., 2005; YABUTA et al., 2006; KURIMOTO et al.,
2008; YAMAJI et al., 2008). Ainda para a especificação de CGP em mamíferos, a expressão do
gene Tcfap2c (também conhecido como AP2g) é necessária (WEBER et al., 2010). Todos os
precursores de CGP Blimp1-positivas inicialmente expressam os genes Hox e reprimem Sox2
(YABUTA et al., 2006; KURIMOTO et al., 2008). No entanto, a partir E6.75, as células Blimp1-
positivas começam a reprimir os genes Hox e expressam Sox2, Stella, e Nanog (YAMAGUCHI
et al., 2005; KURIMOTO et al., 2008). As células Blimp1-positivas continuam a expressar Oct4,
que regula o aumento da expressão de cerca de 500 genes de “especificação de células
germinativas” e reduz a expressão de cerca de 330 genes “somáticos” (KURIMOTO et al., 2008).
Figura 3 - Especificação da linhagem germinativa e eventos moleculares.
Durante o desenvolvimento embrionário in vivo, como resultado da estimulação por BMP-4, BMP-2 e BMP-8b, um
subconjunto de células do epiblasto começa a expressar os genes Fragilis e Blimp1 e tornam-se CGP que migram em
35
direção ao cume gonadal, onde proliferam. Durante a migração destas células, há a expressão de genes como Stella e
c-Kit e iniciam a colonização do cume gonadal. Neste momento, as CGP expressam os marcadores pré-meióticos
Dazl e Vasa. As proteínas SCP1, SCP2 e SCP3 são específicas de células germinativas em meiose, assim como o
gene Dmc1. Nas fêmeas, as células germinativas entram em meiose e param na prófase I. As células germinativas
masculinas não entram em meiose, apenas após o nascimento. Os marcadores pós-meióticos GDF-9 e TEKT1 são
específicos de gametas haplóides maduros. Fonte: adaptado de Marques-Mari (2009).
2.2.1.1 Sinalização da especificação das CGPs
Por volta de 5,5 dias de desenvolvimento embrionário em camundongos, as células
do epiblasto adquirem competência para responder a BMP-4 pelas atividades de NODAL e
WNT3. Por outro lado, a sinalização NODAL mediada por SMAD2/FOXH1 específica de
células da endoderme visceral distal, começam a fornecer sinais agonistas. Sinais do ectoderma
extraembrionário, incluindo BMP8b, aparentemente previne o endoderma visceral proximal de
diferenciar-se em endoderme visceral distal, restringindo assim, a atividade anti-agonista. Por
volta dos dias 6,0-6,25, a endoderme visceral distal move-se para formar endoderme visceral
anterior, e um subconjunto de células do epiblasto recebem altos níveis de sinais da BMP-4 a
partir do ectoderma extraembrionário, os quais são especificados e iniciam a síntese de PRDM1 e
PRDM14, que são marcadores positivos de CGPs (Figura 4). Desta forma, estes dois fatores têm
sido utilizados para induzir a especificação de CGPs a partir de células-tronco embrionárias.
As BMP-4 e BMP-8b induzem a formação de CGPs na região do epiblasto do disco
embrionário (Figura 4, YING; ZHAO, 2001; GÜNESDOGAN; MAGNÚSDÓTTIR; SURANI,
2014). As CGP parecem requerer as proteínas BMP4 ou BMP8b sozinhas ou em combinação. A
proteína BMP-4 secretada a partir do ectoderma extra-embrionário (ExE) ativa a a expressão de
Blimp1 e Prdm14 de maneira dose-dependente. BMP-2 expresso no endoderma visceral proximal
aumenta a via de sinalização, assegurando níveis mais elevados de sinalização de BMP no
epiblasto mais proximal (LAWSON et al., 1999; OHINATA et al., 2005; VINCENT et al.,
2005). Nesse contexto, foi observado que individuos mutantes para os genes Bmp4, Bmp8b,
Bmp2, Smad1, Smad4, Smad5, e Alk2 apresentaram uma redução ou ausência de CGPs AP-
positivas (ZHAO, 2003; SAITOU; YAMAJI, 2010). Ainda não se sabe se as Smads ativam a
transcrição de Blimp1 e Prdm14 direta ou indiretamente (OHINATA et al., 2009). Baseados
nesses achados foi proposta uma via de sinalização para a especificação das CGPs, com base na
influência da expressão de Blimp1 e Prdm14 no epiblasto (Figura 5) (OHINATA et al., 2009).
36
Figura 4 – Representação esquemática do papel da sinalização das BMPs e WNT durante a
formação das células germinativas primordiais (CGPs) em embriões.
A BMP-4 produzida no ectoderma extraembrionário e BMP-2 produzida a partir do endoderma visceral induz a
fosforilação de SMAD1 e SMAD5, que formam um complexo com SMAD4. Este complexo transloca-se para o
núcleo e liga-se, presumivelmente, a reguladores e promotores de genes que são necessários para estabelecimento do
destino das CGP. Além disso, a sinalização das BMPs resulta na ativação direta ou indireta da WNT3, que também é
necessária para induzir o destino das CGP. DVE, endoderme visceral distal; ectoderma extraembrionário (ExE),
endoderme visceral (VE); endoderme visceral distal (DVE); endoderme visceral anterior (AVE). Fonte: adaptado de
YING; ZHAO, 2001; GÜNESDOGAN; MAGNÚSDÓTTIR; SURANI, 2014.
37
Figura 5 – Regulação da transcrição da especificação das CGPs.
Esquema das vias genéticas para a especificação das CGPs. Setas e linhas com barras de terminais as indicam vias de
ativação e vias de repressão, respectivamente, conforme demonstrado por experimentos in vivo (KURIMOTO et al.,
2008; YAMAJI et al., 2008); Setas e linhas pontilhadas com barras de terminais indicam vias de ativação e
repressão, respectivamente, como proposto com base em experimentos in vitro (COVELLO et al., 2006; WEST et
al., 2009; WEBER et al., 2010). Fonte: Adaptado de SAITOU; YAMAJI, (2012).
A sinalização WNT também é essencial para o destino das CGP (Figura 4),
possívelmente através de interações pós-transcricionais, sugerindo que a expressão dos membros
do sistema WNT podem atuar na sinalização das BMPs de maneira pós-transcricional
(OHINATA et al., 2009). Em ~E9,5, as CGP migram para o intestino primitivo, e mais tarde
através do mesentério dorsal, para os cumes genitais em desenvolvimento. Durante o processo de
migração, CGPs ainda expressam Tnap, mas também Oct3/4, o proto-oncogene c-Kit, e SSEA
(antígeno específico da fase embrionária) 1 e 3.
2.2.2 Migração de células germinativas primordiais
A migração através da linha primitiva para o endoderma embrionário posterior
adjacente, endoderma extraembrionário e alantóide inicia a especificação das CGP, as células
38
começam a apresentar morfologia polarizada e extensões citoplasmáticas (ANDERSON et al.,
2000). Em roedores, o primeiro passo na migração das CGP é o movimento das células da linha
primitiva para o endoderma posterior em E7,5. Entre E8,5 e E13,5, as CGP Tnap-positivas
proliferam e migram para as cristas genitais, entrando em meiose nas fêmeas ou em mitose nos
machos, e ainda iniciam a diferenciação em oócitos ou espermatozoides (MOLYNEAUX et al.,
2001; TILGNER et al., 2008). Embriões de camundongos E13,5 devem conter cerca de 24.000
CGPs em suas cristas genitais (TAM; SNOW, 1981). Ainda na fase migratória, as CGPs passam
por uma extensa reprogramação do genoma e alterações epigenéticas, tais como metilação do
DNA e modificações das histonas, além disso, o imprinting genômico pode ser necessário para
restaurar a totipotência das linhagens de células germinativas (TILGNER et al., 2008).
Atualmente, não há evidências sobre as diferenças específicas do sexo durante a
migração das CGP em qualquer espécie. Na gônada, um subconjunto de células germinativas
adquire a capacidade de funcionar como células-tronco de linhagem germinativa, que se
submetem a meiose para produzir oócitos ou espermatozoides e promover a geração do
desenvolvimento embrionário (NIKOLIC et al., 2016).
A proteína Vasa é um componente essencial de germoplasma e representa um
complexo pool de RNA e proteínas que são necessários para a determinação de células
germinativas. Mutações no gene para VASA leva à esterilidade em ratas resultante de defeitos
graves na oogênese (SAFFMAN; LASKO, 1999). Nos seres humanos, a expressão de VASA
começa no fim da fase migratória das CGP (CASTRILLON et al., 2000). Tilgner et al. (2010)
demonstraram que a expressão específica de Vasa nas células-tronco de linhagens germinativas
durante a colonização da crista gonadal sugerem que Vasa é requerido para manter as CGP
funcionais. Por exemplo, ratos machos homozigotos para Mvh (Mouse vasa homologue) são
estéreis e exibem defeitos graves na espermatogênese, enquanto as fêmeas homozigotas são
férteis (MENKE; KOUBOVA; PAGE, 2003; LASKO; ASHBURNER, 1990). Outros sinais
envolvidos na regulação da migração e colonização das CGPs são a molécula de adesão E-
caderina (BENDEL-STENZEL et al., 2000) e a moléculas da matriz extracelular integrina β1
(ANDERSON et al., 1999; CHEN et al., 2013). Infelizmente, a função exata desses fatores e as
vias de sinalização ainda não foram completamente elucidadas.
A migração das CGP mantém um programa genômico associada à pluripotência, em
que elas expressam genes de pluripotência (Oct4, Nanog e Sox2), sendo capazes de formar
39
teratomas após a injeção em ratos (SAITOU; YAMAJI, 2012; CHUMA et al., 2005; HAYASHI;
DE SOUSA LOPES; SURANI, 2007). Além disso, CGPs migratórias expressam antígeno
embrionário específico da fase 1 (stage-specific embryonic antigen 1 - SSEA1) (TILGNER et al.,
2008). Após a chegada na gônada, a proteína de ligação ao RNA específico de células
germinativas Dazl (Deleted in azoospermia-like) é essencial para o desenvolvimento das CGP
(LIN; PAGE, 2005).
Muitos estudos revelaram que o Dazl pode se ligar a proteínas específicas chamadas
de fatores de transcrição e potencializar a transcrição gênica (REYNOLDS et al., 2005; 2007;
ZENG et al., 2009). Diversos trabalhos indicam que a proteína DAZL pode ter papéis adicionais
nas CGP, especialmente estando envolvido com a apoptose, regulando a expressão das enzimas
caspases, agindo como um mecanismo que impede a formação de teratomas, eliminando as CGP
aberrantes (COOKE et al., 1996; RUGGIU et al., 1997; TSUI et al., 2000; CHEN et al., 2014).
Gill et al. (2011) indicam que na ausência de DAZL, as CGP não se desenvolvem para além do
estágio de CGP, mostrado pela expressão contínua de marcadores de pluripotência.
A pós-migração das CGP é marcada pela expressão de várias proteínas de ligação ao
RNA, tais como MVH, DAZL e NANOS3 (REYNOLDS et al., 2005; ZENG et al., 2009;
RUGGIU et al., 1997; TSUI et al., 2000; COOKE et al., 1996; CHEN et al., 2014; GILL et al.,
2011; MCLAREN, 2003). Em camundongos, as CGP femininas rapidamente iniciam a meiose e
param no estágio diplóteno da prófase I da meiose I, enquanto em machos as células se dividem
mitoticamente e entram em repouso quando passam a ser chamados de gonócitos (NIKOLIC et
al., 2016). Especificamente, as CGP aumentam a expressão de genes que lhes permitam sofrer
diferenciação sexual e a gametogênese, como Stra8 (gene requerido para a iniciação meiótica)
junto com Rec8 e Dmc1, enquanto suprimem a pluripotência (LIN et al., 2008; BALTUS et al.,
2006; KOUBOVA et al., 2014; HU et al., 2015).
2.3 Células-tronco: definição, classificação e aplicações
O conceito de células-tronco surgiu a partir de experimentos pioneiros realizados no
início dos anos 1960 por Ernest A. McCulloch e James E. Till que observaram a presença de
colônias hematopoiéticas no baço de camundongos irradiados e que haviam recebido transplante
40
de medula. Essas colônias eram derivadas de uma única célula, a célula-tronco (TILL;
MCCULLOCH; SIMINOVITCH, 1964).
As células-tronco são uma população de células indiferenciadas, caracterizadas pela
capacidade de sofrer auto-renovação e diferenciação em vários tipos de tecido (KOŹLIK;
WÓJCICKI, 2014). Com base no seu potencial de diferenciação, as células-tronco são
classificadas como totipotentes, pluripotentes, multipotentes e unipotentes. As células-tronco
totipotentes estão presentes nas primeiras fases da ontogênese e podem diferenciar-se em todos os
tecidos embrionários e na placenta. As células pluripotentes podem ser coletadas a partir da
camada interna do blastocisto, e podem originar células das 3 camadas germinativas (ectoderma,
endoderma e mesoderma). As células-tronco multipotentes podem ser encontradas em quase
todos os tecido. Durante muito tempo acreditou-se que elas poderiam se diferenciar em células de
uma camada germinativa (por exemplo, as células-tronco hepáticas poderiam diferenciar-se
apenas em hepatócitos ou células dos ductos biliares), mas estudos recentes têm revelado que
algumas células multipotentes têm o mesmo potencial das células-tronco pluripotentes. Já as
células-tronco unipotentes, com menor potencial de diferenciação, são capazes de originar apenas
um tipo de célula (por exemplo, as células-tronco da epiderme podem diferenciar-se apenas em
células epiteliais escamosas queratinizadas superficiais) (BECK; BLANPAIN, 2012; KOŹLIK;
WÓJCICKI, 2014).
As células-tronco também podem ser classificadas em 4 grupos correspondentes às
suas diferentes origens: células-tronco embrionárias (ESC), células-tronco fetais, células-tronco
adultas (ASC) e células-tronco de pluripotência induzidas (iPS) (KOŹLIK; WÓJCICKI, 2014). O
zigoto, resultante da fecundação, é caracterizado ser totipotente. Após ~4 dias de crescimento, o
zigoto se transforma em blastocisto. A camada interna é composta por células-tronco
embrionárias, que são pluripotentes. As células-tronco embrionárias podem proliferar-se de
maneira quase ilimitada, além disso, são identificados pela expressão de fatores de transcrição
específicos, como Nanog, Oct4 e Sox2 (WANG et al., 2012). Com a utilização de meios de
cultivo e fatores de crescimento específicos, as células-tronco embrionárias foram cultivadas in
vitro e diferenciadas em outros tipos celulares, incluindo músculos esqueléticos, células
endoteliais, condrócitos, miocardiócitos, entre outros (ROHWEDEL; MALTSEV; BOBER,
1994; MALTSEV et al., 1993; ZHU et al., 2013; LEYDON et al., 2013). Há uma certa restrição
com os trabalhos envolvendo células-tronco embrionárias e fetais, levando em consideração os
41
dilemas éticos e sociais envolvidos na coleta, cultivo e experimentação dessas células-tronco,
além disso, o uso deste tipo celular é restrito em muitos países (ZARZECZNY; CAULFIELD,
2009). Wakitani et al. (2003) indicam que o uso de células-tronco embrionárias pode levar a
formação de células cancerígenas e de teratomas. Isto provavelmente está relacionada a métodos
falhos e a limitações tecnológicas (WAKITANI et al., 2003).
As ASC são específicas para cada órgão, e estão presentes em pequenas quantidades
em todos os tecidos. Por exemplo, as células-tronco hematopoiéticas compõem cerca de 0,001-
0,01% das células sanguíneas (DA SILVA MEIRELLES; CAPLAN; NARDI, 2008). A
morfologia e os marcadores proteicos permitem que cada células-tronco adulta possa ser
classificada de acordo com o tecido que a origina. As ASC foram previamente classificadas como
multipotentes, mas atualmente são conhecidos por serem pluripotentes. Devido ao fenômeno
conhecido como “plasticidade” ou “transdiferenciação”, algumas destas células-tronco adultas
podem produzir um novo tipo célular, quando transferida para outros tecidos (KOŹLIK;
WÓJCICKI, 2014). Em 2006, Takahashi e Yamanaka, demonstraram que, mesmo células
somáticas maduras, quando expostas a genes associados a pluripotência específica, podem
“recuperar” a pluripotência de maneira semelhante às células-tronco embrionárias, com
capacidade de diferenciação em células de qualquer camada germinativa. Estes experimentos
foram realizados primeiro com fibroblastos de camundongos, e posteriormente com fibroblastos
humanos, sendo estas células denominadas de células-tronco de pluripotência induzida
(TAKAHASHI; YAMANAKA, 2006; TAKAHASHI et al., 2007).
As células-tronco mesenquimais multipotentes têm uma ampla aplicação na prática
clínica. As células-tronco mesenquimais da medula óssea são obtidas rapidamente e tem seu uso
amplamente conhecido, no entanto, a sua obtenção envolve um procedimento invasivo e
ineficiente (KOŹLIK; WÓJCICKI, 2014). Novos procedimentos foram desenvolvidos para a
coleta de vários tipos de células-tronco mesenquimais a partir de outras estruturas do corpo
humano, incluindo o tecido adiposo (células-tronco derivadas do tecido adiposo), o sangue de
cordão umbilical (UCB), o periósteo, os tendões, os músculos (células-tronco derivadas dos
músculos), membranas mucosas e pele (DA SILVA MEIRELLES; CAPLAN; NARDI, 2008).
Uma análise comparativa de todos os tipos de células-tronco mesenquimais não revelou grandes
diferenças entre as suas habilidades de se diferenciar em tecidos específicos (KERN et al., 2006).
Para a diferenciação celular, as células-tronco mesenquimais desenvolvem sinalização parácrina,
42
sendo capazes de induzir neovascularização do tecido a partir da liberação de fatores de
crescimento, tais como fator de crescimento do endotélio vascular (VEGF), fator de crescimento
transformante (TGF) e fator de crescimento de hepatócitos (HGF) (CAI et al., 2009).
2.3.1 Células-tronco embrionárias
As células-tronco embrionárias (ESC) são linhagens de células pluripotentes,
geralmente derivadas a partir da massa celular interna do blastocisto. Estas células são
consideradas como um modelo para estudar a embriogênese (Figura 6, DE PAEPE et al., 2014).
Podem ser propagadas indefinidamente em cultura, permanecendo em estado indiferenciado.
Essa pluripotência refere-se à capacidade de uma célula em diferenciar-se em células das três
camadas germinativas in vitro e in vivo. Os fatores de transcrição OCT4 (POU5F1), SOX2 e
NANOG desempenham um papel importante na manutenção deste estado indiferenciado
(BOYER et al., 2005; 2006).
Em ratos, células-tronco embrionárias, as células-tronco do trofoblasto (TS) e células-
tronco do endoderma extra-embrionário (XEN) foram derivadas do blastocisto (YAMANAKA et
al., 2006). As linhagens de ESC e mTS (células-tronco do trofoblasto murinos) têm sido
derivadas de blastômeros no estágio de 1 a 8 células (CHUNG et al., 2006).
Em humanos, as linhagens de células-tronco embrionárias pluripotentes (hESC)
(THOMSON et al., 1998; REUBINOFF et al., 2000) foram derivadas de blastocistos pré-
implantados, sendo caracterizadas por marcadores de superfície celular, capacidade de
diferenciação, transcriptômica e (epi)-genômica. As hESC são capazes de se diferenciar em
células do endoderma extra-embrionário in vitro (THOMSON et al., 1998; LEE et al., 2013), e
em células trofoblásticas (THOMSON et al., 1998; XU et al., 2002; GERAMI-NAINI et al.,
2004; HARUN et al., 2006). A diferenciação de linhagens de hESC em células trofoblásticas
induz a expressão de fatores de transcrição específicos (Cdx2 e Gata3), genes associados com o
citoesqueleto (Krt7 e Krt8) e a matriz extracelular (Cola4), genes envolvidos na invasão (Igf2,
Cdh1 ou E-caderina) e hormônios (β-hCG) (MARCHAND et al., 2011).
