CONTRIBUTIONS OF ART TECHNOLOGY IN NEW THERAPEUTIC APPROACHES S.I.S.ME.R. Reproductive Medicine Unit...

CONTRIBUTIONS OF ART TECHNOLOGY IN NEW THERAPEUTIC APPROACHES

S.I.S.ME.R. Reproductive Medicine Unit - Via Mazzini, 12 - 40138 Bologna

[email protected]

L. Gianaroli, MC Magli, AP Ferraretti

Since the birth of the first baby conceived using IVF techniques in 1978 over three million babies have been born worldwide as the result of ART.

The initial goal was treatment of infertility. However, assisted reproduction techniques are no longer used only to help infertile couples, but they have a great significance in the field of therapeutic medicine.

S.I.S.ME.R.VISION 2000

- Gamete and tissue cryo-banking- PGD – HLA matching


Therapeutic medicine

- Stem cells

- Gamete and tissue cryo-banking



S.I.S.ME.R.

VISION 2000

OOCYTE CRYO-BANKINGFERTILITY RESTAURATION

Premature ovarian failure - POF

defined as menopause before the age of 40 years or

hypergonadotropic hypogonadism

defined as menopause consequent to chemotherapy

Depending on the extent of damage to the ovaries

Acute Ovarian Failure (AOF) = loss of ovarian function during or shortly after the end of chemotherapy

Premature menopause = loss of ovarian function that occurs years after the end of chemotherapy (before age 40 yr)

Mattle et al, 2005

The chemotherapeutic destruction of an already low follicular reserve, reduces the number of follicles below a certain ‘threshold’ number required to sustain ovarian function, resulting in menopause.


OOCYTE CRYO-BANKINGRISK OF POF ACCORDING TO AGE AFTER CHEMOTHERAPY

Sonmezer, M. et al. Oncologist 2006;11:422-434

Alkylating agents are extremely gonadotoxic because they are not cell cycle-specific and can damage resting primordial follicles.

Cycle-specific agents such as MTX and 5-FU do not have any effect on ovarian reserve.


OOCYTE CRYO-BANKINGRISK OF POF ACCORDING TO CHEMOTHERAPEUTIC AGENTS

Dose of Cyclophosphamide (mg/kg)

No. of primordial

follicles

Meirow et al, 1999

Follicular damage from alkylating agents is dose-dependent.A dose of chemotherapy strong enough to destroy 50% of the ovarian primordial reserve does not affect the reproductive performance in a murine model.


OOCYTE CRYO-BANKINGDOSE-EFFECT DAMAGE ON OVARIAN FUNCTION

• Ovary(s) removed laparoscopically, divided into small strips, frozen and stored

• Females, before and after puberty

• Outpatient surgical procedure

• Experimental, one possible live birth to date

• Re-implantation can restore hormone function


OOCYTE CRYO-BANKINGOVARIAN TISSUE FREEZING

S.I.S.ME.R.

VISION 2000

OOCYTE CRYO-BANKINGOVARIAN TISSUE FREEZING

• A transient restoration of spontaneous ovarian follicular development and estrogen production, but not ovulation, was observed after autotransplantation of frozen/thawed ovarian tissue that had been harvested and banked before chemotherapy and radiation therapy for lymphoma (Redford et al., 2001).

• Embryo development was obtained after autotransplantation of frozen/thawed ovarian tissue (Oktay and Sommezer, 2004).


OOCYTE CRYO-BANKINGOVARIAN TISSUE FREEZING – CLINICAL RESULTS

• Livebirth resulted after orthotopic transplantation of cryopreserved ovarian tissue (Donnez, 2004).

• A pregnancy was obtained after transplantation of cryopreserved ovarian tissue and IVF in a patient with ovarian failure after chemotherapy (Meirow, 2005).

• Actually, follicular growth, hormonal production and some pregnancies (5 as reviewed by Fabbri et al., 2008), spontaneous or after IVF treatments, have only been achieved after the autotransplantation technique.


OOCYTE CRYO-BANKINGOVARIAN TISSUE FREEZING – CLINICAL RESULTS

Oocyte cryopreservation could be a clinical tool for:

• Women at risk of losing ovarian function

• Women desiring fertility preservation (e.g. delayed maternity)

• Eliminating ethical concerns of embryo cryopreservation

• Solving the dilemma of abandoned frozen embryos in the IVF laboratory


OOCYTE CRYO-BANKINGOOCYTE FREEZING – CLINICAL APPLICATIONS

TOTAL

March 2004 -

December 2007

No. cycles 434

No. Transferred cycles (%)

358 (82.5)

No. Clinical pregnancies (%)

55 (15.4)

Implantation rate (%)

65/688 (9.5)

Abortions (%) 12 (22)

LBR (%) 43 (9.9)

OHSS

March 2004 -December 2007

168

149 (88.7)

32 (21.5)

38/320 (12)

8 (25)

24 (14.3)S.I.S.ME.R.

VISION 2000

OOCYTE CRYO-BANKINGOOCYTE FREEZING – SISMER EXPERIENCE

S.I.S.ME.R.

VISION 2000

OOCYTE CRYO-BANKINGOOCYTE FREEZING – VITRIFICATION

SISMER EXPERIENCE (2004-2008)

0

5

10

15

20

25

30

35

Clinical pregnancy rate

Implantation rate (FHB)

Spontaneous abortion rate

%




GENETIC INVESTIGATIONPGD

specific disease mutation (including X-linked diseases)chromosome aberration

Diagnosis implies looking for:

implies looking for a genetic defect in all members of a population at risk being the risk dependent on the incidence and severity of the defect

Screening


MAIN INDICATIONSESHRE PGD consortium data collection 1997-2005

PGD


High risk of genetic disease

Single gene disordersChromosomal abnormalitiesLate onset diseases

(i.e. Huntington Chorea)

HLA typing

Predisposition to late onset diseases (i.e. cancer)

Sex selection for family balancing

14.419 cycles

Aneuploidy

Goossens et al. (2008) ESHRE PGD Consortium data collection VIII: cycles from January to December 2005 with pregnancy follow-up to October 2006. Hum Reprod 23,2629-2645.