43
Figura 6 – Isolamento e diferenciação das células-tronco embrionárias.
As células-tronco embrionárias são removidas do blastocisto cerca de 4-5 dias após a fertilização. O processo ocorre
quando o embrião em desenvolvimento apresenta aproximadamente 150 células. Essas células, quando estimuladas,
apresentam o potencial de diferenciação de linhagens celulares. As células coletadas são cultivadas sob condições
especiais de laboratório, podendo ser usadas para o tratamento de diferentes tipos de doenças, bem como para o
transplante em órgãos vitais. Fonte: Adaptado de
<http://sgugenetics.pbworks.com/w/page/38198357/Embryonic%20Stem%20Cells>.
Inicialmente, as hESC foram obtidas a partir da massa celular interna (THOMSON et
al., 1998; REUBINOFF et al., 2000) com uma alta taxa de derivação a partir do 6º dia de
blastocisto (CHEN et al., 2009). As hESC também têm sido derivadas de blastômeros no estágio
de 4 a 8 células (KLIMANSKAYA et al., 2006, 2007; FEKI et al., 2008; GEENS et al., 2009;
ILIC et al., 2009). As hESC derivados da massa celular interna e derivados de blastômeros têm
perfis semelhantes de transcrição (GIRITHARAN et al., 2011; GALAN et al., 2013). As hESC
apresentam expressão de marcadores como Dazl e Stella, que são característicos de células
germinativas iniciais (ZWAKA; THOMSON, 2005). No entanto, a diferenciação de hESC em
44
células germinativas ainda é um processo falho (GEIJSEN et al., 2004; NAYERNIA et al., 2006;
AFLATOONIAN et al., 2009).
2.3.2 Células-tronco adultas
Em organismos multicelulares, grupos de células semelhantes especializadas são
agrupadas em tecidos e órgãos para executar funções específicas. No decorrer da vida adulta,
estas células podem perder de maneira progressiva as suas funções. Para compensar essa perda
contínua de células diferenciadas, novas células funcionais devem ser geradas de modo que
tecidos permaneçam em homeostase. A manutenção e o reparo deste ciclo nos tecidos adultos,
geralmente dependem de uma pequena população de células, as chamadas células-tronco adultas,
que possuem a capacidade de se auto-renovar, dando origem a células diferenciadas, mantendo o
seu número de células constante nos tecidos (WATT; HOGAN, 2000; FUCHS; CHEN, 2013).
A capacidade de auto-renovação tem sido considerada a definição característica de
ASC (CLERMONT; LEBLOND, 1952, 1953; LEBLOND; STEVENS, 1948). Para a
manutenção da homeostase, a proliferação e diferenciação de ASC deve ser perfeitamente
equilibrada, de modo que, a divisão seguinte, uma das células- filha permanece como célula-
tronco, ao passo que as outras se diferenciam diretamente, ou ainda através de uma série de
divisões (KRIEGER; SIMONS, 2015).
O processo pelo qual as células-tronco dão origem a diferentes tipos celulares não é
completamente compreendido e é denominado de diferenciação celular. Acredita-se que entre os
fatores responsáveis por este fenômeno, estejam substâncias secretadas por células vizinhas, além
de componentes do microambiente, presentes de forma solúvel ou ligados a matriz-extracelular
(CARMO; SANTOS, 2009). As ASC estão presentes em praticamente todos os tipos de tecidos,
no entanto, sua proporção em relação aos outros tipos celulares é baixa, o que torna estas células,
difíceis de identificar, isolar e purificar (CARMO; SANTOS, 2009).
As ASC podem ser divididas em dois tipos principais: as células-tronco
hematopoiéticas (responsáveis pela formação dos diferentes tipos celulares sanguíneos), e as
células-tronco mesenquimais (MSC) (pode formar tecido cartilaginoso, ósseo, adiposo e
muscular). As MSC são um tipo de células-tronco multipotentes que podem ser isoladas de vários
tecidos e membranas fetais ou adultas (BIANCHI et al., 2001; FILIOLI URANIO et al., 2011)
45
incluindo tecido adiposo, medula óssea, sangue do cordão umbilical (KERN et al., 2006;
TAKEMITSU et al., 2012; STRIOGA et al., 2012), polpa dentária (XIAO; TSUTSUI, 2013),
placenta e músculos (KISIEL et al., 2012). Ambas podem ser encontradas na medula óssea, mas
a última também pode ser encontrada em outros tecidos (ZAGO; COVAS, 2006). A figura 7
ilustra um exemplo de diferenciação de células-tronco mesenquimais.
Devido a sua função de manutenção de tecidos adultos e de resposta a lesões do
organismo, atribui-se as ASC um grande potencial terapêutico, no tratamento das mais variadas
injúrias, como diabetes, doenças auto-imunes, na hematologia, na oftalmologia e na regeneração
de lesões provocadas por acidentes. Devido a este provável potencial, tais células têm se tornado
alvo de inúmeras pesquisas nos últimos anos, muitas das quais obtiveram resultados favoráveis
(CARMO; SANTOS JÚNIOR, 2009).
Figura 7 – Isolamente e diferenciação de células-tronco mesenquimais.
As MSC podem ser isoladas de diferentes tecidos como adiposo, medula óssea, sangue do cordão umbilical, polpa
dentária, placenta e músculos. Essas células, quando estimuladas podem se diferenciar tipos celulares, como
adipócitos, condrócitos e osteócitos. Fonte: Disponível em: <http://www.eurostemcell.org/factsheet/mesenchymal-
stem-cells-other-bone-marrow-stem-cells>.
Além de classificadas como multipotentes, as MSC expressam um nível
relativamente elevado de marcadores de pluripotência semelhantes às ESC, como Oct4, Nanog e
Sox2 (SACHS et al., 2012; TAKEMITSU et al., 2012; KISIEL et al., 2012). Estes fatores de
transcrição estão envolvidos na regulação da multipotência, auto-renovação e proliferação das
MSC (TAKEMITSU et al., 2012; KISIEL et al., 2012). Oct4 está envolvido no desenvolvimento
inicial dos mamíferos e é essencial para a formação de massa celular interna de embriões e para a
46
manutenção de ESC (NICHOLS et al., 1998). Sox2 regula a expressão de Oct4, e mantém o
estado pluripotente de ESC, enquanto Nanog é necessário para a manutenção do estado
indiferenciado e para a auto-renovação das células-tronco (TAKEMITSU et al., 2012; ZOMER et
al., 2015).
In vivo, as MSC proporcionam um suporte estrutural em diferentes órgãos e regulam
o fluxo de algumas substâncias. Além disso, elas apresentam uma alta e rápida taxa de
proliferação em meio de cultura simples, e podem ser mantidas in vitro, sem alterações do
cariótipo por várias passagens (WEBSTER et al., 2012). As MSC têm a capacidade de se
diferenciar em vários tipos de células, tais como adipócitos, osteócitos e condrócitos, a partir da
camada germinativa mesodérmica (SACHS et al., 2012; DU et al., 2010). Esta plasticidade
depende do ambiente da matriz extracelular e da presença de fatores de crescimento solúveis
(VIDANE et al., 2013). Alguns autores induziram a diferenciação de MSC em células de outras
camadas germinativas, como neurônios (KRAMPERA et al., 2007), que são originados do
ectoderma, e hepatócitos que são derivados a partir do endoderma (AURICH et al., 2009). No
entanto, a diferenciação em tecidos não-mesodérmicos ainda é controversa, devido a falta de
resultados in vivo (STRIOGA et al., 2012).
Devido à sua plasticidade, as MSC são consideradas o tipo de célula mais importante
para medicina regenerativa, e são as mais extensamente estudadas em ensaios pré-clínicos e
clínicos. Suas vantagens para aplicação clínica incluem o fácil isolamento e alto rendimento, alta
plasticidade, bem como a capacidade para mediar a inflamação e promover o crescimento e
diferenciação celular, reparação de tecidos por imunomodulação e imunossupressão, e ainda,
estão isentas de implicações éticas (LAGE-CONSIGLIO et al., 2013; PLOCK et al., 2013;
INSAUSTI et al., 2014). Além disso, as MSC não formam teratomas após o transplante,
garantindo a segurança para o receptor (ZOMER et al., 2015).
As MSC derivadas a partir da medula óssea têm sido as mais intensivamente
estudadas. No entanto, procedimentos invasivos são necessários para o seu isolamento, e a
quantidade e a qualidade das células isoladas variam de acordo com a idade do doador (ZOMER
et al., 2015). Um número pequeno de MSC está presente em aspirados de medula óssea em
comparação com as células totais da composição do estroma da medula óssea (BYDLOWSKI et
al., 2009). Devido à heterogeneidade da população de células, as suas propriedades imunogênicas
dependem de várias configurações, tais como métodos de isolamento, de superfície e meio de
47
cultura e suplementação química (WEBSTER et al., 2012). Portanto, a identificação de fontes
alternativas de MSC tem sido o ponto focal de pesquisas recentes. Entre diferentes fontes de
MSC, o tecido adiposo destaca-se pela sua acessibilidade e pela abundância de células isoladas
(LAM; LONGAKER, 2012; DU et al., 2010; HOUSMAN et al., 2002; CHERUBINO et al.,
2011; KIM et al., 2011). Cada isolamento resulta em aproximadamente 100 vezes mais células do
que o isolamento da medula óssea (DEY; EVANS, 2011), e o processo é muito menos invasivo
(SACHS et al., 2012).
2.3.2.1 Células-tronco ovarianas
Diversos trabalhos indicam a existência das células-tronco ovarianas (OSC) nos
ovários das fêmeas adultas (ZOU et al., 2009; WHITE et al., 2012; ZHANG et al., 2012;
BHARTIYA et al., 2013; PARTE et al., 2014). Esta tese refuta as observações de Zuckerman et
al. (1951), que postularam o dogma de que nos ovários de mamíferos na vida pós-natal não há
OSC germinativas renováveis, assim, apoiando a hipótese de que durante a vida há um pool
numericamente fixo de oócitos que serão direcionados para a fertilidade. Este pool contaria com
cerca de 106 oócitos na puberdade. No entanto, esse número diminui com o envelhecimento até a
completa exaustão na menopausa (ZUCKERMAN et al., 1951; SILVESTRIS et al., 2015). Esta
hipótese foi posteriormente refutada por Johnson et al. (2004), que identificaram a presença de
OSC mitoticamente ativas em ovários murinos jovens e adultos, capazes de garantir a
disponibilidade de oócitos após o nascimento.
Em seus experimentos, Johnson et al. (2004) observaram uma discordância entre a
taxa de depleção folicular e a vida útil reprodutiva, e ainda, encontraram aspectos histológicos
normais nos ovários de ratos que foram esterelizados quimicamente por busulfan (quimioterápico
usado para o tratamento de leucemia), mostrando folículos saudáveis em maturação e corpo lúteo
após o receberem fragmentos de ovários de animais saudáveis. No entanto, Bristol-Gould et al.
(2006) simularam a dinâmica da progressão folicular murina durante a vida útil de camundongos
através de dois modelos matemáticos, chamados de “células-tronco” e “modelo de pool fixo”,
que argumentam que o declínio fisiológico da população folicular apoia a teoria da “população
fixa de oócitos” como reserva ovariana. Na verdade, foi descrito a expressão de marcadores de
linhagens de células germinativas incluindo Oct4, MVH, Dazl, Stella e Fragilis na medula óssea
48
de camundongas fêmeas adultas, e depois transplantadas para a medula óssea de fêmeas adultas
pré-esterilizadas com ciclofosfamida e bussulfan, detectando-se assim a geração significativa de
novos folículos contendo oócitos e também a formação de corpo lúteo (JOHNSON et al., 2005).
Assim, essas observações preliminares deram origem a novos estudos que visam o isolamento
das OSC e sua transferência para animais estéreis com o intuito de recuperar a sua fertilidade
(SILVESTRIS et al., 2015).
A primeira tentativa de isolar e cultivar as OSCs germinativas em mamíferos foi
realizada por Zou et al. (2009) que purificaram OSCs de camundongas fêmeas neonatais e
adultos, que foram infectadas com vírus GFP (proteína fluorescente verde) e transplantadas para
ovários de camundongas inférteis. As células transplantadas participaram da oogênese e geraram
descendentes (ZOU et al., 2009). White et al. (2012) isolaram e purificaram OSC humanas
baseados na detecção imunológica de um marcador de superfície celular, o Dead box polypeptide
4 (Ddx-4). Essas células mostraram um padrão de expressão de genes de linhagens germinativas
e, portanto, foram estabelecidas em cultura, injetadas em biópsias no tecido cortical de ovários
humanos adultos. Foi observada a formação de novos folículos contendo oócitos do tecido
transplantado (WHITE et al., 2012). Em contrapartida, Zhang et al. (2012) através de imagens de
células vivas e de experimentos de neofoliculogênese, mostram que as células de ovários de ratas
pós-natais, que expressam Ddx4, não entraram em mitose, e nem contribuem para renovação de
oócitos durante a neofoliculogênese.
Virant-Klun et al. (2008; 2009) isolaram OSC a partir do epitélio de superfície do
ovário (OSE) de mulheres normais, de mulheres na pós-menopausa e em mulheres com falha
ovariana precoce (FOP). As células obtidas expressaram Ssea-4, Oct-4, Nanog, Sox-2 e c-Kit.
Durante o cultivo, foi observada a diferenciação em células semelhantes à oócitos, que atingiram
o diâmetro de até 95 µm e expressaram Oct-4, c-Kit, Vasa e Zp2. Os resultados indicam que as
células expressaram marcadores de células-tronco embrionárias, e esses dados podem ser usados
para estudos que visem o tratamento autólogo da infertilidade ovariana e a cura doenças
degenerativas como FOP (VIRANT-KLUN et al., 2008; 2009).
Bhartiya et al. (2013) descreveram a presença de pequenas células-tronco
embrionárias (small embryonic-like stem cells - VSEL) e OSC germinativas no OSE de
mamíferos adultos. As OSC foram coletadas e analisados quanto à expressão de Oct-4a, Ssea-4,
Fragilis, Cd133, dentre outros. Estas células foram cultivadas por 21 dias e sofreram
49
diferenciação espontânea em estruturas semelhantes à oócitos (BHARTIYA et al., 2013). Com
base nestas observações, Parte et al. (2014) isolaram células-tronco do OSE de ovários de
ovelhas usando SSEA-4 como marcador de superfície. Essas células foram analisadas por
imunocitoquímica, imunofluorescência e por RT-PCR para detectar os marcadores específicos de
linhagens de células-tronco e linhagens germinativas. Além disso, durante o cultivo, as células
sofreram mudanças em suas características e diferenciaram-se espontâneamente em estruturas
semelhantes à oócitos (PARTE et al., 2014). Hayashi e colaboradores (2012) induziram células-
tronco embrionárias de camundongas para se diferenciarem em células semelhantes a células
germinativas primordiais (PGCLC) que foram utilizadas para reconstituir o córtex ovariano.
Após o transplante na bursa, os ovários foram reconstituídos, e em seguida, as PGCLCs foram
isoladas e histologicamente analisadas, indicando que as PGCLCs contribuiram para a formação
de oócitos. Os oócitos destes animais foram fertilizados, gerando filhotes que atingiram a idade
adulta (HAYASHI et al., 2012).
Stimpfel et al. (2013) isolaram OSC no OSE de mulheres que expressavam
marcadores de pluripotência como fosfatase alcalina, Ssea-4, Oct4, Ddx4. Essas células exibiram
um elevado grau de plasticidade, uma vez que, quando adequadamente estimuladas,
diferenciaram-se em várias células somáticas derivadas dos 3 folhetos germinativos (mesoderma,
ectoderma e endoderma), e ainda sem formar teratomas em camundongos imunodeficientes
(STIMPFEL et al., 2013).
2.3.3 Células-tronco de pluripotência induzidas e reprogramação celular
Em 2006, Yamanaka et al. identificaram quatro fatores de transcrição, Klf4, Sox2,
Oct4 E c-Myc, capazes de transformar fibroblastos de camundongos (TAKAHASHI;
YAMANAKA, 2006) e humanos (TAKAHASHI et al., 2007), em clones pluripotentes através de
transdução retroviral. As células iPS são geradas a partir da indução da expressão de fatores de
transcrição associados à pluripotência, permitindo que uma célula somática diferenciada possa
inverter a sua condição para o estágio embrionário (ZOMER et al., 2015), ver figura 8.
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Figura 8 – Reprogramação de células adultas e formação das células iPS.
Na formação das células iPS, genes de pluripotêmcia são introduzidos nas células adultas de maneira a induzir a
reprogramação. As células iPS resultantes se assemelham a células-tronco embrionárias e podem ser diferenciadas
em qualquer tipo de célula, que podem ser utilizadas para estudos de doenças, teste de medicamentos ou correção de
genes defeituosos e desenvolvimento de terapias com células. Fonte: Adaptado de
<http://www.eurostemcell.org/factsheet/ips-cells-and-reprogramming-turn-any-cell-body-stem-cell>.
A descoberta de tal tecnologia foi baseada na hipótese de que a reprogramação
nuclear é um processo conduzido por fatores que desempenham um papel crítico na manutenção
da pluripotência das células ESC (TAKAHASHI; YAMANAKA, 2006; YAMANAKA, 2008).
As iPS poderiam implicar na eliminação de problemas éticos e problemas de rejeição após
transplantes, uma vez que podem ser coletadas do próprio paciente (trasnplante autólogo),
ampliando as possibilidades de pesquisa (LAM; LONGAKER, 2012; TAKAHASHI;
YAMANAKA, 2006). Já é bem conhecido que um ou vários fatores de transcrição podem
converter uma célula em outra. Entretanto, os mecanismos pelos quais fatores exógenos alteram o
estado epigenético permanece desconhecida. Os fatores de Yamanaka são os mais utilizados,
51
outras combinações de fatores foram testadas com sucesso, tais como a substituição de c-Myc e
Klf4 por Nanog e Lin28 (YU et al., 2007), ou a exclusão de c-Myc (XU et al., 2013).
Nas células somáticas, os promotores de genes de pluripotência são altamente
metilados, refletindo um estado transcricional reprimido (ZOMER et al., 2015). Mikkelsen et al.,
(2008) mostraram que a geração de células iPS envolve a ativação destes genes, e a sua
desmetilação é utilizada para determinar o sucesso da reprogramação. Quando os genes de
pluripotência exógenos são introduzidos na célula, induzem a expressão de genes pluripotência
endógenos (JAENISCH; YOUNG, 2008). Por sua vez, o aumento da produção de fatores
endógenos induz o silenciamento de genes exógenos por metilação dos promotores (HOTTA;
ELLIS, 2008).
O valor terapêutico de células iPS é a presença de integrações provirais abrigando
oncogenes conhecidos, especialmente c-Myc, bem como Oct4 e Klf4. c-Myc foi dispensável para
a geração de iPS a partir de fibroblastos, e os camundongos quiméricos produzidos por células
iPS que receberam esses três fatores (Oct4, Sox2 e Klf4), não apresentaram formação do tumor,
enquanto as células derivadas dos quatro fatores (Klf4, Sox2, Oct4 e c-Myc) apresentaram
características tumorigênicas (NAKAGAWA et al., 2008; WERNIG et al., 2007). As técnicas de
reprogramação convencionais dependem da integração estável de transgenes, mas podem
introduzir o risco de mutagênese de inserção. Assim, diversas técnicas de reprogramação não-
integrativas foram desenvolvidas para melhorar a qualidade das células geradas (DIECKE et al.,
2014). Os sistemas integrativos consistem em vetores virais, tais como retrovírus (TAKAHASHI;
YAMANAKA, 2006) e lentivírus (PICANÇO-CASTRO et al., 2011). Já vetores não-integrativos
também tem sido utilizados, tais como adenovírus (STADTFELD et al., 2008) ou sistemas não
virais, como plasmídeos (OKITA; YAMANAKA, 2006), proteínas (ZHOU et al., 2009) e
RNAm, que não promovem a integração de DNA complementar (DNAc) dos fatores OSKM para
o genoma na célula (OKITA; YAMANAKA, 2006; WARREN et al., 2010; YU et al., 2009).
Recentemente, novas abordagens foram testadas para induzir a pluripotência, usando moléculas
químicas exógenas (HOU et al., 2013; PENNAROSSA et al., 2013) ou vetores epissomais (uso
de DNA plasmidial) (YU et al., 2009; DIECKE et al., 2014).
Diferentes razões tem levado os pesquisadores a desenvolver estratégias para gerar
células iPS sem a utilização de lentivírus. Em primeiro lugar, a introdução de DNA exógeno
capaz de se integrar em posições aleatórias do genoma representam um risco para a fisiologia da
52
célula, podendo dar origem a uma mutagênese de inserção. O DNA exógeno poderia destruir o
quadro aberto de leitura (ORF) de genes ou influenciar a regulação gênica. Além disso o
silenciamento de transgenes de lentivírus é incompleto, levando a reativação de vetores lentivirais
nas células iPS (WERNIG et al., 2007).
Há diversos estudos sobre a ação de moléculas que poderiam substituir os vetores
virais durante o processo de reprogramação. A maioria dessas moléculas podem melhorar a
eficiência da reprogramação (LIN; WU, 2015). Durante a reprogramação, as células sofrem
alterações a nível transcricional global e também passam por mudanças epigenéticas em seu
DNA (metilação e desmetilação), juntamente com modificações nas histonas, como acetilação e
desacetilação, ou ainda metilação e desmetilação (CHIN et al., 2009; MAHERALI et al., 2007;
HUANGFU et al., 2008; MIKKELSEN et al., 2008; NIE et al., 2012; HAWKINS et al., 2010;
SHU et al., 2013; ZHU et al., 2010; MALI et al., 2010). Huangfu et al. (2008) foram os
primeiros a estudar a aplicação de um composto na geração de iPS, em que avaliaram o efeito do
ácido valpróico (VPA), um inibidor de enzimas histona-desacetilase (HDAC), e descobriram que
a eficiência da reprogramação foi aumentada em 100 vezes, em relação ao método tradicional,
usando vetores de transcrição (HUANGFU et al., 2008). Um inibidor da DNA metiltransferase, o
5-aza-citidina (5-Aza), é um análogo da citosina que se converte em um nucleotídeo-trifosfato in
vivo, possibilitando a sua incorporação no DNA, o que influência a sua estrutura e estabilidade
(JUTTERMANN; LI; JAENISCH, 1994). Assim, o 5-aza-citidina é considerado um agente da
inibição da metilação do DNA, pois inibe a enzima DNA metiltransferase 1 (DNMT1), e é
amplamente utilizado no estudo da regulação epigenética da expressão gênica (JONES; TAILOR,
1980). Há ainda outros compostos que podem ser utilizados na reprogramação, como a
tricostatina A (TSA) que é um potente inibidor de HDAC, induz o acúmulo de histonas acetiladas
no núcleo e subsequente ativação de genes alvo (TADDEI et al., 2005; DOKMANOVIC;
CLARKE; MARKS, 2007; KIM et al., 2009). O butirato de sódio (NaB) aumenta a acetilação
das histonas e é um potente inibidor de HDAC provocando hiperacetilação de histonas H3 e H4
em células de mamíferos (CANDIDO; REEVES; DAVIE, 1978; NÖR et al., 2013).
Zomer et al. (2015) indicam que o estado de pluripotência nas células iPS pode ser
avaliado pela capacidade de formar teratomas in vivo e a formação de corpos embrionárias in
vitro. Além disso, as iPS podem apresentar morfologia semelhante as ESC, como formato
arredondo, grande nucléolo e escasso citoplasma (ZOMER et al., 2015). A capacidade de
53
diferenciação das iPS parece assemelhar-se a das células ESC. Vários estudos descrevem a
diferenciação de uma variedade de tipos de células a partir de células iPS murinas e humanas,
entre eles, cardiomiócitos (CHRISTOFOROU et al., 2013), células de músculo liso (WANG et
al., 2014), células hepáticas (TAKEBE et al., 2013) e neurônios (HEILKER et al., 2014). Além
disso, essas células apresentam comportamento semelhante, quando da diferencição de iPS e
células ESC.
Diversos trabalhos indicam que as células iPS são muito semelhantes, mas não
idênticas a ESC (TAKAHASHI; YAMANAKA, 2006; CHIN et al., 2010; PFANNKUCHE et
al., 2010). Chin et al. (2010) ao compararem o padrão de expressão de ESC e de células iPS em
humanos, observaram modificações das histonas e a expressão de miRNA não codificantes em
ambos os tipos celulares, e construíram um padrão de expressão, que distingue as iPS das ESC
(PFANNKUCHE et al., 2010). Pfannkuche et al. (2010) identificaram 318 genes
diferencialmente expressos entre ESC e iPS humanas e de células em qualquer fase. Além disso,
foi demonstrado que os genes que foram mais expressos em iPS também tiveram uma expressão
aumentada em fibroblastos, quando comparados à expressão em ESC. A mesma conclusão foi
observada quando foram avaliados os genes menos expressos, sendo eles menos expressos em
células iPS do que em ESC, também tendo uma baixa expressão em fibroblastos
(PFANNKUCHE et al., 2010). Dentre os genes expressos em ambas as células, tanto em iPS
como em ESC, há a expressão de inúmeros marcadores de pluripotência como, Oct4, Nanog,
Sox2, Ssea-1, Ssea-3, Ssea-4, Tra1-60, Tra1-81, bem como a atividade da fosfatase alcalina
(LEWITZKY; YAMANAKA, 2007; WANG et al., 2010; SCHEPER; COPRAY, 2009).
2.4 Proteínas morfogenéticas ósseas e seu papel durante a formação das CGPs
Os membros da superfamília Fator de Crescimento Transformante-β (TGF-β),
especialmente as BMPs, regulam uma grande variedade de funções celulares nas células-tronco
pluripotentes (MISHRA; DERYNCK; MISHRA, 2005; ZHANG; LI, 2005). Os membros destas
famílias se ligam especificamente aos seus respectivos complexos receptores, consistindo em
subunidades do tipo I e tipo II. Após a ligação do ligante ao receptor do tipo II, este induz a
fosforilação do receptor do tipo I, o qual por sua vez, transmite o sinal para moléculas de
transdução de sinal intracelular, chamadas receptores ativados ou R-Smads. As BMPs
54
tipicamente utilizam como transdutores de sinal as R-Smads 1/5/8. A fosforilação mediada por
receptores desencadeia a associação dessas R-Smads com um mediador comum ou co-Smad-4, e
a subsequente translocação deste complexo fator de transcrição para o núcleo. Adicionalmente,
os receptores ativados podem influenciar vias Smad-independente, tais como a via MAPK
(Proteína cinase ativada por mitógenos) (TERADA et al., 1999). Para confirmar se as BMPs
atuam no processo de diferenciação, Park; Woods; Tilly, (2013) demonstraram que a BMP-4
regula diretamente a diferenciação de células-tronco oogoniais de mamíferos através da via de
sinalização das SMADs 1/5/8.
Em camundongos, a formação e a proliferação dos precursores das CGP são
dependentes de BMP-2, BMP-4, BMP-8b (LAWSON et al., 1999; DE SOUSA LOPES et al.,
2004; YING et al., 2000; YING; ZHAO, 2001). Camundongos mutantes para BMP-4
apresentaram defeitos graves no desenvolvimento das células germinativas, com uma quase
completa ausência de CGP, enquanto camundongos knockouts para BMP-7 e BMP-8b
demonstraram uma grave redução no número de células germinativas (ZHAO et al., 1996; 2001;
YING et al., 2000; ZHAO, 2003). Lawson et al. (1999) utilizando camundongos mutantes para
BMP-4 demonstraram que esses animais eram desprovidos de CGP e alantóide. Já em mutantes
para Bmp8b foi observada uma redução acentuada no número de células germinativas. Estudos in
vitro estabeleceram que as vias de sinalização da BMP-4 e BMP8b atuam sinergicamente (YING;
QI; ZHAO, 2001).
A proteína morfogenética óssea 2 (BMP-2) é um membro da superfamília BMP, que
desempenha um papel crucial na produção de CGPs (PERA; TROUNSON, 2004). Em ratos, esta
proteína é produzida pelo endoderma visceral e exerce um importante papel na formação de
células germinativas primoridiais positivas para fosfatase alcalina (SAITOU; YAMAJI, 2010). A
BMP-2 também é responsável por estimular a expressão de Prdm1 e Prdm14 no epiblasto
durante a formação das CGPs (SAITOU; YAMAJI, 2010).
Já foi demonstrado que a BMP-4 aumenta o número de CGP migratórias em culturas
de embrião (DUDLEY et al., 2007). Em um trabalho com células-tronco embrionárias, McLaren
(1999) mostrou que a BMP-4 foi requerida no ectoderma extraembrionário, mais do que nas
células epiblásticas (MCLAREN, 2003). O papel da sinalização das BMP na regulação do
comportamento pós-migratório das CGP é menos claro, pois o cultivo de ovários fetais de
camundongos com BMP-2 ou BMP-4 reduziu o número de células germinativas em meiose
55
(ROSS et al., 2003). Além disso, a BMP-4 pode promover a proliferação de CGP pós-migratórias
isoladas de camadas alimentadoras cultivadas in vitro (PESCE et al., 2002). Childs et al. (2010)
identificaram que a sinalização das BMPs está associada ao desenvolvimento do ovário fetal
humano, além disso, a BMP-4 apresenta um papel pró-apoptótico na regulação das CGP pós-
migratórias em humanos. A combinação de LIF e BMP-4 ou LIF e BMP-2 induzem um aumento
na auto-renovação das células-tronco embrionárias, resultando em populações altamente puras e
não diferenciadas após 2 ou 3 passagens (YING et al., 2003).
A BMP-4 atua pela via de sinalização de mensageiros intracelulares (SMADs 1/5)
que se ligam a domínios específicos nas células, chamados de Prdm14 e Prdm1. Este mecanismo
de ação mostra os caminhos genéticos para a ativação e inibição gênica. Prdm1 é essencial para
inibir todos os genes somáticos, podendo também ser importante para a criação de um estado
epigenético para a expressão de genes específicos de CGPs. O Pdm14 é também um regulador
crítico para a especificação CGPs (YAMAJI et al., 2008). É importante ressaltar que a expressão
inicial de Prdm14 em CGPs é independente de Prdm1, mas a sua manutenção e/ou regulação
positiva subsequente é estritamente dependente Prdm1. Assim, Prdm1 e Prdm14 são os dois
principais reguladores transcricionais para o desenvolvimento da linhagem de células
germinativas em ratos.
As BMPs promovem uma melhoria na derivação das CGPs a partir de células-tronco
embrionárias de humanos (KEE et al., 2006) e camundongos (YOUNG; DIAS; LOVELAND,
2010). Childs et al. (2010) relataram que a BMP-4 aumenta a expressão de marcadores pré-
migratórios (Oct4, Nanog e c-Kit) e pós-migratórios (Dazl e Vasa) em CGPs na diferenciação de
células-tronco embrionárias. Por outro lado, o tratamento com BMP-4 diminuiu a expressão Vasa
em células-tronco embrionárias humanas após um cultivo longo (21 dias) em culturas em
monocamada (TILGNER et al., 2008). VASA é expresso apenas por células germinativas em
diferenciação, assim a BMP-4 tem um efeito negativo sobre a derivação de células germinativas
derivadas a partir de células-tronco embrionárias humanas, resultando em perda de CGP pós-
migratórias em ovários tratados com BMP. A exposição contínua a BMPs pode ser prejudicial
para a sobrevivência de células germinativas derivadas de células-tronco embrionárias (CHILDS
et al., 2010).
Estudos em murinos e humanos demonstram que as BMPs promovem a diferenciação
de células germinativas a partir de células-tronco embrionárias humanas (hESCs) ou células-
56
tronco pluripotentes induzidas (iPS) in vitro (KEE et al., 2009; PANULA et al., 2011; WEI et al.,
2008). Para determinar se proteínas morfogenéticas ósseas recombinantes humanas (rhBMPs)
podem induzir a diferenciação de células germinativas a partir de células-tronco embrionárias,
Kee et al. (2006) ao utilizarem a de BMP-4 recombinante humana verificaram que ela aumentou
a expressão de marcadores específicos de células germinativas tais como: Vasa, Sycp3. Além
disso, BMP-7 e BMP8b associada à BMP-4 demonstraram efeitos sinérgicos na indução de
células germinativas. Deste modo, a adição de BMPs na diferenciação de células-tronco
embrionárias humanas tem aumentado o percentual de células marcadas positivamente para
VASA, bem como a diferenciação de células germinativas humanas (KEE et al., 2006). Shah et
al. (2015a,b) demonstraram que a BMP-4 induz a diferenciação de células-tronco embrionárias
de búfalos em células germinativas.
O papel das BMPs na especificação de células germinativas foi demonstrado in vitro,
Ying et al. (2001) adicionaram BMP-4 e BMP-8b em culturas de epiblastos e foi observada a
indução da formação das CGP. Toyooka et al. (2003) ao promoverem o co-cultivo de células
produtoras de BMP-4 com células-tronco embrionárias de camundongos, aumentou o número de
CGP formadas. Nesse contexto, foi sugerido que as BMPs recombinantes podem induzir
diferenciação de células germinativas in vitro, especialmente células-tronco embrionárias de
murinos. Xu et al. (2002) indicaram que quando as células-tronco embrionárias humanas foram
incubadas com BMP4 recombinante, houve a indução na transcrição de diversos genes
relacionados com o desenvolvimento do trofoblasto.
2.5 Fluido folicular e seu papel durante a formação das CGPs e oócitos
O fluido folicular serve como uma importante fonte de substâncias regulatórias ou
moduladoras derivadas do sangue ou das secreções de células foliculares (BARNETT et al.,
2006). Atualmente, a principal hipótese sobre o mecanismo de formação do antro folicular,
sugere que as células da granulosa geram um gradiente osmótico ao produzir substâncias de alto
peso molecular, como os glicosaminoglicanos e os proteoglicanos, acumulando líquido entre as
células (SCHOENFELDER; EINSPANIER, 2003; CLARKE et al., 2006). O gradiente formado
atrai fluido derivado dos vasos presentes na teca e induz um relativo movimento das células da
granulosa para permitir que o fluido se acumule. Este movimento envolve a remodelação das
57
junções gap entre as células, ou mesmo a morte de algumas células da granulosa (RODGERS;
IRVING-RODGERS, 2010). Em adição, estudos têm demonstrado que a expressão de proteínas,
como as aquaporinas, nas células da granulosa facilitam o transporte de água através das células
(McCONNELL et al., 2002; SKOWRONSKI; KWON; NIELSEN, 2009).
O fluido folicular humano contém uma variedade de substâncias bioquímicas
transferidas a partir do plasma sanguíneo e segregadas a partir de células da teca e da granulosa
(FORTUNE, 1994). Como os oócitos secretam fatores de crescimento parácrinos que regulam o
desenvolvimento de células da granulosa, as células da granulosa, por sua vez regulam o
crescimento de oócitos durante a formação folicular (GILCHRIST et al., 2004). Desta forma, é
evidente que as substâncias bioquímicas das células da granulosa podem desempenhar um papel-
chave na formação de células germinativas e desenvolvimento do oócito. Estudos mostram que o
fluido folicular contém diversos fatores biativos tais como: GDF-9 (PROCHAZKA et al., 2004;
WANG; ROY, 2004), BMP-15 (BERTOLDO et al., 2013) e gonadotrofinas (WANG; ROY,
2004). Vários estudos têm relatado que o fluido folicular suíno promove efetivamente a formação
de células germinativas a partir de células-tronco (DYCE; WEN; LI, 2006; CHENG et al., 2012;
DYCE et al., 2011). Vários trabalhos (DYCE et al., 2004; DYCE; WEN; LI, 2006; LINHER;
DYCE; LI, 2009) mostraram que células-tronco isoladas a partir da pele de fetos suínos tiveram a
capacidade de se diferenciar em células semelhantes a oócitos após cultivo em meio contendo
fluido folicular. Estas células expressaram marcadores de células germinativas (Dazl e Vasa), e
marcadores de oócitos e meiose (Gdf-9 e Oct4) (DYCE; WEN, 2006).
Em suínos, para a indução da diferenciação, as células-tronco isoladas a partir da pele
de fetos suínos foram cultivadas por até 42 dias na presença de fluido folicular e, em seguida,
observou-se a presença de marcadores específicos de células germinativas, tais como Oct4, Bmp-
15, Dazl e Vasa, comprovando a formação de células germinativas. Logo, estas células formaram
estruturas semelhantes a folículos ovarianos secretando estradiol e progesterona, e respondiam ao
estímulo de gonadotrofinas (DYCE et al., 2011), e expressavam marcadores de oócitos, como por
exemplo, proteínas da zona pelúcida e Scp3 (DYCE et al., 2011).
Um estudo mais detalhado sobre os mecanismos de indução da diferenciação de
células germinativas primordiais e estruturas semelhantes a oócitos a partir de células-tronco deu
origem ao artigo de revisão que será mostrado a seguir.
58
3 ARTIGO I
In vitro differentiation of primordial germ cells and oocytes-likes cells from stem cells
(Diferenciação in vitro de celulas germinativas primordiais e células semelhantes à oócitos a
partir de células-tronco)
Artigo submetido ao periódico Histology and Histopathology
(Qualis B1 – Biotecnologia)
59
In vitro differentiation of primordial germ cells and oocytes-likes cells from stem cells
José J.N.Costa1; Glaucinete B. Souza1; Maria A.A Soares1; Regislane P. Ribeiro1; Robert van den
Hurk2, José R.V. Silva1
1Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042-280,
Sobral, CE, Brazil. 2Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands.
*Corresponding address (J. R. V. Silva): Biotechnology Nucleus of Sobral - NUBIS, Federal
University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041-040, Sobral,
CE, Brazil. [[email protected]]
Abstract
Infertility is the result of failure due to an organic disorder of the reproductive organs, especially
their gametes. Recently, much progress has been made on generating germ cells, including
oocytes, from various types of stem cells. This review focuses on advances in female germ cell
differentiation from different kinds of stem cells, with emphasis on embryonic stem cells, adult
stem cells, and induced pluripotent stem cells. The advantages and disadvantages of the
derivation of female germ cells from several types of stem cells are also highlighted, as well as
the ability of stem cells to generate mature and functional female gametes. This review shows
that stem cell therapies have opened new frontiers in medicine, especially in the reproductive
area, with the possibility of regenerating fertility.
Keywords: Adult stem cells. Embryonic stem cells. Induced pluripotent stem cells. Primordial
germ cells. Oocytes-likes cells
1. Introduction
It has been estimated that human infertility affects approximately 14% of couples (Boivin
et al., 2007) and thus, in recent years, new reproductive medicine technologies have been
developed to help to reduce this problem. Women are born with a finite complement of eggs and
60
an ovulated oocyte in a woman has the same age as she. It is known that cellular DNA is not
completely invulnerable to the passage of years, the impact of age on oocytes being consistent
with its effect on the risk of congenital abnormalities (Balen, 2011). Recently, it was
demonstrated that the ovaries contain cells which can be isolated and propagated, and have the
characteristics of oogonial stem cells. After reintroduction into the ovary, or in reaggregation
models, these primitive cells can form new oocytes and follicles, that can generate healthy
offspring (Johnson et al., 2004; White et al., 2012). Stem cell-based strategies for ovarian
regeneration and oocyte production have been proposed as future clinical therapies for treating
infertility in women (Volarevic et al., 2014). Stem cells are undifferentiated cells that are present
in embryonic, fetal, and adult stages of life and give rise to differentiated cells that make up
tissues and organs (Volarevic et al., 2014). From literature, it is known that oocyte-like cells
expressing different oocyte-specific genes can be developed in vitro from mouse embryonic stem
cells (mESCs) (Psathaki et al., 2011) or human embryonic stem cells (hESCs) (Richards et al.,
2010; Medrano et al., 2012). In addition, stem cells from human amniotic fluid (Cheng et al.,
2012) and from porcine fetal skin (Dyce et al., 2011) were also found to differentiate into oocyte-
like structures. Furthermore, human primordial germ cells have been differentiated from induced
pluripotent stem cells (iPS) (Panula et al., 2011; Eguizabal et al., 2011). Hayashi et al. (2012)
recently proposed that hiPS can potentially be utilized to give rise to de novo oocytes for use in
in-vitro fertilization (IVF) clinics, thus allowing sterile women to conceive a child.
In this review, we discuss the advances in our knowledge about the derivation of female
differentiated germ cells from several types of stem cells, including ES cells and iPS cells, as
well as, the mechanisms of reprogramming somatic cells into pluripotent stem cells.
2. Differentiation of primordial germ cells and oocytes-likes cells from embryonic stem cells
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ESCs are derived from totipotent cells of the early mammalian embryo from the inner cell
mass (ICM) of blastocysts and can give rise to all three germ layers (ectoderm, endoderm and
mesoderm) of the developing embryo (Evans and Kaufman, 1981; Martin, 1981; Singhal et al.,
2014). ESCs are capable of self-renewing, but also of exhibiting pluripotency, a feature
maintained by the core network of transcription factors comprising OCT4, SOX2 and NANOG
(Boiani and Schöler, 2005; Singhal et al. 2014). Dyce et al. (2014) found that expression of the
gap junction protein, CONNEXIN43 (Cx43), begins early during embryogenesis and is
maintained in many different cell types. Thus, CX43 can play an important role in somatic stem
cell proliferation and is required in at least some cases for maintaining a pluripotent,
undifferentiated stem cell population. Therefore, ESCs have been used in an in-vitro model of
early embryogenesis to investigate the detailed molecular mechanisms for developmental
processes (Itoh et al., 2014).
Female reproductive potential is limited in the majority of species due to oocyte depletion.
Functional human oocytes are restricted in number and accessibility. A robust system to
differentiate oocytes from stem cells would need a thorough investigation of the genetic,
epigenetic, and environmental factors affecting human oocyte development. The differentiation
of functional oocytes from stem cells may help to restore fertility in women. In mice, Hayashi et
al. [20] demonstrated the generation of primordial germ cell-like cells (PGCLCs) from ESCs with
high capacity for differentiation. Hayashi et al. (2011) have demonstrated that female ESCs were
induced into PGCLCs, which underwent proper development in reconstituted ovaries in vitro and
matured further into fully functional GV oocytes upon transplantation in vivo. Upon
transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into
germinal vesicle-stage oocytes, which then contribute to fertile offspring after in-vitro maturation
62
and fertilization. This system serves as a robust foundation to investigate and further reconstitute
female germline development in vitro in mammals (Hayashi et al., 2012), including the human.
Several studies in different species such as mouse (Nagano et al., 2002; Hübner et al.,
2003) and human (Clark et al., 2004; Park et al., 2009), indicate that ESCs can differentiate in
vitro into oocyte- or sperm-like cells. Nicholas et al. (2009) observed endogenous and ESC-
derived human oocyte development. Hübner et al. (2003) were the first to report that
differentiating mouse ESCs can spontaneously form oocyte-like cells in vitro, when cultivated
without LIF and feeder cells. Psathaki et al. (2011) indicated that follicle-like structures formed
by mouse ESCs in vitro consist of a single oocyte-like cell that can grow as large as 70 mm in
diameter, surrounded by one or more layers of tightly adherent somatic cells. Besides presenting
the expression of genes associated with steroidogenesis, these structures are connected via
intercellular bridges with their enclosed germ cells, which may serve to facilitate cell-to-cell
interaction required for normal follicle development (Hübner et al., 2003; Novak et al., 2006;
Albertini et al., 2001). Lacham-Kaplan et al. (2006) have cultured mouse ESC-derived embryoid
bodies (EBs), which formed ovarian-like structures containing oocyte-like cells, surrounded by
one or two layers of flattened cells, which expressed specific oocyte marker genes (FIGα and
ZP3). Kerkis et al. (2007) induced oocyte differentiation by stimulating EBs with retinoic acid.
Yu et al. (2009) reported that Deleted in Azoospermia-Like (DAZL) is a master gene controlling
germ cell differentiation and that ectopic expression of DAZL promotes the dynamic
differentiation of mouse ES cells into gametes in vitro. Furthermore, transient overexpression of
DAZL led to suppression of NANOG, but induced germ cell nuclear antigen in mESCs, whereas
DAZL knockdown resulted in reduction in the expression of germ cell markers, including
STELLA, MVH and PRDM1 (Yu et al., 2009).
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In humans, Clark et al. (2004a) demonstrated that differentiation of ESCs into EBs in
vitro results in formation of cells that express markers specific for gonocytes and thus are capable
to form germ cells in vitro. In undifferentiated cells, NANOG, STELLA, OCT4 and DAZL
appeared expressed, whereas after differentiation in EBs, mRNAs and proteins for VASA, SCP1,
SCP3, BOULE and GDF3 were found, which are germ cell specific markers (Clark et al., 2004b).
Kee et al. (2006) demonstrated that addition of recombinant human BMP4 increased the
expression of the germ cell-specific markers VASA and SCP3 during differentiation of hESCs to
embryoid bodies. In addition, BMP2 and BMP8b associated with BMP-4 demonstrated additive
effects on germ cell induction. Thus, several BMPs could induce differentiation of germ cells
from human hESCs. Medrano et al. (2012) examined whether intrinsic germ cell translational,
rather than transcriptional factors might drive germline formation and/or differentiation from
human pluripotent stem cells in vitro. They found that overexpression of VASA and/or DAZL
promoted differentiation of both hESCs into primordial germ cells. Additionally, maturation and
progression through meiosis was enhanced. Over the last decade, much progress has been made
in the differentiation of human germ cells from both hESCs (Ishii et al., 2013). Nicholas et al.
(2009) established fundamental parameters of oocyte development during ESC differentiation.
They demonstrated a timeline of definitive germ cell differentiation from ESCs in vitro that
initially parallels endogenous oocyte development in vivo by single-cell expression profiling and
analysis of functional milestones, including responsiveness to defined maturation media, shared
genetic requirement of DAZL, and entry into meiosis (Nicholas et al., 2009). Thus, ESCs can be
used in an in-vitro system to study oocyte development and could be of help in the treatment of
female infertility. Figure 1 shows the differentiation of stem cells in oocytes-like cells, as well as
the stimulating substances and markers for each stage of differentiation.
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3. Differentiation of primordial germ cells and oocytes from adult stem cells
After embryonic development, adult stem cells (ASCs) exist in almost all tissues of the
body. They are ready for emergencies after tissue injury, as well as for maintenance of tissue
homeostasis (Itoh et al., 2014). Recent literature (Johnson et al., 2004; 2005; Lee et al., 2007;
Bukovsky et al., 2008; Niikura et al., 2009; Selesniemi et al., 2009; Gong et al., 2010; Bukovsky,
2011; White et al., 2012; Tilly and Sinclair, 2013; Gheorghisan-Galateanu et al., 2014;
Hummitzsch et al., 2015) suggests the presence of germ line stem cells in mammalian ovary,
however, their ability to replenish the germ cell pool at an adult or postnatal stage during the
reproductive phase is not very clear. In this context, Zou et al. (2009) isolated female germline
stem cells (FGSCs) in postnatal mammalian ovaries from adult mice. After culture, these stem
cells underwent oogenesis, where after the recipient mice produced offspring. White et al. [4]
demonstrated that both adult mice and human ovaries possess mitotically active germ cells that
can differentiate into oocytes both in vitro and in vivo. Germ-line stem cells have been reported in
mice and human ovaries by several research groups and have been recently reviewed elaborately
(Virant-Klun et al., 2011; Telfer and Albertini, 2012; Woods et al., 2012; 2013; Dunlop et al.,
2014; Hanna and Hennebold, 2014; Hummitzsch et al., 2015); but, studies on human ovarian
stem cells are relatively few in number because of scarcity of ovarian tissue for research.
Bukovsky et al. (2005) demonstrated differentiation of surface epithelium of post-menopausal
human ovary and development into oocytes and blastocysts in vitro. Thus, those stem cells
generated functional oocytes in vitro irrespective of age and condition (menopausal and
premature ovarian failure) (2012). Already McLaughlin et al. (2015) developed a model of
estimated a woman’s ovarian reserve by non-invasive means, which is calculated a non-growing
65
follicle density in the human ovarian cortex, using ovarian biopsies, and could be used in a
variety of experimental and pathological situations.
By using various techniques, Virant-Klun et al. (2008; 2009; 2013) and Virant-Klun and
Skutella (2010) identified putative stem cells in ovarian sections and in scraped ovarian surface
epithelium (OSE) from post-menopausal women and from those with premature ovarian failure
(POF), which could differentiate into oocyte-like structures and parthenotes in vitro. Parte et al.
(2011) reported two distinct stem cell populations namely, pluripotent very small embryonic-like
stem cells expressing nuclear OCT4 and slightly bigger progenitor stem cells, termed ovarian
germ stem cells (OGSCs), which express cytoplasmic OCT4 in peri-menopausal women and
other higher mammalian species. Similar results were observed in adult human (Bhartiya et al.,
2010) and mice (Bhartiya et al., 2012) ovaries. The putative stem cells differentiated
spontaneously in cultures to give rise to oocyte-like structures and parthenotes (Parte et al.,
2011), and expressed both pluripotent (OCT4, OCT4A, SSEA4, NANOG, SOX2, TERT, STAT3)
and germ cell (c-KIT, DAZL, GDF9, VASA) markers.
Bhartiya et al. (2012) reported that mouse ovarian stem cells lodged in the ovarian surface
epithelium (OSE) are modulated by pregnant mare serum gonadotropin (PMSG) resulting in neo-
oogenesis and postnatal follicular assembly. There is evidence that FSH modulates ovarian stem
cells through an alternatively spliced variant of FSH receptor. After in in-vitro cultured sheep
OSE, stem cells underwent potential self-renewal and clonal expansion as "germ cell cysts"
(Patel et al., 2013). In mice, PMSG treatment resulted in augmented stem cell activity, neo-
oogenesis and primordial follicle assembly (Bhartiya et al., 2012). Stem cells isolated from the
skin of porcine fetuses also have the ability to differentiate into oocyte-like cells (OLCs) after
stimulation with follicular fluid (Dyce et al., 2006). These cells i) express germ-cell markers,
(OCT4, GDF9B, DAZL and VASA), ii) form follicle-like aggregates that secrete oestradiol and
66
progesterone, iii) respond to gonadotropin stimulation, and iv) express oocyte markers, such as
zona pellucida (ZP), and the meiosis marker, synaptonemal complex protein 3 (SCP3) (Dyce et
al., 2006). In mice, Dyce et al. (2011) reported that newborn mice skin-derived stem cells are also
capable of differentiating into early OLCs that express oocyte markers. Linher et al. (2009) also
demonstrated that stem cells isolated from fetal porcine skin have the potential to form primordial
germ cells (PGCs) that express specifc markers (OCT4, FRAGILIS, STELLA, DAZL and VASA)
and originate putative oocytes after culture.
Stem cells were also isolated from adipose tissue and ovarian stroma from pigs and
cultured in presence of follicular fluid (Song et al., 2011). These cells expressed transcription
factors, such as OCT4, NANOG and SOX2 and exhibited their potential for in-vitro oogenesis,
since expressed markers like OCT4, GDF9B, C-MOS, VASA, DAZL, ZPC and FSHR after
culture. The described results indicate that stem cells from ovarian tissue may differentiate into
oocytes (Song et al., 2011). Figure 1 shows the differentiation of adults stem cells into oocytes-
like cells, as well as the stimulating substances and markers for each stage of differentiation. The
function of markers for stem cells, PGCs and oocytes are shown in Table 1.
4. Differentiation of primordial germ cells and oocytes from the iPS cells
The differentiation of induced pluripotent stem cells (iPS) has been observed in several
studies. Park et al. (2009), demonstrated the formation of PGCs from reprogrammed skin
fibroblasts of human. However, PGCs derived from iPS cells did not start imprint erasing
efficiently, suggesting that more studies are needed to elucidate the germ cell induction
mechanisms from iPS. In another study, Panula et al. (2011) compared the efficiency of human
67
iPS derived from adult and fetal somatic cells to form primordial and meiotic germ cells. They
showed that around 5% of human iPS have the potential to differentiate into PGCs after induction
with BMPs. In addition, they had the ability to express specific markers of germ cells (DAZ,
DAZL and BOULE) and the iPS cells formed meiotic cells with extensive synaptonemal
complexes. In human species, Eguizabal et al. (2011) reported generation of postmeiotic cells
from human hiPS from different origin (keratinocytes and cord blood). These cells demonstrated
complete and robust meiotic competence, opening the way for the production of in-vitro gametes
in the human species.
Hayashi et al. (2011) demonstrated that mouse iPS cells differentiate into epiblast-like
cells, primordial germ cell-like cells (PGCLCs), and fertile oocytes, successively. After
reconstitution of ovaries with PGCLCs, these cells differentiated into germinal vesicle-stage
oocytes and contributed to fertile offspring after in-vitro maturation and fertilization (Hayashi et
al., 2012). Eguizabal et al. (2011) demonstrated the differentiation of female iPS into haploid
cells following the detection of SCP3 and H2AX proteins, which are indicators of meiotic
competence. Singhal et al. (2015) demonstrated the generation of germ cell-like cells and oocyte-
like cells from goat iPS (giPS) from fibroblast cells. These germ cell-like cells were characterized
by expression of germ cell specific markers (VASA, DAZL, STELLA and SCP3) at transcription
and protein level. These giPS differentiated into primordial germ cell-like cells in the presence of
retinoic acid and BMP4. Among the differentiated germ cell-like cells, oocyte-like structures
were observed. Figure 2 shows the strategy to restore fertility by differentiating iPS into oocytes.
4.1. Mechanisms of cellular reprogramming to produce iPS
Efficient reprogramming methods have been explored since the first report of the
generation of human induced pluripotent stem cells (Takahashi et al., 2007; Yu et al., 2007). In
68
differentiated cells (adult cells), absence of expression of the pluripotency genes may be due to
changes in chromatin due to DNA methylation or histone acetylation in the promoter region of
these genes (Li, 2002; Li et al., 2007; Cedar and Bergman, 2009). Currently, new methods have
been applied in the cellular reprogramming process, especially in the production of iPS cells,
which include the use of episomal plasmids (Yu et al., 2009) or excisable expression systems
(Soldner et al., 2009), recombinant cell-penetrating reprogramming proteins (Kim et al., 2009;
Zhou et al., 2009), reprogramming mRNAs (Warren et al., 2010; Yakubov et al., 2010), or
microRNAs (Anokye-Danso et al., 2011; Miyoshi et al., 2011). Furthermore, a growing number
of compounds have been identified that can functionally replace reprogramming transcription
factors, enhance efficiency of iPS generation and accelerate the reprogramming process (Zhang et
al., 2012).
Takahashi and Yamanaka (2006) tested 24 different candidate factors that play important
roles in the maintenance of pluripotency. They found that four transcription factors (OCT4,
SOX2, C-MYC and KLF4) are involved in the generation of iPS, which are similar to ESCs in
morphology, proliferation, and teratoma formation. In regard to aging and rejuvenation, the
reprogramming process resets an aged, somatic cell to a more youthful state, elongating
telomeres, rearranging the mitochondrial network, reducing oxidative stress, restoring
pluripotency, and making numerous other alterations (Rohani et al., 2014). In goats, Singhal et al.
(2013) reprogrammed adult female goat fibroblast cells into induced pluripotent stem cells using
ectopic expression of OCT4, NANOG and SOX2 genes and the germ-cells-like cells, generated
from reprogrammed giPS, could be differentiated into goat oocytes-like structure. However, there
have been concerns over the use of integrating retroviruses to deliver the iPS factors, which could
potentially compromise the quality of or even cause tumorigenicity in the resultant iPS (Zhang et
al., 2012). The efficiency of iPS cell induction is quite low: less than 1% of human fibroblasts
69
that received transcription factors OCT4/SOX2 and the oncogenes KLF4/C-MYC (together
abbreviated to OSKM) become iPS cells. However, Tanabe et al. (2013) showed that, after
receiving OSKM (Tanabe et al., 2013; 2014), the reprogramming process initiates in more than
20% of human fibroblasts.
There are several genes related to cell reprogramming, like OCT4. This gene is encoded
by the gene POU5F1 (Wu and Schöler, 2014) and is restricted in the blastomeres of the
developing mouse embryo, the ICM of blastocysts, the epiblast, germ cells and oocytes. It is also
expressed in pluripotent stem cells, including ESCs (Pesce et al., 1998; Yamanaka, 2007).
Singhal et al. (2014) demonstrated that Brahma-Related Gene 1 (BRG1) is essential for early
embryonic development, but also enhances the efficiency of reprogramming somatic cells in
murine species.
During the reprogramming process, remodeling of the epigenome and modulations of
the epigenetic processes may facilitate conversion of cell fate by making cells more permissive to
these epigenomic changes. Enzymes that stand out in this process are histone deacetylase
(HDAC), histone methyltransferase (HMT), histone demethylase (HDM) and DNA
methyltransferase (DNMT) (Zhang et al., 2012).
Among the compounds that can be used in reprogramming, 5-azacytidine, N-Phthalyl-L-
Tryptophan (RG108), Valproic acid (VPA), Trichostatin A (TSA), Sodium butyrate (NaB), and
other molecules (Federation et al., 2014) can be highlighted. Demethylation of one or more
(unknown) loci of DNA is a critical step in the late stages of direct reprogramming. Inhibition of
DNMT1 lowers this kinetic barrier, thereby facilitating the transition to pluripotency (Mikkelsen
et al., 2008).
70
DNA demethylation has been recently reported after use of 5-Azacytidine (5-Aza) or
RG108 during cellular reprogramming (Shi et al., 2008; Okita and Yamanaka, 2011; Pennarossa
et al., 2013; 2014; Federation et al., 2014). 5-Aza is a chemical derivative of the DNA nucleoside
cytidine, the only difference being the presence of a nitrogen atom at position 5 of the cytosine,
the same site at which DNA methylation occurs. Thus, 5-Aza causes DNA demethylation or
hemi-demethylation. DNA demethylation can regulate gene expression by relaxing chromatin
structure. This is detectable as an increase in nuclease sensitivity. This remodeling of chromatin
structure allows transcription factors to bind to the promoter regions, assembly of the
transcription complex, and gene expression, particularly genes associated with cell pluripotency.
As an analog of cytosine, DNA polymerase does not recognize the difference between 5-Aza and
cytosine and will incorporate 5-Aza during DNA replication (Christman, 2002; Federation et al.,
2014). Pennarosa et al. (2014) exposed pig dermal fibroblasts to 5-Aza for 18 h, followed by a
protocol for the induction of endocrine pancreatic differentiation. The results demonstrated
changes in cell morphology and expression of pluripotency genes (OCT4, NANOG, SOX2 and
REX1) (Pennarosa et al., 2014). The cells expressed insulin and were able to release it in response
to a physiological glucose challenge.
Valproic acid (VPA), 2-propyl-pentanoic acid, is a short-chain branched fatty acid
(Almutawaa et al., 2014). This VPA, an HDAC inhibitor, increases reprogramming efficiency of
human fibroblasts, enabling the efficiency of primary human fibroblasts infected with the
transcription factors OCT4, SOX2 and KLF4 (Huangfu et al., 2008). The molecular mechanisms
of VPA action involves several protein kinase pathways that have been suggested to be the
targets for this drug (Monti et al., 2009). VPA has been shown to inhibit the activity of histone
deacetylases (HDACs), resulting in chromatin remodelling and changes in gene expression (Phiel
et al., 2001; Gotfryd et al., 2011). Trichostatin A (TSA) is one of the most potent known histone
71
deacetylase inhibitor. This hydroxamic acid is in vitro active at nanomolar concentrations and
inhibits HDACs with zinc-containing catalytic sites, leading to accumulation of acetylated
histones in the nucleus and subsequent activation of target genes (Taddei et al., 2005;
Dokmanovic et al., 2007; Kim et al. 2009;). Sodium butyrate (NaB) increases histone acetylation
and is a potent HDAC inhibitor that causes hyperacetylation of histones H3 and H4 in
mammalian cells (Candido et al., 1978; Nör et al., 2013).
5. Clinical potential
Pluripotent stem cells hold great promise in the field of regenerative medicine, because
they can propagate indefinitely. In addition, these cells can lead to every other cell type (sperm,
oocytes, neurons, heart, pancreatic, and liver cells), they represent a single source of cells that
could be used to replace those lost to damage or disease.
Nearly 72.4 million people have fertility problems, caused by various underlying
pathologies, with exposure to toxicants or immune-suppressive treatments, in cases with gonadal
insufficiency due to POF or azoospermia (Boivin et al., 2007; Bhartiya et al., 2014; Volarevic et
al., 2014). Currently, several advancements have been made in assisted reproduction treatment,
especially the generation of gametes derived from pluripotent stem cells (Bhartiya et al., 2014).
Considering that the use of ESCs has led to deliberate ethical controversy (Klimanskaya et al.,
2006), ASCs or iPS can be considered the most suitable type of cells to produce patient-matched
oocytes that can be used to recover fertility.
6. Final considerations
72
This review emphasizes the potential of embryonic stem cells, adult stem cells and
induced pluripotent stem cells to produce germ cells and oocytes. Recent advances in cellular
therapies have led to the understanding how stem cells can give rise to gametes and how somatic
stem cells differentiate into germ cells and oocytes. This knowledge gives significant insight into
the regulation of developmental gametes and has important implications for female fertility and
regenerative medicine. More studies are needed in this area, to clarify what problems affect the
achievement of female germ cells. Various studies addressed the potential of stem cells to
differentiate into oocytes. However, more information is necessary for obtaining mature gametes
of good quality, which subsequently have to go through a process of fertilization to produce
viable offspring. It is expected that, in the near future, stem cells can be isolated that are able to
differentiate into viable oocytes in a reproducible manner. The findings will not only contribute
to enhancement of female germ cell induction but also to reduction of female infertility rates.
Acknowledgments
This work was financially supported by CNPq (Grant N° 478198/2013-2), and the authors thank
the members of the Laboratory of Animal Reproduction of the Biotechnology Nucleus of Sobral.
Compliance with Ethical Standards
Conflict of Interest
All authors have no conflicts of interest, including employment, financial ownership, and grants
or other funding.
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Figures and tables
Table 1. Molecular markers for stem cells, PCGs, oocytes and their functions.
Figure 1. Schematic presentation of the development of stem cells into oocytes-like cells, and the
stimulating substances and markers for each stage of differentiation.
Figure 2. Procedure for restoring fertility by differentiating iPS into oocytes.
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Table 1. Molecular markers for stem cells, PCGs, oocytes and their functions.
Markers Functions References
Stem cells
OCT4 Transcription factor associated with
self-renewal of undifferentiated embryonic
stem cells, used as a marker for
undifferentiated cells.
Lavial et al., 2007;
Hu et al., 2015
NANOG Transcription regulator involved in ICM and
ESC proliferation and self-renewal, and
prevents the differentiation of ESC in towards
extraembryonic endoderm and trophectoderm
lineages.
Lavial et al., 2007;
Singhal et al., 2015
SOX2 Transcription factor associated with controls
the expression of genes involved in embryonic
development such as YES1, FGF4, UTF1 and
ZFP206. Furthermore , it is essential for early
embryogenesis and for embryonic stem cell
pluripotency.
Yu et al., 2014
REX1 Marker of pluripotency, is usually found in
undifferentiated embryonic stem cells, its
regulation is also critical in maintaining a
pluripotent state, as the cells begin to
differentiate, Rex1 is severely and abruptly
downregulated
Lavial et al., 2007
Klf4 Mediator to LIF-Stat3 signal changes, and
directly binds to the promoter of Nanog to help
Oct4 and Sox2 in regulating the expression of
Nanog
Singhal et al., 2015
c-Myc Regulator the self-renewal and
pluripotency of mESC as well as pluripotent
cells of the early
embryo
Singhal et al., 2015
SSEA4 Cell membrane protein of ESC Zhao et al., 2012
STAT3 Main extracellular signal that sustains mouse
ESCs self-renewal and pluripotency is the
activation of leukemia inhibitory factor
(LIF) pathway
Niwa et al., 2009
BRG1 Important role in maintaining pluripotency by
fine-tuning the expression level of Oct4 and
other pluripotency-associated genes.
Singhal et al., 2014
Cx43 Mouse ESC express this connexin and form
functional gap junctions during growth
Dyce et al., 2014
PGC
DAZL Protein involved in early germ cell
differentiation
Panula et al., 2011;
Clark et al., 2004;
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Kerr and Cheng,
2010
BLIMP1 or
PRDM1
First indicator of germ cell fate Yu et al., 2009
STELLA Gene initial germline Hu et al., 2015;
West et al., 2006
c-KIT Migration and survival of PGCs Marqués-Marí et al.,
2009; Cai et al.,
2013
MVH Germ pre-meiotic cell-specific marker Cai et al., 2013;
Toyooka et al., 2000
FRAGILIS Early indicator of germ cell fate Medrano et al., 2012
VASA Germ pre-meiotic cell-specific marker Hu et al., 2015;
Medrano et al., 2010
Zhu et al., 2012
BOULE Genes modulate primordial germ-cell and
haploid gamete formation
Kee et al., 2009
GDF3 Protein that modulate TGF-β superfamily
members, e.g. potentiates the activity of
NODAL
Levine and
Brivanlou, 2006
Oocytes
SCP1, SCP3 Component of the synaptonemal complex, and
this complex is involved in synapsis,
recombination and segregation of meiotic
chromosomes.
Hu et al., 2015;
Yuan et al., 2000
Figa Factor required for progression of germ cells
through the pachytene stage of MI
Marqués-Marí et al.,
2009;
Liang et al., 1997
ZP1, ZP2 e
ZP3
Glycoproteins are markers of female germ cells in
post-meiotic development which are only
secreted by oocytes within primary follicles, to
form a glycocalyx known as the zona pellucida
Hu et al., 2015;
El-Mestrah et al.,
2002
GDF9 Key factor regulating germ cell development,
and act as master regulators oocyte-specific
transcription factors
Salvador et al., 2008;
Sriraman et al., 2015
GDF9B Expressed in the human fetal ovary at the time
of oocyte nest breakdown and primordial
follicle formation
Bayne et al., 2015
Nobox Essential for follicle formation and oocyte
survival, and regulates the expression of GDF9
in humans
Singhal et al., 2015
c-MOS Specifically expressed in male and female germ cells where it appears to play a central role in
regulating the meiotic cell cycle
Cooper, 1994
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FSH-R Stimulates proliferation and differentiation of
progenitors ovarian germ stem cells to oocytes
and primordial follicle assembly
Bhartiya et al., 2012
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Figure 1. Schematic presentation of the development of stem cells into oocytes-like cells, and the stimulating substances and markers
for each stage of differentiation.
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Figure 2. Procedure for restoring fertility by differentiating iPS into oocytes.
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4 PROBLEMA
Conforme mostrado na revisão de literatura, vários estudos têm demonstrado a
diferenciação in vitro de oócitos a partir da utilização de células-tronco embrionárias, células-
tronco adultas, células-tronco ovarianas, células da linhagem germinativa isoladas de ovários de
indivíduos adultos, e ainda, células-tronco de pluripotência induzidas. Diante disto, levantou-se
os seguintes questionamentos:
1) Será que a utilização do 5-aza-citidina em fibroblastos bovinos é capaz de
promover a expressão de RNAs mensageiros para marcadores de pluripotência?
2) Será que a BMP-2, BMP-4 e fluido folicular estimulam a formação das células
germinativas primordiais e posterior diferenciação em oócitos a partir de fibroblastos tratados
com 5-aza-citidina e aumentam os níveis de RNA mensageiros para os marcadores de células
germinativas (VASA, DAZL e C-KIT), de meiose (SCP3) e de oócitos (ZPA e GDF-9) após o
cultivo in vitro?
3) Será que a BMP-2, BMP-4 e fluido folicular contribuem para a formação das
células germinativas primordiais e posterior diferenciação em oócitos a partir de células-tronco
isoladas de ovários bovinos e aumentam os níveis de RNA mensageiros para os marcadores de
células germinativas (VASA, DAZL e C-KIT), de meiose (SCP3) e de oócitos (ZPA e GDF-9)
após o cultivo in vitro?
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5 JUSTIFICATIVA
Nos últimos anos, a reprogramação de células somáticas por meio da transdução viral
de fatores de transcrição em animais de laboratório e em humanos está associada com grandes
perspectivas para o desenvolvimento da medicina regenerativa (TAKAHASHI; YAMANAKA,
2006). No entanto, há uma grande preocupação relacionada com a introdução retroviral ou
lentiviral nas células alvo, pois isto pode causar instabilidade gênica a longo prazo, bem como o
desenvolvimento de tumores (OKITA; ICHISAKA; YAMANAKA, 2007). Desta forma, é
essencial realizar a indução de pluripotência sem a introdução de modificações genéticas. Neste
contexto, a avaliação do efeito de componentes quimicamentes definidos, como o 5-aza-citidina,
pode trazer grandes benefícios para o desenvolvimento de terapias celulares que visam à cura de
doenças e também a resolução de problemas de fertilidade.
Nos países desenvolvidos tem se observado um aumento progressivo dos problemas
de concepção em humanos devido ao fato de a maioria dos casais estarem adiando cada vez mais
a concepção do primeiro filho (SCHMIDT et al., 2012). Associado a isto, a qualidade de vida dos
casais inférteis tem sido grandemente reduzida devido à ausência de capacidade de conceber
(REIJO PERA, 2013). Assim, a partir do advento da fecundação in vitro, novas intervenções
clínicas vêm sendo desenvolvidas para a resolução de problemas de fertilidade em humanos.
Recentemente, vários estudos demostraram avanços significativos na diferenciação de óocitos a
partir de células- tronco embrionárias ou adultas em animais de laboratório (PSATHAKI et al.,
2011; WHITE et al., 2012). No entanto, até o momento, a diferenciação de oócitos a partir da
reprogramação de fibroblastos ainda não foi descrita. Devido ao fato de ter mais similaridades
com humanos do que animais de laboratório, os bovinos podem ser utilizados como modelos para
o desenvolvimento desta biotecnologia que possibilitará o estudo de fatores genéticos e
epigenéticos envolvidos com a formação oocitária. Além disso, os oócitos oriundos da
diferenciação de fibroblastos podem ser utilizados para fins biomédicos, contribuindo assim para
incrementar o desenvolvimento de terapias celulares que visem à promoção da capacidade de
concepção em casais inférteis.
No tocante a reprodução animal, a obtenção de oócitos a partir de células-tronco ou
de fibroblastos reprogramados pode contribuir para a produção de um grande número de
embriões a partir de animais de alto valor zootécnico ou em via de extinção. No caso de espécies
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já em extinção, mas que deixaram tecidos criopreservados, o desenvolvimento desta técnica
possibilitará a diferenciação de gametas a partir de células somáticas, abrindo uma nova
perspectiva de recuperação de espécies extintas. Considerando que durante a meiose, ocorre a
recombinação genética, todos os embriões produzidos seriam geneticamente diferentes e evitaria
problemas de redução de variabilidade genética dos rebanhos, que é um problema da clonagem
com fins reprodutivos. Além disso, no futuro, a diferenciação de oócitos a partir de células
somáticas pode contribuir para reduzir os tratamentos hormonais ou a punção oocitária semanal
visando à produção de embriões in vitro. Apesar de estas técnicas serem largamente utilizadas,
elas apresentam várias implicações relacionadas ao bem-estar animal (FIGUEIREDO;
MOLENTO, 2008).
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6 HIPÓTESES CIENTÍFICAS
1) O 5-aza-citidina promove a expressão de RNAs mensageiros para marcadores de
pluripotência (OCT-4, NANOG, REX-1, SOX2) em fibroblastos bovinos .
2) A BMP-2, a BMP-4 e o fluido folicular influenciam positivamente na
diferenciação de fibroblastos tratados com 5-aza-citidina e de células-tronco isoladas de ovários
bovinos, em células germinativas primordiais e oócitos.
3) A BMP-2, a BMP-4 e o fluido folicular afetam positivamente a expressão de
RNAs mensageiros para os marcadores de células germinativas (VASA e DAZL), de meiose
(SCP3) e de oócitos (ZPA e GDF-9), após o cultivo in vitro de fibroblastos tratados com 5-aza-
citidina e de células-tronco isoladas de ovários bovinos.
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7 OBJETIVOS
7.1 Objetivos gerais
1) Induzir a expressão de marcadores de pluripotência em fibroblastos bovinos e
diferenciá-los em células germinativas primordiais e em oócitos.
2) Isolar células-tronco da linhagem germinativa a partir de ovários de bovinos
adultos e diferenciá-las em células germinativas primordiais e em oócitos.
7.2 Objetivos específicos
1) Estudar os efeitos da 5-aza-citidina na indução da aquisição pluripotência em
fibroblastos bovinos, através da expressão de marcadores de pluripotência (OCT-4, NANOG,
REX-1, SOX2).
2) Avaliar os efeitos da BMP-2, BMP-4 e do fluido folicular na diferenciação de
fibroblastos tratados com 5-aza-citidina em células germinativas primordiais e em oócitos.
3) Identificar a capacidade de células-tronco isoladas de ovários de animais adultos
se diferenciarem em células germinativas primordiais e em oócitos após estímulo com BMP-2,
BMP-4 e fluido folicular.
4) Quantificar a expressão de marcadores de células germinativas (VASA e DAZL),
de meiose (SCP3) e de oócitos (ZPA e GDF-9) após o cultivo in vitro de fibroblastos tratados
com 5-aza-citidina e de células-tronco isoladas de ovários bovinos em meio suplementado com
BMP-2, BMP-4 e fluido folicular.
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8 ARTIGO II
Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-
azacytidine and cultured in presence of BMP-2, BMP-4 or follicular fluid
(Expressão de marcadores de células germinativas e oócitos em fibroblastos da pele bovina
tratados com 5-aza-citidina e cultivados na presença de BMP-2, BMP-4 ou fluido folicular)
Artigo submetido ao periódico Molecular Biology Reports
(Qualis B1 - Biotecnologia)
100
Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-
azacytidine and cultured in presence of BMP-2, BMP-4 and follicular fluid
José Jackson do Nascimento Costa1, Glaucinete Borges de Souza1, Joyla Maria Pires Bernardo1,
Regislane Pinto Ribeiro1, José Renato de Souza Passos1, Francisco Taiã Gomes Bezerra1, Márcia
Viviane Alves Saraiva1, José Roberto Viana Silva1*
1Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042-280,
Sobral, CE, Brazil.
*Corresponding address (J. R. V. Silva): Biotechnology Nucleus of Sobral - NUBIS, Federal
University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041-040, Sobral,
CE, Brazil. [[email protected]]
Abstract
I. Background: This study aims to investigate the effect 5-azacytidine during induction of
pluripotency in bovine fibroblast skin, evaluate the effects of culture for 7 or 14 days in a
medium containing BMP-2, BMP-4 or follicular fluid in the differentiation of reprogrammed
fibroblasts in primordial germ cells and oocytes, as well as to analyze the mRNA levels for OCT-
4, NANOG, REX-1, SOX2, VASA, DAZL, c-KIT, SCP3, ZPA and GDF-9, after induction of
pluripotency or differentiation in bovine fibroblasts.
II. Methods and Results: Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of
5-Aza for 18 h, 36 h or 72 h. Then, cultured in DMEM/F12 supplemented with with 10 ng/mL
BMP-2, or 10 ng/mL BMP-4 or 5% follicular fluid. After the cell culture, were evaluated
101
morphological characteristics, viability and gene expression by qPCR. Treatment of skin
fibroblasts at 2.0 μM 5-Aza for 72 h results in changes in the morphology (oval or round shape)
and cellular proliferation rate, and significantly increased expression of pluripotency factors. The
culture in medium supplemented with BMP-2, BMP-4 or follicular fluid, for 7 or 14 days,
altering cellular morphology, inducing formation of cell, and gene expression of germ cells and
oocytes markers.
III. Conclusions: This study describes the possibility to convert bovine skin fibroblasts into
another cell type, oocyte-likes cells, contribute to increase the development of therapies aimed at
solving the problems of infertility in humans, as well as enhance the reproductive efficiency of
animals.
Keywords: Gene expression. Fibroblast. 5-Azacytidine. BMP-2. BMP-4. Follicular fluid.
Introduction
The development of new regenerative therapies adapted to reproductive needs of female
has been challenging in the last years [1]. Stem cell-based strategies for ovarian regeneration and
oocyte production have been proposed as future clinical therapies for treating infertility in women
[2]. Various studies have been conducted to identify, characterize, and differentiate cells from
various sources [3-5], that can potentially be used for clinical studies, including embryonic stem
cells (ESC) [6], stem cells isolated from adult tissues like the mesenchymal stem cells (MSC) [7],
and induced pluripotent stem cells (iPS) which are adult somatic cells reprogrammed to
pluripotency [8].
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In 2006, Takahashi and Yamanaka [9] generated pluripotent cells by reprogramming
somatic cells, this discovery it is now possible to convert differentiated somatic cells into
pluripotent stem cells that have the capacity to generate all cell types of adult tissues [10]. iPS
were generated by using a combination of 4 reprogramming factors, including OCT4 (Octamer
binding transcription factor-4), SOX2 (Sex determining region Y)-box 2, KLF4 (Kruppel Like
Factor-4), and c-MYC and were demonstrated both self-renewing and differentiating like ESCs
[11]. Conventional reprogramming techniques depend on the stable integration of transgenes,
using viral vectors, such as retroviruses [9] and lentiviruses [12], but it can introduce the current
risk of insertional mutagenesis [13]. A chemical reprogramming is a promising strategy for safety
and efficiency of iPS generation, many small molecules have been identified that can be used in
place of exogenous transcription factors and significantly improve iPS reprogramming efficiency
and quality [14]. Small molecules promote a chemical reprogramming, accompanied by
remodeling of the epigenome, modulations of the epigenetic processes may facilitate such
conversion of cell fate by making cells more permissive to these epigenomic changes [15].
To this purpose, 5-Azacytidine (5-Aza), a DNA methyltransferase inhibitor, can also
improve reprogramming efficiency, by activating the expression of silent genes and altering the
differentiation state of cells [16, 17]. Taylor and Jones, (1979) [18], used the 5-Aza in the
conversion of a mesenchymal cell line into muscle cells, adipocytes and chondroblasts.
Pennarossa et al. [19, 20] reprogrammed human and pig dermal fibroblast into insulin secreting
cells by a brief exposure to 5-Aza. However, the use of 5-Aza to reprogram bovine skin fibroblast
followed by a differentiation protocol, have not yet been reported.
The efficiency of differentiation of iPS cells into female germ cells has been considered
low [21], since Eguizabal et al. (2011) [22] observed between 1.0%–2.0% haploid cells
differentiated from a human female iPS cell line. The formation of iPS-derived germ cells
103
requires a strategy that involves differentiating iPS cells into primordial germ cells (PGCs), and
subsequently directing the PGCs to undergo meiosis to form functional gametes [23, 21]. Several
studies indicate that efficiency of differentiation to PGC can be increased with the addition of
bone morphogenetic proteins (BMP-4, -7 and -8b) in human [24] and murine [25, 26] species.
Günesdogan, et al. (2014) [27] indicated the BMP signals acts through a receptor complex
including BMP receptor type II and ALK3/6, which results in SMAD1/5 phosphorylation, which
form a complex with SMAD4 and translocate to the nucleus to control target gene expression.
Mice carrying null mutations for BMP4, BMP8B, SMAD1 and SMAD5 showed impaired PGC
development [28-30]. Ying et al. (2001) [31], Ying and Zhao (2001) [32] indicated mutation in
BMP2 affects the size of the PGC founding population rather than PGC proliferation and/or
survival, besides that, BMP2 produced by the endoderm (together with BMP4 and BMP8B) acts
as a part of the first signal in PGC precursor generation. In human, Kee et al. (2006) [24] founded
that addition of BMP-4 increased the expression of the germ cell-specific markers VASA and
SYCP3 during differentiation of hES cells. These authors also showed that BMP-7 and BMP8b
have additive effects on germ cell induction when added together with BMP4. West et al. (2010)
[33] reported that BMP-4 increased the expression of pre-migratory (OCT-4, NANOG, c-KIT)
and post-migration (DAZL, VASA) markers in PGC differentiated from ESC. Studies have also
shown that follicular fluid contains many bioactives factors such as GDF9b, stem cell factor,
basic fibroblast growth factor and oestrogen [34-36]. Dyce et al. (2006) [37] used follicular fluid
in porcine skin-derived cells, and observed the induction of germ-cell formation and supported
the expression of the markers, as OCT4, GDF9B, VASA and DAZL. In woman, the addition of
follicular fluid, rich in substances for oocyte growth and maturation, to the culture medium
triggers development of putative stem cells from the ovarian surface epithelium of women, and
express several genes related to pluripotency and oocytes [38].
104
The aims of the present study were to investigate the expression of pluripotency markers
(OCT-4, NANOG, REX-1, SOX2) in bovine fibroblast treated with different concentration of 5-
Aza, as well as to evaluate the effects of BMP-2, BMP-4 or follicular fluid on mRNA levels for
germ cell (VASA, DAZL, c-KIT), meiosis (SCP3) and oocytes (ZPA and GDF-9) markers in 5-
Aza treated fibroblasts cultured for 7 and 14 days.
Materials and Methods
Skin Fibroblasts
All biological material were collected from animals at the local abattoir. Primary bovine
skin fibroblast cultures were established from fresh biopsies from fetal ear skin. Fragments of
tissues were washed twice in saline solution (0.9% NaCl) that contained antibiotics (100 IU/mL
penicillin and 100 mg/mL streptomycin) (Sigma, St. Louis, MO, USA) and then, transported
within 1 h to the laboratory in this same solution.
In the laboratory, fragments of tissues of approximately 2 mm3 were transferred into 24-
well culture dishes (Corning, Lowell, MA, USA) that contained 1 mL of culture media. The basic
culture medium consisted of D-MEM (pH 7.2–7.4) supplemented with 20% FBS (Sigma), 2 mM
glutamine (Sigma) and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) (Sigma).
Cells were cultured at 38.5 °C in 5% CO2 in a humidified incubator. After 7 days, primary
fibroblast cultures started to grow out of the tissue fragments which were carefully removed and
passaged twice a week in a 1:3 ratio. All experiments were performed on at least three lines.
Treatment of Skin Fibroblasts with 5-Aza and cell viability
105
Dermal fibroblasts (1.5×105 cells/well) were cultured in 0.1 % gelatin (Sigma) precoated
4-well multidish (Nunc) and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza (Sigma) for 18 h, 36 h or 72
h. The basic culture medium consisted of D-MEM (pH 7.2–7.4) supplemented with 20% FBS
(Sigma), 2 mM glutamine (Sigma) and antibiotics (100 IU/mL penicillin and 100 mg/mL
streptomycin) (Sigma). Cells were cultured at 38.5 °C in 5% CO2 in a humidified incubator. At
the end of the culture period, the proportion of living and dead cells was assessed with calcein
AM (Molecular Probes) and ethidium homodimer-1 (Molecular Probes). Calcein AM (4 µM) and
ethidium homodimer-1 (2 µM) was added to wells and incubated protected from light for a
period of 15 min at room temperature. Determination of calcein and ethidium fluorescence at
excitation/emission wave lengths of 488/568 nm was performed using an inverted microscopy
(NIKON, Eclipse, TS100). Besides morphological evaluation, from each treatment, samples of
cells were collected and stored at –80 ºC until RNA extraction to analyze the expression of
markers for pluripotency (OCT4, NANOG, SOX2 and REX1).
Influence of follicular fluid and BMPs on the differentiation of germ cells
After treatment with 5-Aza (concentration and time of incubation determined previously),
the bovine fibroblast cells were cultured in DMEM/F12 medium supplemented with 0.1 mM β-
mercaptoetanol (Sigma), 2 mM glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 1 mM Non-
Essential Amino Acids (Sigma ), antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin)
(Sigma), 20% Knockout Serum Replacement (KSR) (Life, Grand Sland, NY, USA). For the
treatments, this medium was supplemented with 10 ng/mL BMP-2 (R&D systems, Minneapolis,
MN, USA), or 10 ng/mL BMP-4 (R&D systems) or 5% follicular fluid [39]. Cells were cultured
106
at 38.5 °C in 5% CO2 in a humidified incubator. Every 2 days, the culture medium was replaced
with fresh medium. After 7 and 14 days of culture, morphological analysis was performed and
cellular viability was determined by immunofluorescence analysis (Calcein AM and ethidium
homodimer-1) as described previously. Besides morphological evaluation, from each treatment,
samples of cells were collected and stored at –80 ºC until RNA extraction to analyze the
expression of markers for germ cells (VASA, DAZL and c-KIT), and oocytes (GDF-9, SCP3 and
ZPA).
RNA extraction and cDNA synthesis
Isolation of total RNA was performed using the Trizol® Plus purification kit (Invitrogen,
São Paulo, Brazil). According to the manufacturer’s instructions, 800 µL of Trizol solution was
added to each frozen samples and the lysate was aspirated through a 20-gauge needle before
centrifugation at 10,000 g for 3 min at room temperature. Thereafter, all lysates were diluted 1:1
with 70% ethanol and subjected to a mini-column. After binding of the RNA to the column, DNA
digestion was performed using RNAse-free DNAse (340 Kunitz units/mL) for 15 min at room
temperature. After washing the column three times, the RNA was eluted with 30 µL RNAse-free
water. The RNA concentration was estimated by reading the absorbance at 260 nm and was
checked for purity at 280 nm in a spectrophotometer (Amersham Biosciences, Cambridge,
England). For each sample, RNA concentrations were adjusted and used to synthesize cDNA.
Before the reverse transcription reaction, samples of RNA were incubated for 5 min at 70 ºC and
then cooled in ice. The reverse transcription was performed in a total volume of 20 µL composed
of 10 µL of sample RNA, 4 µL reverse transcriptase buffer (Invitrogen, São Paulo, Brazil), 8
units RNase out, 150 units of reverse transcriptase Superscript III, 0036 U random primers, 10
107
mM DTT and 0.5mMof each dNTP (Invitrogen, São Paulo, Brazil). The mixture was incubated at
42 ºC for 1 h, subsequently at 80 ºC for 5 min, and finally stored at –20ºC. The negative control
was prepared under the same conditions, but without the addition of reverse transcriptase.
qPCR
Quantification of mRNA was performed using GoTaq® qPCR Master Mix. PCR
reactions were composed of 1μL cDNA as a template in 7.5 μL of GoTaq® qPCR Master Mix
(Promega Corporation, Madison, WI, USA), 5.5 µL of ultra-pure water, and 0.5 μM of each
primer. The primers were designed by using the PrimerQuestSM program
(http://www.idtdna.com). Primers used in this study are shown in table 1. The specificity of each
primer pair was confirmed by melting curve analysis of PCR products. The thermal cycling
profile for the first round of PCR was: initial denaturation and activation of the polymerase for 10
min at 95 oC, followed by 40 cycles of 15 sec at 95 oC, 30 sec at 58 oC, and 30 sec at 72 oC. The
final extension was for 10 min at 72 oC. All reactions were performed in StepOne Real-Time
PCR (Applied Biosystems, Foster, CA, USA). Relative quantifications of mRNA were carried
out using the comparative threshold (Ct) cycle method. The delta-delta-Ct method was used to
transform the Ct values into normalized relative expression levels [40].
Statistical analysis
Levels of mRNA for pluripotency (SOX2, NANOG, OCT4, REX1), germ cells (VASA,
DAZL, c-KIT) and oocytes (ZPA, GDF9, SCP3) genes were analyzed by using the non-parametric
Kruskal–Wallis test and Dunn’s test for post hoc pair-wise comparisons (P<0.05). Data were
expressed as mean ± s.e.m.
108
Results
Cellular morphology and viability after treating fibroblasts with 5-Aza
Fibroblasts obtained from biopsies from ear skin fetuses grew out of the original explants
within 7 days and formed a monolayer, displaying a standard elongated morphology and a
vigorous growth in culture typical of this cell population. After the exposure to 5-Aza, cell
phenotype changed and fibroblast elongated morphology (Figure 1A) was replaced by an oval or
round shape (Figure 1B), furthermore, there was a reduction in cell number.
During the exposure to 5-Aza, the total cell number remained substantially unstable,
resulting from the effect of different concentrations of 5-Aza at different times, in manner time x
concentration-dependent, it can be observed in immunofluorescence analysis (calcein AM and
ethidium homodimer-1) (Figure 1C and Figure 1D). Cell proliferation rapidly decreased after
exposure to 5-Aza, and can observed a high apoptotic index, especially with the treatment with
2.0 μM of 5-Aza for 72 h showed a greater reduction in the number of cells when compared to
other treatments.
Expression of mRNA for markers of pluripotency after treating fibroblasts with 5-aza-cytidine
After treatment with 5-Aza for 18 h, real-time PCR analysis demonstrated that the levels
of mRNA for SOX2 (Figure 2A) and REX (Figure 2D) did not significant changes in any of the
treatments tested. However, NANOG expression was significantly higher after treatment with 2.0
μM of 5-Aza, when compared to 0.5 μM (Figure 2B). While the treatment with 1.0 μM of 5-Aza
109
significantly increased the levels of mRNA for OCT4, when compared to 0.5 μM of 5-Aza
(Figure 2C).
Figure 3 shows the expression of pluripotency genes after treatment with different
concentrations of 5-Aza for 36 h. The expression of mRNA for SOX2 (Figure 3A) did not
significant change in any of the treatments tested. The expression of mRNA for NANOG (Figure
3B), was significantly increased after treatment with 1.0 μM of 5-Aza when compared to 0.5 μM
of 5-Aza. The expression of mRNA for OCT4 (Figure 3C) was significantly increased after
treatment with 2.0 μM of 5-Aza when compared to 0.5 μM of 5-Aza. In addition, expression of
mRNA for REX (Figure 3D) was significantly increased after treatment with 1.0 or 2.0 μM of 5-
Aza when compared to 0.5 μM of 5-Aza.
After culture for 72 h in different treatments with 5-Aza, the expression of SOX2 (Figure
4A), NANOG (Figure 4B), OCT4 (Figure 4C), and REX (Figure 4D) was significantly higher
after supplementation of the medium with 2.0 μM of 5-Aza compared to 0.5 μM of 5-Aza.
Regarding the effects of incubation time on the expression of pluripotency genes, the
culture with 0.5 (Figure 5Ai), 1.0 (Figure 5Aii) or 2.0 µM (Figure 5Aiii) 5-Aza for 72 h increased
SOX2 mRNA levels, when compared to those seen after 18h or 36h. Fibroblasts cultured with 5-
Aza (0.5 μM or 2.0 μM) for 72 h had higher levels of mRNA for NANOG than those cultured for
18 and 36 h (Figure 5Bi and 5Bii). However, no effect of incubation time on expression of
NANOG was observed after culturing in presence of 1.0 μM of 5-Aza (Figure 5Bii).
After culturing cells with 0.5 μM and 1.0 μM of 5-Aza, a significant reduction in OCT4
expression was observed when the incubation time was increased from 18 to 72h (Figures 5Ci
and 5Cii). On the other hand, fibroblasts cultured with 2.0 μM of 5-Aza for 72h had higher levels
of OCT4 mRNA than those cultured for 18 and 36h a (Figure 5Ciii). The mRNA levels for REX1
in fibroblasts cultured either with 0.5 μM or 2.0 μM of 5-Aza for 72h were significantly higher
110
than those observed after 18 and 36h (Figures 5Di and 5Diii). When the cells were cultured with
1.0 μM of 5-Aza, a progressive and significant increase in REX1 expression was observed when
the incubation time was increased from 18 to 36 and 72h (Figure 5Dii).
Cellular morphology and viability after culturing 5-Aza treated cells with BMP-2, BMP-4 or
follicular fluid
After a series of preliminary experiments, we established that 2.0 μM of 5-Aza for 72 h
represented the optimal combination for bovine skin fibroblasts. At the end of this treatment,
cells were cultured in differentiation medium supplemented with BMP-2, or BMP-4 or follicular
fluid, for 7 or 14 days.
Initially, growing in differentiation medium with BMP-2, BMP-4 or follicular fluid,
induced the cells to present the ability to form colony. The culture in differentiation medium
associated with 10 ng/mL of BMP-2 (Figure 6A, B, C, D) or BMP-4 (Figure 6E, F, G, H) for 14
days, resulted in a resumption of proliferative index, and morphological changes, gradually cells
organized in clusters. The culture in BMP-2 or BMP-4 for 7 days did not affect the cell
proliferation rate. The culture in 5% follicular fluid for 7 or 14 days (Figure 6I, J, K, L),
promoted an large increase in cell proliferation rate, with vigorous growth and large rate of
apoptosis.
Expression of mRNA for markers of germ cells and oocytes after culturing 5-Aza treated cells
with BMP-2, BMP-4 or follicular fluid
111
Since exposure of fibroblasts to 2.0 µM of 5-Aza for 72h promoted higher expression of
pluripotency genes, this concentration and incubation time were chosen to treat the cells before
culture in differentiation medium containing BMP-2, BMP-4 or follicular fluid for 7 or 14 days.
After 7 days of culture, BMP-4 stimulated a significant increase in the expression of
VASA, when compared to control medium. However, BMP-4 or follicular fluid had no effect on
VASA expression (Figure 7A). While the culture for 14 days in medium containing follicular
fluid, significantly increased the expression of VASA, compared to the culture in medium control
(Figure 7B). DAZL expression was significantly increased after culture in medium containing
BMP-2 for 7 or 14 days (Figure 7C, D), when compared to other treatments. The culture for 7
days in medium supplemented with BMP-2, stimulated mRNA expression of c-KIT (Figure 7E),
when compared to the culture in to other treatments. The culture for 14 days, did not significantly
modify the expression of c-KIT (Figure 7F).
Regarding the expression of oocyte markers, after 7 days of the culture, ZPA expression
was significantly reduced when cells were cultured in medium containing BMP-2 (Figure 8A).
Already, the culture in medium containing follicular fluid for 14 days, significantly increased
ZPA expression, when compared to the culture in medium containing BMP-4 or control medium
only (Figure 8B). Follicular fluid increased significantly mRNA levels for GDF-9 (Figure 8C),
when compared to culture in control medium. After 14 days of culture in medium supplemented
with BMP-2, increased significantly GDF-9 expression, when compared to other treatments
(Figure 8D). An increase in SCP3 mRNA expression was observed after culture in presence of
BMP-4 for 7 days, when compared to other treatments (Figure 8E). After 14 days culture in
medium supplemented with BMP-2, promoted an increase in expression of SCP3 (Figure 8F),
when compared to other treatments.
112
Figures 9, 10, 11 and 12, showed the expression of makers for germ cells and oocyte after
0 h, 7 or 14 days in the different treatments. For cells cultured in control medium, VASA
expression was significantly increased after 7 days, when compared to the time 0 or after 14 days
(Figure 9A). DAZL expression was not altered after 7 or 14 days culture (Figure 9B). An increase
in the mRNA levels for c-KIT was observed after 7 or 14 days of culture, when compared to 0h
(Figure 9C). Culture for 14 days significantly increased ZPA (Figure 9D) and GDF-9 (Figure 9E)
expression, compared to culture for 0 h or 7 days. A progressive and significant increased in
SCP3 expression was observed when increasing culture time from 0 h to 7 and 14 days (Figure
9F). Regarding the cells cultured in medium supplemented with BMP-2, an increase in the
mRNA levels for VASA (Figure 10A), DAZL (Figure 10B), c-KIT (Figure 10C) and GDF-9
(Figure 10E) was observed after 7 days of culture, when compared to 0 h and 14 days. While,
after 14 days of culture in medium supplemented with BMP-2, the levels of mRNA for ZPA
(Figure 10D) and SCP3 (Figure 10F) were significantly higher when compared to other times of
culture.
For cells cultured in presence of BMP-4, an increased in the mRNA levels for VASA
(Figure 11A), c-KIT (Figure 11C), GDF-9 (Figure 11E) and SCP3 (Figure 11F) was observed
after 7 days of culture, when compared to 0h or 14 days. However, a reduction in DAZL levels
(Figure 11B), was seen when increasing culture time from 7 or 14 days. A progressive and
significant increased in mRNA levels for ZPA (Figure 11D) was observed when increasing
culture time from 0 h to 7 or 14 days.
The culture for 7 days in medium containing 5% follicular fluid, significantly increased
VASA expression (Figure 12A), when compared to 0 h or 14 days. After 14 days of culture, and
significantly increased in the levels of mRNA for DAZL (Figure 12B) were observed, when
compared with 0 h or 7 days. A progressive and significant increased in mRNA levels for c-KIT
113
(Figure 12C), ZPA (Figure 12D) and SCP3 (Figure 12F) was observed when increasing culture
time from 0 h to 7 or 14 days. The culture with follicular fluid for 7 or 14 days, significantly
increased the levels of mRNA for GDF-9 (Figure 12E).
Discussion
This study shows for the first time that 5-Aza promotes changes in cell morphology and
induces expression of genes related with pluripotency in cultured bovine fibroblasts. The 5-Aza is
able to modify cell morphology [18, 41] in tumor-derived cell lines [18, 42], embryonic and adult
cells [41]. Pennarossa et al. (2013) [19] used 5-Aza to induce conversion of human skin
fibroblasts into insulin-secreting cells. 5-Aza is a chemical derivative of the DNA nucleoside
cytidine that causes DNA demethylation or hemi-demethylation, binds covalently and
irreversibly to DNA methyltransferase 1 (DNMT1), and regulate gene expression by relaxing
chromatin structure. Consequently, 5-Aza allows transcription factors to bind to the promoter
regions of genes associated with cell pluripotency [43-45]. Previous studies have reported that 5-
Aza alter cell, as mouse fibroblasts and B lymphocytes [46], human and porcines fibroblasts [19,
20, 44], as well as phenotype and gene expression and promotes the generation of iPS in mouse
[15, 47]. Efficient reprogramming methods have been explored since the first report of the
generation of human induced pluripotent stem cells [9, 48, 49]. Currently, new methods have
been applied in the production of iPS cells, which include the use of episomal plasmids [50] or
excisable expression systems [51], recombinant cell-penetrating reprogramming proteins [52,
53], reprogramming mRNAs [55, 56, or microRNAs [57, 58]. Besides 5-Aza, a growing number
of compounds have been identified that can functionally replace reprogramming transcription
factors, enhancing efficiency of iPS generation and accelerating the reprogramming process [15].
114
Among these compounds, it can be highlighted N-Phthalyl-L-Tryptophan (RG108), valproic acid
(VPA), trichostatin A (TSA), sodium butyrate (NaB) and other molecules [44].
In this study we demonstrate that exposure of bovine skin fibroblasts to 2.0μM of 5-Aza
for 72h lead to highest expression of mRNA for SOX2, NANOG, OCT4 and REX. OCT4,
NANOG and REX are considered the main pluripotency markers, and the expression of these
genes supported the pluripotency of iPS in goat species [59]. Exposure of in porcine fetal
fibroblasts to 5-Aza reduced the expression of DNA methyltransferase 1 (DNMT1) and altered the
expression of genes involved in imprinting (IGF2) and apoptosis (BAX, BCL2L1) [60]. In
addition, Pennarosa et al. (19, 20] exposed human and pig dermal fibroblasts to 5-Aza for 18 h,
followed by a protocol for the induction of endocrine pancreatic differentiation. The results
demonstrated changes in cell morphology and expression of OCT4, NANOG, SOX2 and REX, and
after differentiation, the cells expressed insulin and was able to release it in response to a
physiological glucose challenge [19, 20]. OCT4, a regulator of pluripotency, it is expressed in
pluripotent stem cells, in iPS and in germ cells (during embryogenesis) [9, 61, 62]. It has been
demonstrated that germline stem cells derived from ovaries also possessed pluripotency as
evidenced by the expression of OCT4 [54, 63]. The transcription factor NANOG is essential for
the establishment of pluripotency during the derivation of ESC and iPS. However, NANOG is not
essential to maintain pluripotency [64, 65]. The repression of OCT4 or SOX2 in ESCs promotes
differentiation [66, 67], besides that, OCT4 and SOX2 proteins dimerize on DNA to activate their
target genes in order to specify pluripotency [68]. NANOG directly transactivates the REX1
promoter and positively regulates REX expression. OCT4 and SOX2 can either activate or repress
the REX promoter, depending on the cellular environment [69-71]. REX is a stringent
pluripotency marker that specifies a subpopulation of the most undifferentiated ESC [72].
115
The culture of 5-Aza treated fibroblasts in differentiation medium supplemented with
BMP-2 and BMP-4 induced the expression of mRNA for germ cell and oocyte markers. In vivo
and in vitro evidences demonstrate that BMP4 induce the differiantion of PGC in mouse
developing embryos [28, 73, 74]. BMP signals through ACVRI in the visceral endoderm ant is
necessary for normal induction of PGCs in the epiblast [75]. In vitro differentiation of human
PGCs was increased by addition of BMP-4, BMP-7 and BMP-8b, in co-culture with human or
mouse fetal gonad stromal cells [24, 76-79). Several studies have PGC absence in BMP4 mutant
mouse embryos and a reduction in these cells in BMP2 and BMP8 mutant embryos [25, 28, 31,
80-81]. Addionally, BMP-4 promotes mammalian oogonial stem cell differentiation in humans
[82-84], buffaloes [85], and goats [11]. BMP-2 also plays a crucial role in the differentiation of
primordial germ cells [86].
The presence of follicular fluid in differentiation medium also influences the expression
of mRNA for germ cell and oocyte markers in cultured 5-Aza treated fibroblasts. The follicular
fluid is a select ultrafiltrate of plasma that has been modified by secretion and uptake of specific
components by the cells within the follicle itself [87, 88]. The follicular fluid may contain several
proteins, such as BMP that are involved in the formation of germ cells [37, 89-91]. In porcine
species, follicular fluid successfully simulate differentiation of oocyte during culture of stem
cells obtained from skin, adipose and ovarian tissues [37, 39, 92], and this cells expressed DAZL,
VASA and SCP3 [39]. Follicle-like structures were successfully differentiated in vitro from
hESCs in the presence of follicular fluid [94]. Cheng et al. (2012) [93] promoted differentiation
human amniotic fluid stem cells (hAFSCs) into oocyte-like cells after culturing those cells in
presence of porcine follicular fluid. The differentiated OLCs expressed EGFP under the BMP15
promoter which was co-localized with ZP2 expression [93].
116
Our results indicate that BMP-2, BMP-4 or bovine follicular fluid influenced the
expression of markers for germ cells (VASA, DAZL and c-KIT), meiosis (SCP3) and oocyte
(GDF-9, ZPA). Studies indicated that VASA is expressed in post-migratory PGCs until the post-
meiotic stage of oocytes [95, 96]. DAZL is also considered essential for PGC development, since
DAZL knockout mice lack a germ cell population [76, 97]. DAZL and VASA have been used as
markers for germ cell differentiation in various species (human: [76, 98], murine: [99], and
porcine [37]. Clark et al. (2004) [100] indicated that c-KIT is one of the most used markers for
PGCs, and this receptor is important for migration and survival of PGC [101]. GDF-9 is
expressed specifically in oocytes [102], and have been used as oocyte marker after its
differentiation from stem cells [37, 91]. GDF-9 was expressed in populations of cultured mouse
embryonic stem cells with an oocyte-like phenotype [103]. SCP3 expression is frequently used as
a meiotic marker [104]. The zona pellucida glycoprotein is only expressed in oocytes [105], and
earlier studies reported ZP-like structures surrounding OLCs differentiated in vitro [106, 107].
Singhal et al. (2015) [108] recently showed that expression of germ cell markers, like zona
pellucida and VASA, were stimulated by BMP-4.
Conclusions
In conclusion, treatment of bovine skin derived fibroblasts with 2.0 μM 5-Aza for 72h
induces the expression of mRNAs for SOX2, OCT4, NANOG and REX. The cultured of these
cells in differentiation medium culture supplemented with BMP-2, BMP -4 or follicular fluid
induce morphological changes and promotes expression of markers for germ cells (VASA, DAZL
and c-KIT), meiosis (SCP3) and oocyte (GDF-9, ZPA). This method can be effective for
production of oocytes from differentiation of fibroblasts and can thus contribute, in the future, for
117
the development of new biotechnologies to solve fertility problems in human species, and to
increase the reproductive potential of genetically superior animals or endangerous species.
Acknowledgements
This work was financially supported by CNPq (Grant N° 478198/2013-2), and the
authors thank the members of the Laboratory of Animal Reproduction of the Biotechnology
Nucleus of Sobral.
Compliance with ethical standards
Conflict of interest: The authors declare that they have no conflict of interest.
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List of tables and figures
Table 1. Primer pairs used in real-time PCR for quantification of markers of pluripotency, germ
cells and oocytes genes expressed in cells cultured.
Figure 1. Representative pictures of the morphological changes in bovine skin fibroblasts
exposed to 5-Aza. (A) The fibroblast cells isolated from bovine fetal ear skin (untreated cells).
(B) Fibroblasts exposed to 2.0 µM of 5-Aza for 72 h. (C) Fluorescence staining of viable cells for
Calcein AM. (D) Fluorescence staining of apoptotic cells for ethidium homodimer-1. Scale bar =
100 µm.
Figure 2. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in fibroblasts
cultured for 18 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).
Figure 3. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in fibroblasts
cultured for 36 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).
Figure 4. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in fibroblasts
cultured for 72 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).
Figure 5. Levels of mRNA for of pluripotency genes, (A) SOX2, (B) NANOG, (C) OCT4 and (D)
REX, in fibroblasts cultured for 18 h, 36 h or 72 h in with different concentrations of 5-Aza (0.5,
1.0 or 2.0 µM).
Figure 6. Representative pictures of the morphological characterization in bovine skin fibroblasts
exposed to 5-Aza and cultured in differentiation medium for 14 days. Fibroblast cultured for 14
days in differentiation medium supplemented with 10 ng/mL of BMP-2 (line 1), 10 ng/mL of
BMP-4 (line 2), 5% follicular fluid (line 3), (A, D, G) cell analyzed by light microscopy, (B, E,
H) Fluorescence staining of viable cells for Calcein AM; (C, F, I) Fluorescence staining of
apoptotic cells for ethidium homodimer-1. Scale bar = 100 µm.
Figure 7. Levels of mRNA for markers of germ cells [VASA (A, B), DAZL (C, D), C-KIT (E, F)]
in cells cultured for 7 (A, C, E) or 14 (B, D, F) days in control medium or supplemented with
BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular fluid.
Figure 8. Levels of mRNA for markers of oocytes [ZPA (A, B), GDF-9 (C, D), SCP3 (E, F)] in
cells cultured for 7 (A, C, E) or 14 (B, D, F) days in control medium or supplemented with BMP-
2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular fluid.
Figure 9. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and
oocytes [ZPA (D), GDF-9 (E), C-KIT (F)] after culture cells for 0 h (5-Aza), 7 or 14 days in
control medium.
Figure 10. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and
oocytes [ZPA (D), GDF-9 (E), C-KIT (F)] after culture cells for 0 h (5-Aza), 7 or 14 days in
medium supplemented with 10 ng/mL of BMP-2.
131
Figure 11. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and
oocytes [ZPA (D), GDF-9 (E), C-KIT (F)] after culture cells for 0 h (5-Aza), 7 or 14 days in
medium supplemented with 10 ng/mL of BMP-4.
Figure 12. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and
oocytes [ZPA (D), GDF-9 E), C-KIT (F)] after culture cells for 0 h, 7 or 14 days in medium
supplemented with 5% follicular fluid.
132
Table 1. Primer pairs used in real-time PCR for quantification of markers of pluripotency, germ
cells and oocytes genes expressed in cells cultured.
Target
gene
Primer sequence (5´ 3´)
Sense (s),
anti-sense
(As)
GenBank number
GAPDH
CACCCTCAAGATTGTCAGCA
GGTCATAAGTCCCTCCACGA
S
As
NM_001034034.2
SOX2
TGGATCGGCCAGAAGAGGAG
CAGGCGAAGAATAATTTGGGGG
S
As
NM_001105463.2
NANOG
CGTGTCCTTGCAAACGTCAT
CTGTCTCTCCTCTTCCCTCCTC
S
As
DQ069776
OCT-4
GAGAAAGACGTGGTCCGAGTG
GACCCAGCAGCCTCAAAATC
S
As
NM_174580.2
REX
CCTGTGAGGGGAGCTCTAGT
TTTTCAGCAAGCACCCATGC
S
As
XM_003587951.1
VASA
TGGTCCTGGCTTCAGTGGTA
TCTTGCCGGGGTAATTCTTTCT
S
As
NM_001007819.1
DAZL
TACCCGCCTCTGACTCTCTC
GTGTTCACTCAGAGGGGCTC
S
As
EF501823.2
c-KIT
TCGTGGATGGCTGTGAATACAA
CAAGTGAGAGAATGCCGGGT
S
As
GI: 16580734
ZPA
TCGTGGATGGCTGTGAATACAA
CAAGTGAGAGAATGCCGGGT
S
As
NM_173973.2
GDF-9
ACAACACTGTTCGGCTCTTCACCC
CCACAACAGTAACACGATCCAGGTT
S
As
GI:51702523
SCP3
GTTGGCAAAACCATCCGTGG
GGGGTCTTCTCTTCAATGGCA
S
As
NM_001040588.2
133
Figure 1. Representative pictures of the morphological changes in bovine skin fibroblasts
exposed to 5-Aza. (A) The fibroblast cells isolated from bovine fetal ear skin (untreated cells).
(B) Fibroblasts exposed to 2.0 µM of 5-Aza for 72 h. (C) Fluorescence staining of viable cells for
Calcein AM. (D) Fluorescence staining of apoptotic cells for ethidium homodimer-1. Scale bar =
100 µm.
134
Figure 2. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in fibroblasts cultured for 18 h in with different
concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).
135
Figure 3. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in fibroblasts cultured for 36 h in with different
concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).
136
Figure 4. Levels of mRNA for SOX2 (A), NANOG (B), OCT4 (C) and REX (D) in fibroblasts cultured for 72 h in with different
concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).
137
Figure 5. Levels of mRNA for of pluripotency genes, (A) SOX2, (B) NANOG, (C) OCT4 and (D) REX, in fibroblasts cultured for 18
h, 36 h or 72 h in with different concentrations of 5-Aza (0.5, 1.0 or 2.0 µM).
138
Figure 6. Representative pictures of the morphological characterization in bovine skin fibroblasts exposed to 5-Aza and cultured in
differentiation medium for 14 days. Fibroblast cultured for 14 days in differentiation medium supplemented with 10 ng/mL of BMP-2
(line 1), 10 ng/mL of BMP-4 (line 2), 5% follicular fluid (line 3), (A, D, G) cell analyzed by light microscopy, (B, E, H) Fluorescence
staining of viable cells for Calcein AM; (C, F, I) Fluorescence staining of apoptotic cells for ethidium homodimer-1. Scale bar = 100
µm.
139
Figure 7. Levels of mRNA for markers of germ cells [VASA (A, B), DAZL (C, D), C-KIT (E, F)] in cells cultured for 7 (A, C, E) or 14
(B, D, F) days in control medium or supplemented with BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular fluid.
140
Figure 8. Levels of mRNA for markers of oocytes [ZPA (A, B), GDF9 (C, D), SCP3 (E, F)] in cells cultured for 7 (A, C, E) or 14 (B,
D, F) days in control medium or supplemented with BMP-2 (10 ng/mL), BMP-4 (10 ng/mL) and 5% follicular fluid.
141
Figure 9. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and oocytes [ZPA (D), GDF-9 (E), C-KIT
(F)] after culture cells for 0 h (5-Aza), 7 or 14 days in control medium.
142
Figure 10. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and oocytes [ZPA (D), GDF-9 (E), C-
KIT(F)] after culture cells for 0 h (5-Aza), 7 or 14 days in medium supplemented with 10 ng/mL of BMP-2.
143
Figure 11. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and oocytes [ZPA (D), GDF-9 (E), C-
KIT (F)] after culture cells for 0 h (5-Aza), 7 or 14 days in medium supplemented with 10 ng/mL of BMP-4.
144
Figure 12. Levels of mRNA for markers of germ cells [VASA (A), DAZL (B) and C-KIT (C)] and oocytes [ZPA (D), GDF-9 (E), C-
KIT(F)] after culture cells for 0 h, 7 or 14 days in medium supplemented with 5% follicular fluid.
145
9 ARTIGO III
Bovine ovarian stem cells differentiate into germ cells and oocyte-like structures after
culture in vitro
(Células-tronco ovarianas bovinas diferenciam-se em células germinativas e estruturas
semelhantes a oócitos após cultivo in vitro)
Artigo a ser submetido ao periódico Reproduction in Domestic Animals
(Qualis B2 - Biotecnologia)
146
Bovine ovarian stem cells differentiate into germ cells and oocyte-like structures after
culture in vitro
Oocyte-like structures formation from from ovarian stem cells bovine
G B. de Souza1, J J N Costa1, E V da Cunha1, J R S Passos1, R P Ribeiro1, M V A Saraiva1, R van
den Hurk2, J R V Silva1*
1Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042-280,
Sobral, CE, Brazil; 2Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands
*Corresponding address (J. R. V. Silva): Biotechnology Nucleus of Sobral - NUBIS, Federal
University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041-040, Sobral,
CE, Brazil. [[email protected]]
Contents
The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the
effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation
of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured
in α-MEM+ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were
147
evaluated for their morphological appearance, viability, expression of alkaline phosphatase, and
for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and
SCP3) by qPCR. Levels of mRNA were analysed by using ANOVA and Bonferroni test
(P<0.05). The results showed that at day 0, ovarian stem cells expressed specific markers of
pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase. After
the period of differentiation, cells had morphological features that resemble primordial germ cells
(PGCs) and oocyte-like cells (OLCs). The PGC-like cells expressed VASA and DAZL, while the
OLCs expressed mRNAs for GDF9, ZPA and SCP3. In conclusion, OLCs can be differentiated in
vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the
expression of mRNAs for germ cell and oocyte markers.
Keywords: BMP2, BMP4, gene expression, follicular fluid
Introduction
In recent years, reproductive biotechnology has been developed in order to solve female
reproductive problems or to increase reproductive efficiency in several species of domestic
animals or in endangered species. In humans, it is well-known that between 10 and 15% of
couples are infertile, the main cause being associated with the default of appropriate release of
fertilizable oocytes (Nicholas et al. 2009a). This information shows that in-vitro differentiation of
germ cells in oocytes from stem cells may have an important therapeutic use. In addition, it may
provide a valuable model for identifying factors involved in germ cell formation and
differentiation.
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In the last years, researches have provided evidences for the existence of female germ
line cells that contribute to postnatal oogenesis (Pan et al. 2015). Several other studies have
shown the presence of germ line cells and potential postnatal oogenesis using various techniques
in mice as well as in other species, including human (Parte et al. 2011). The isolation and
manipulation of ovarian stem cells have tremendous application in medical, veterinary and
animal production fields (Mooyottu et al. 2011).The stem cells have been isolated using different
strategies, propagated in vitro for several generations and their ability to differentiate into oocytes
in vivo has been demonstrated (White et al. 2012). Multiple attempts have been made over the
past decade to determine whether murine or human embryonic stem cells (ESC) are able to
differentiate into primordial germ cells (PGCs) or oocyte-like cells (OLCs) in vitro (Parte et al.
2011). Moreover, it has been reported that germ cell-like cells can be derived in vitro from
mesenchymal stem cells (MSCs) derived from porcine fetal skins (Dyce et al. 2011), mouse bone
marrow (Johnson et al. 2005), or human adult ovaries (Bukovsky et al. 2005). Additionally,
certain studies have reported that human or murine ESCs can spontaneously differentiate into
OLCs (Clark et al. 2004).
Several studies show the role of exogenous factors on the formation of germ cells and
oocyte differentiation. Bone morphogenetic protein 4 (BMP4) promotes mammalian oogonial
stem cell differentiation in humans (Park et al. 2013), buffalo (Shah et al. 2015) and goat (Singhal
et al. 2015). BMP2 also plays a crucial role in the differentiation of primordial germ cells (Pera et
al. 2004). In rats, this protein plays an important role in the formation of alkaline phosphatase
positive primordial germ cells and induces expression of markers for PGCs (Saitou and Yamaji,
2010). Other studies have shown that porcine follicular fluid effectively promotes the formation
of germ-like cells from stem cells (Cheng et al. 2012). Hong et al. (2014) reported that human
follicular fluid stimulated differentiation into oocyte-like cells in vitro from stem cells (Hong et
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al. 2014). However, the differentiation of OLCs from ovarian stem cells isolated from adult
bovine ovaries has not yet been demonstrated. Thus, we hypothesize that bovine BMP2, BMP4
and bovine follicular fluid stimulated ovarian stem cells to form germ cells and OLCs in vitro.
The aim of the study was to evaluate the effects of BMP2, BMP4 and follicular fluid on
differentiation of ovarian stem cells into germ cells and oocytes, and to evaluate the effects on
expression of markers for primordial germ cells (VASA, DAZL) and oocytes (SCP3, GDF9, ZPA)
in vitro.
Materials and Methods
Unless otherwise stated, all chemicals and reagents used in this study were purchased
form from Sigma Chemical Co. (St Louis, MO, USA). All the animal procedures and protocols
were approved by the Local Ethics Committee for Animal Experiments of the Federal University
of Ceara (2015/18).
Ovary Collection
Fresh ovaries (n = 20) from adult mixed breed cows (Bos taurus) were collected at a
slaughterhouse. Immediatelly, the ovaries were washed (for approximately 10 sec) in 70%
alcohol, then washed twice in 0.9% saline solution, and transferred to minimum essential
medium-alpha (α-MEM – Sigma-Aldrich, Saint-Louis, Missouri, USA) supplemented with 100
IU/mL penicillin and 100 mg/mL streptomycin. Subsequently, ovarian pairs from each cow were
transported to the laboratory within 1 h at 4 ºC (Chaves et al. 2008).
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Primary ovarian cortical tissue culture
In the laboratory, to perform a primary culture, ovarian cortical tissue from the same
ovarian pair was cut (1 mm × 1 mm × 1 mm) using a scissor and scalpel under sterile conditions.
To isolate ovarian stem cells, cortical slices were transferred to 24-well culture dishes (Corning,
Germany) that contained 1 mL of culture medium. The basic culture medium consisted of α-
MEM (pH 7.2–7.4) supplemented with ITS (10 µg/mL insulin, 5.5 µg/mL transferrin, and 5
ng/mLselenium), 2 mM glutamine (Sigma-Aldrich), antibiotics (100 IU/mL penicillin and 100
mg/mL streptomycin) (Sigma), 2 mM pyruvate and 3.0 mg/mL of bovine serum albumin (α-
MEM+) as described by Garor et al. (2009). Culture was performed at 38.5 °C in 5% CO2 in a
humidified incubator. The cortical tissue pieces were discarded from the cell culture, where after
ovarian stem cells grow and migrate from the margin of the ovarian tissue piece and adhered onto
the plastic surface of the culture plates to form cell colonies. These ovarian stem cells were
processed to analyse the morphology, viability, activity of alkaline phosphatase and expression of
specific markers of pluripotency stem cells or placed in culture for 14 days in the presence of
BMP2, BMP4, both BMP2 and BMP4, and follicular fluid.
Influence of follicular fluid and BMPs on cellular differentiation
In order to induce differentiation into germ cells and oocytes, ovarian stem cells
obtained from ovarian tissues were plated onto 24-well culture plates (Corning, Germany), and
cultured in five treatments, i.e., in control medium (α-MEM+) alone or supplemented with 50
ng/mL BMP2 (R&D Systems, Inc., Minneapolis, MN), 50 ng/mL BMP4 (R&D Systems, Inc.,
Minneapolis, MN), both BMP2 (50 ng/mL) plus BMP4 (50 ng/mL) (Panula et al. 2011), or 5%
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cow follicular fluid (Dyce et al. 2011) at 38.5 °C in 5% CO2 in a humidified incubator. The
culture medium was replaced every 4 days with fresh medium. After 14 days of culture in
differentiation medium, the cells were destined to analyse viability and expression of alkaline
phosphatase or stored at –80 ºC until RNA extraction to evaluate the expression of specific
markers for germ cells and oocytes.
Viability analysis
On days 0 and 14 of culture, the proportion of living and dead cells was assessed with
calcein AM (Molecular Probes) and ethidium homodimer-1 (Molecular Probes). Calcein AM (12
µM) and ethidium homodimer-1 (120 µM) was added to wells and the cells were incubated
protected from light for a period of 10 min at room temperature. Determination of calcein and
ethidium fluorescence at excitation/emission wave lengths of 488/568 nm a fluorescence
microplate reader was used, using an inverted microscopy (NIKON, Eclipse, TS100).
Alkaline Phosphatase Assay
Alkaline phosphatase (AP) activity was evaluated according to manufacturer instructions
(Alkaline Phosphatase Detection Kit, Sigma-Aldrich) in ovarian stem cells before (day 0) and
after differentiation (day 14) in the different treatments. In short, cells were fixed (citrate,
buffered acetone 60%) for 30 sec and then incubated in a working solution of reagents which
consisted of Salt RR Blue, Naphtol AS-MX phosphate solution and water in a 2:1:1 ratio. After
30 min, the cells were rinsed with deionized water, stained with hematoxylin, rinsed with
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deionized water and after 15 min the cells were observed under an inverted microscope (NIKON,
Eclipse, TS100). The cells or cell colonies expressing AP activity were stained in red.
RNA extraction and cDNA synthesis
To analyze the expression of markers for pluripotency (OCT4 and SOX2), in germ cells
(DAZL and VASA) and oocytes (SCP3, GDF9, ZPA), isolation of total RNA was performed using
the TRIzol® Reagent Plus purification kit (Invitrogen, São Paulo, Brazil). According to the
manufacturer’s instructions, 800 µL of TRIzol® solution was added to each of the frozen samples
and the respective lysates were aspirated through 20-gauge needles before centrifugation at
10,000 g for 3 min at room temperature. Thereafter, all lysates were diluted 1:1 with 70% ethanol
and subjected to a mini-column. After binding of the RNA to the column, DNA digestion was
performed using RNAse-free DNAse (340 Kunitz units/mL) for 15 min at room temperature.
After washing the column three times, the RNA was eluted with 30 µL RNAse-free water. The
RNA concentration was estimated by reading the absorbance at 260 nm and was checked for
purity at 280 nm in a spectrophotometer (Amersham Biosciences, Cambridge, England). For each
sample, RNA concentrations were adjusted and used to synthesize cDNA. Before the reverse
transcription reaction, samples of RNA were incubated for 5 min at 70 ºC and then cooled in ice.
The reverse transcription was performed in a total volume of 20 µL composed of 10 µL of
sample RNA, 4 µL reverse transcriptase buffer (Invitrogen, São Paulo, Brazil), 8 units RNase
out, 150 units of reverse transcriptase Superscript III, 0036 U random primers, 10 mM DTT and
0.5 mM of each dNTP (Invitrogen, São Paulo, Brazil). The mixture was incubated at 42 ºC for 1
h, subsequently at 80 ºC for 5 min, and finally stored at –20 ºC. The negative control was
prepared under the same conditions, but without the addition of reverse transcriptase.
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Real time PCR
The quantification of mRNA was performed with the use of SYBR® Green. Each
reaction in real time (20 µL) contained 10 µL of SYBR® Green Master Mix (Applied
Biosystems, Warrington, UK), 7,3 µL of ultra-pure water, 1 µL of cDNA and 0.5 μM of each
primer, and real-time PCR was performed in StepOne Real-Time PCR (Applied Biosystems,
Warrington, UK) thermocycler. The thermal cycling profile for the first round of PCR was initial
denaturation and activation of the polymerase for 10 min at 95 oC, followed by 40 cycles of 15
sec at 95 oC, 30 sec at 58 oC, and 30 sec at 72 oC. The final extension was for 10 min at 72 oC.
The primers were designed by using the primerquestsm program (http://www.idtdna.com) primers
used in this study are shown in Table 1. The specificity of each primer pair was confirmed by
melting curve analysis of PCR products. Relative quantifications of mRNA were carried out
using the comparative threshold (CT) cycle method. The delta-delta-Ct method was used to
transform the Ct values into normalized relative expression levels (Livak and Schmittgen, 2001).
Statistical analysis
Levels of mRNA were analysed by using ANOVA and Bonferroni test (P<0.05). Data
were expressed as mean ± standard error of the mean (SEM).
Results
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Morphological characteristics of cells before and after culture with BMP-2, BMP-4 and
follicular fluid
During culture of ovarian cortical tissue, cells migrated from the margin of the ovarian
tissue piece and adhered onto the plastic surface of the culture plates to form cell colonies. These
cells showed fusiform shapes and, morphologically, resembled undifferentiated stem cells (Fig.
1A-B). As illustrated in Figure 2, alkaline phosphatase activity was detected in these cells. After
seven days of culture in differentiation medium, some subpopulations of the cells became
morphologically different from the starting cultures, increased their volume and formed
aggregates (Fig. 1C-D). These aggregates gradually became structures similar to oocytes after 14
days of culture in medium supplemented with BMP2 (Fig. 1E-F), BMP4 (Fig. 1G-H), BMP2 and
BMP4 (Fig. 1I-J) and follicular fluid (Fig. 1K-L).
On days 0 and 14 of culture, the proportion of living and dead cells was assessed with
calcein AM and ethidium homodimer-1 staining of cells, respectively. Fluorescence analysis
shows ovoid cells clusters positive for calcein (green) and ethidium homodimer (red) in control
medium (α-MEM), suggesting that the ovarian stem cells proliferated and form clusters (Fig. 1B).
After 14 days of culture in control medium and other treatments, cells became morphologically
distinct, suggesting that they are germ cell clusters. These cells aggregated and gradually became
structures similar to oocytes that were labeled positive for calcein (Fig.1. D, F, H, L).
Quantification of mRNA for specific markers of pluripotency, germ cells and oocytes
At day 0, real-time PCR showed that cells that migrated from the margin of the ovarian
tissue piece expressed mRNA for OCT4 and SOX2 (Fig. 3A). Melting curve analysis confirmed
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the specificity of the products after amplification. After 14 days of culture, OLCs formed after
culture in medium, supplemented with BMP2, both BMP2 and BMP4, and follicular fluid, had
significantly higher (P<0.05) levels of mRNA for VASA than those cultured in control medium
(α-MEM+, Fig. 4A). Addition of BMP-4 alone to cell cultures had no effect on VASA expression
(Fig. 4A). Cells cultured in presence of follicular fluid had significantly increased levels of
mRNA for VASA compared to those cultured with BMP2, BMP4 or both BMP2 and BMP4 (Fig.
3A). Presence of BMP2, BMP4, both BMP2 and BMP4, and follicular fluid in culture medium
significantly (P<0.05) increased the levels of mRNA DAZL in OLCs, compared to those found in
control medium (Fig. 4B). Additionaly, lower levels of mRNA for DAZL were observed in OLCs
cultured with both BMP2 and BMP4 than in those cultured in presence of both BMP4 alone or
follicular fluid (Fig. 4B). Regarding the oocyte-specific genes, mRNA levels for SCP3 were
significantly increased in OLCs cultured in medium containing BMP2, BMP4, both BMP2 and
BMP4 or follicular fluid, when compared to control medium (Fig. 4C). Furthermore, compared to
control medium, BMP4, both BMP2 and BMP4 and follicular fluid significantly increased GDF9
mRNA expression in OLCs (Fig. 4D). Moreover, OLCs cultured with follicular fluid had higher
levels of mRNA for GDF9 than those cultured in presence of BMP2 alone (Fig. 4D). Both BMP2
and BMP4, as well as follicular fluid increased the expression of mRNA for ZPA, compared to
those cultured in control medium alone or in medium supplemented with BMP2 (Fig. 4E). In
addition, OLCs cultured in presence of follicular fluid had higher levels of mRNA for ZPA than
those cultured in control medium supplemented with BMP2, BMP4 or both (Fig. 4E).
Discussion
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This study shows for the first time that bovine ovarian stem cells have an intrinsic ability
to differentiate into OLCs. Isolated ovarian stem cells expressed mRNAs for OCT4, and SOX2,
which are transcription factors that act in maintainance of cellular pluripotency (Takahashi and
Yamanaka, 2006). Foregoing studies have shown that OCT4 and SOX2 regulate the expression of
NANOG (Rodda et al. 2005), while NANOG regulates REX1 expression (Shi et al. 2006). OCT4
and NANOG activate transcription of non encoding RNAs that promotes self-renewal of stem
cells and avoid differentiation (Mohamed et al. 2010).
Bovine ovarian stem cells also had shown alkaline phosphatase activity. This enzyme is a
key marker to identify pluripotent embryonic stem, since blastocysts present alkaline phosphatase
activity strictly in inner cell mass, but not in trophoblast cells (Štefková et al. 2015). Various
studies have also reported that alkaline phosphatase is an enzyme commonly used to identify
primordial germ cells (Clark et al. 2004; Parte et al. 2011).
As previously has been demonstrated, germ cell development requires a series of
multiple well-orchestrated steps, which involve up and down regulation of the expression of
specific genes (Takai et al. 2003). Earlier studies have also shown that PGC-like cells and OLCs
can be generated from embryonic, differentiated pluripotent and adult stem cells in vitro (Clark et
al. 2004; Panula et al. 2011). In mouse, pigs and human, the differentiation of ESCs or somatic
stem cells into OLCs was generally performed by culturing the cells with growth factors (Hübner
et al. 2003; Wei et al. 2008), estrogenic stimuli (Clark et al. 2004), conditioned medium from
testicular cell cultures (Lacham-Kaplan et al. 2006), follicular fluid, gonadotrophins (Hong et al.
2014) or with ovarian granulosa cells (Qing et al. 2007). The present study shows that BMP2 and
follicular fluid increase the expression of VASA in bovine OLCs formed in vitro. VASA is
expressed in post-migratory PGCs until the post-meiotic stage of oocytes (Castrillon et al. 2003).
Previous studies did show that follicular fluid promotes VASA expression and diffentiation of
157
skin-derived stem cells into OLCs (Dyce et al. 2011; Linher et al. 2009). Apart from VASA
expression, that of DAZL appears currently increased after culture of stem cells in presence of
BMP2 and follicular fluid, but also in presence of BMP4. DAZL is considered essential for PGC
development, as knockout mice lack a germ cell population (Kee et al. 2009). DAZL and VASA
have been used as markers for germ cell differentiation in various species (human: Kee et al.
2006; murine: Niikura et al. 2009).
Expression of SCP3 is frequently used as a meiotic marker, while GDF9 is expressed
specifically in oocytes. This study showed that their expression in in-vitro differentiated bovine
OLCs is promoted by BMPs and follicular fluid. SCP3-positive oocytes were previously
considered to be direct evidence of postnatal oogenesis through differentiation of germline stem
cells in mice (Pan et al. 2015). GDF9 is expressed in bovine oocytes (Vasconcelos et al. 2013)
and it has been used as marker to identify oocytes differentiated from stem cells cultured in vitro
(Linher et al. 2009). Both BMP2 and BMP4, as well as follicular fluid are currently found to
stimulate expression of ZPA in OLCs. The latter zona pellucida glycoprotein is only expressed in
oocytes (Lefièvre et al. 2004). Earlier studies reported ZP-like structures surrounding OLCs
differentiated in vitro (Hong et al. 2014). Singhal et al. (2015) recently showed that expression of
germ cell markers, like ZP1, ZP2, ZP3 and VASA, were stimulated by BMP4. In the present
study, BMP4 and BMP2 had no synergistic interaction to stimulate mRNA expression of genes
related to germ cells and oocytes. This is probably due to binding of these BMPs to the same
receptor, i.e., the BMP type II receptor.
In this study, it can be emphasized that follicular fluid stimulates the expression of
markers for both PGCs and oocytes, while most of these markers are also activated by BMPs.
Various studies have previously shown that BMP4 promotes oogonial stem cell differentiation in
human (Park et al. 2013), buffalo (Shah et al. 2015) and goat (Singhal et al. 2015). BMP2 also
158
plays a crucial role in the differentiation of primordial germ cells (Pera et al. 2004) and influence
the expression of oocyte-specific genes, like GDF9 in bovine follicles (Rossi et al. 2016).
Follicular fluid contains numerous bioactive factors secreted from granulosa cells, theca
cells, and oocytes and play important roles in regulating folliculogenesis and oogenesis (Jiang et
al. 2003; Webb et al. 2003). As oocytes secrete soluble paracrine growth factors that can regulate
granulosa cell development, and granulosa cells in turn regulate oocyte growth during follicle
formation (Van den Hurk and Zhao, 2005), it is apparent that the biochemical substances from
granulosa cells may play a key role in the initiation of germ cell formation and oocyte
development. The inducing role of follicular fluid in the formation of germ cells from stem cells
was demonstrated in swine species (Cheng et al. 2012; Linher et al. 2009). Dyce et al. (2011)
showed that follicular fluid induces the expression of markers of germ cells (DAZL and VASA),
meiosis (SCP3) and oocyte (GDF9, ZP1, ZP2, ZP3).
Conclusions
OLCs can be differentiated in vitro from ovarian stem cells, while BMPs and follicular
fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.
This study opens new possibilities for in-vitro production of bovine oocytes from ovarian stem
cells, which can have a positive impact on the production of embryos from genetically superior
animals or endangered species.
Acknowledgements
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This work was financially supported by CNPq (Grant N° 478198/2013-2), and the
authors thank the members of the Laboratory of Animal Reproduction of the Biotechnology
Nucleus of Sobral.
Conflict of interest
None of the authors have any conflict of interest to declare.
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List of tables and figures
Table 1. Primer pairs used in real-time PCR.
Figure 1. Fluorescence staining of ovarian stem cells before (A-B) and after 14 days culture in
minimum essential medium (α-MEM) (C-D) or supplemented with BMP2 (E-F), BMP4 (G-H),
both BMP2 and BMP4 (I-J) and follicular fluid (K-L). Scale bar = 100 µm. Green (calcein)
staining cells are viable and red (ethidium homodimer) staining cells are not.
Figure 2. Alkaline phosphatase activity in ovarian stem cells at day 0. Scale bar = 100 µm.
Figure 3. Cycle threshold (CT) after amplification of mRNA for pluripotency stem cell markers
(OCT4 and SOX2) by real time RT-PCR.
Figure 4. Levels of mRNA for specific germline cell [VASA (A), DAZL (B)] and oocyte [SCP3
(C), GDF9 (D) and ZPA (E)] markers in ovarian stem cells cultured for 14 days in MEM
supplemented with BMP2, BMP4, BMP2 and BMP4 and follicular fluid. Significant differences
at P<0.05.
166
Table 1. Primer pairs used in real-time PCR.
Target
gene
Primer sequence (5´ 3´)
Sense (s),
anti-sense
(As)
GenBank number
GAPDH
CACCCTCAAGATTGTCAGCA
GGTCATAAGTCCCTCCACGA
S
As
NM_001034034.2
SOX2
TGGATCGGCCAGAAGAGGAG
CAGGCGAAGAATAATTTGGGGG
S
As
NM_001105463.2
OCT-4
GAGAAAGACGTGGTCCGAGTG
GACCCAGCAGCCTCAAAATC
S
As
NM_174580.2
VASA
TGGTCCTGGCTTCAGTGGTA
TCTTGCCGGGGTAATTCTTTCT
S
As
NM_001007819.1
DAZL
TACCCGCCTCTGACTCTCTC
GTGTTCACTCAGAGGGGCTC
S
As
EF501823.2
ZPA
TCGTGGATGGCTGTGAATACAA
CAAGTGAGAGAATGCCGGGT
S
As
NM_173973.2
GDF-9
ACAACACTGTTCGGCTCTTCACCC
CCACAACAGTAACACGATCCAGGTT
S
As
GI:51702523
SCP3
GTTGGCAAAACCATCCGTGG
GGGGTCTTCTCTTCAATGGCA
S
As
NM_001040588.2
s: sense; as: anti-sense.
167
Figure 1. Fluorescence staining of ovarian stem cells before (A-B) and after 14 days culture in
minimum essential medium (α-MEM) (C-D) or supplemented with BMP2 (E-F), BMP4 (G-H),
both BMP2 and BMP4 (I-J) and follicular fluid (K-L). Scale bar = 100 µm. Green (calcein)
staining cells are viable and red (ethidium homodimer) staining cells are not.
168
Figure 2. Alkaline phosphatase activity in ovarian stem cells at day 0. Scale bar = 100 µm.
Figure 3. Cycle threshold (CT) after amplification of mRNA for pluripotency stem cell markers
(OCT4 and SOX2) by real time qRT-PCR.
169
Figure 4. Levels of mRNA for specific germline cell [VASA (A), DAZL (B)] and oocyte [SCP3 (C), GDF9 (D) and ZPA (E)] markers
in ovarian stem cells cultured for 14 days in MEM supplemented with BMP2, BMP4, BMP2 and BMP4 and follicular fluid.
Significant differences at P<0.05.
170
10 CONCLUSÕES
O tratamento de fibroblastos com 2.0 μM de 5-aza-citidina por 72 h induz mudanças
na morfologia e na taxa de proliferação celular, e induz a expressão de RNAs mensageiros para
fatores de pluripotência (OCT-4, SOX2, NANOG e REX1).
O cultivo de fibroblastos, previamente tratados com 5-aza-citidina, em meio de
diferenciação suplementado com BMP-2, BMP-4 ou fluido folicular, durante 7 ou 14 dias,
promove alterações na morfologia celular e induz a expressão de RNAs mensageiros para para
marcadores de células germinativas (VASA, DAZL e C-KIT) e de oócitos (SCP3, GDF-9 e ZPA).
Células-tronco da linhagem germinativas, que expressam marcadores de
pluripotência (OCT-4 e SOX2) e fosfatase alcalina, podem ser isoladas eficientemente do epitélio
ovariano de bovinos, e podem ser utilizadas para a diferenciação em estruturas semelhantes a
oócitos.
O cultivo de células-tronco da linhagem germinativas em meio de diferenciação
suplementado com BMP-2, BMP-4 ou fluido folicular promove modificações na morfologia
celular e estimula a expressão de marcadores par células germinativas primordiais (VASA e
DAZL) e de oócitos (SCP3, GDF-9 e ZPA).
171
11 PERSPECTIVAS
Os resultados obtidos no presente trabalho poderão ser utilizados para o
desenvolvimento de novas tecnologias que podem revolucionar a reprodução assistida, tanto em
humanos, como em animais de alto padrão zootécnico. Nos últimos anos, a diferenciação de
células somáticas está associada com grandes perspectivas para o desenvolvimento da medicina
regenerativa. Adicionalmente, este trabalho contribuirá para a formação de gametas a partir de
células somáticas, abrindo uma nova perspectiva de recuperação de espécies em via de extinção.
A reprogramação celular e a presença de células-tronco mitoticamente ativas no
epitélio da superfície ovariana podem viabilizar a formação de uma quantidade de oócitos
suficientes para iniciar um novo capítulo na biotecnologia e na medicina reprodutiva. Com base
na disponibilidade dessas novas tecnologias no campo da medicina reprodutiva, os resultados
deste trabalho poderão contribuir para a solução dos problemas relacionados com a infertilidade
no futuro.
172
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