Normally fertile couples whose children might inherit

- a severe disease- a predisposition to a pathology

PGDWHY TO GO FOR IT?

Normally fertile couples who wish to save a sibling’s life (HLA-typing)


UNESCO’s report on preimplantation genetic diagnosis (PGD) and Germ-Line Intervention, 2003.

“IVF aims at having a child, PGD aims at having a healthy child and PGD/HLA testing aims at having a healthy and helpful child”.


PGDWHY TO GO FOR IT?

PGD FOR HLA - MATCHING

• Healthy embryos are selected for transfer

avoids the need for termination of an ongoing pregnancy in cases of an affected fetus

• HLA – matching with an affected childS.I.S.ME.R.

VISION 2000

PREIMPLANTATION HLA MATCHING

One of the most recent applications in reproductive medicine.

Viable option for couples with children needing haematopoietic stem cell (HSC) transplantation.

Selection of embryos both free of disease and HLA matched with the existing child.

PGD is used not only to avoid the birth of affected children, but also to conceive healthy children who may also be potential HLA-identical donors of HSC

At delivery of the newborn, cord blood HSC can be used to treat the affected sibling.


Allogeneic HSC transplantation

Only curative option for severe cases of haematopoietic disorders.

A critical factor associated with a favourable outcome is the use of HLA identical donors

HSC from HLA identical siblings provide the higher success rate (~90%)

Reduced incidence of graft rejection and other serious complications associated with transplantation.

Transplantation using non HLA-identical donors is associated with higher morbidity and poorer survival.

Limited availability of HLA-matched unrelated donor, identified from national or international registers.


Indication for preimplantation HLA matching

Severe cases of haematopoietic disorders requiring a HLA compatible HSC donor.

Thalassemia Fanconi anaemia Wiskott-Aldrich syndrome Diamond-Blackfand Anemia X-linked Hyper IgM Syndrome X-linked adrenoleukodystrophy X-linked Hypohidrotic Ectodermal Dysplasia with immune deficiency Aplastic anemia

For diseases such as Acute Lymphoid Leukemia, in which HLA matching becomes the primary indication.


Patient’s acceptance policy for HLA program

HSC transplantation is the best treatment option for the affected child (advise transplantation hematologist is required);

HSC transplantation is not urgent;

The family cares unconditionally about all the children.


Simplified map of the HLA region

Class IIIMHC Class II

DP

LMP/TAPDM

DQ

DR

1B C A

MHC Class I

CLASS ICLASS I: 3 types HLA-AHLA-A, HLA-BHLA-B, HLA-C.HLA-C.

CLASS IICLASS II: 3 types HLA-DP,HLA-DP, HLA-DQ,HLA-DQ, HLA-DRHLA-DR.

4 53

3 extra DR genes in some individuals can allow 3 extra HLA-DR molecules

(also MHC class II genes for HLA-DM, and TAP, and LMP)


Polymorphism in the MHCPolymorphism in the MHC

In the human population, over 1,200 HLA alleles have been identified

2

317

19

89

2045

DR DP DQ

Class II

381

185

91

A B C

No

of

po

lym

orp

his

ms

Class I657 alleles 492 alleles

The HLA ComplexHLA Complex (Human Leukocyte AntigenHuman Leukocyte Antigen) - located on chromosome 6 - represents one of the most polymorphic regions of human genome. Comparative

DNA sequence analysis of HLA complex has shown the presence of a high number of alleles in this region.


Inheritance of MHC haplotypes Inheritance of MHC haplotypes

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

X

ParentsDP-1,2DQ-3,4DR-5,6B-7,8C-9,10A-11,12

DP-9,8DQ-7,6DR-5,4B-3,2C-1,8A-9,10

DP-1,8DQ-3,6DR-5,4B-7,2C-9,8A-11,10

DP-1,9DQ-3,7DR-5,5B-7,3C-9,1A-11,9

DP-2,8DQ-4,6DR-6,4B-8,2C-10,8A-12,10

DP-2,9DQ-4,7DR-6,5B-8,3C-10,10A-12,9

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

Children

all persons: have 2 haplotypes2 haplotypes (1 maternal, 1 paternal in origin)

Genes in the MHC are tightly LINKED and usually inherited in a group. The combination of alleles on a chromosome is an MHC HAPLOTYPE


HLA - AHLA - A HLA - BHLA - B HLA - CHLA - CIVS-I-110 IVS-I-110 G/AG/A

FatherFather


MotherMother


ChildChild

Minisequencing-based Preimplantation HLA Minisequencing-based Preimplantation HLA matching on single blastomeresmatching on single blastomeres



ChildChild

Blast. 8Blast. 8


HLAHLA

Non Non IdenticalIdentical

NormalNormal



HLAHLA

IdenticalIdentical

Blast. 3Blast. 3

Blast. 4Blast. 4HomozygoteHomozygote

HeterozygotHeterozygotee

Minisequencing-based Preimplantation HLA Minisequencing-based Preimplantation HLA matching on single blastomeresmatching on single blastomeres

Overall Results and Outcome of Preimplantation Diagnosis for Single Gene Disorders & Preimplantation HLA testing

RGI Experience

Testing Patient/Cycle

# of Transfers

# Embryos Transferred

Pregnancy /Birth

HLA97 / 242 149 228 46 / 37

31 %

Single Gene Disorders 735 / 1220 1061 2168 423 / 452

39.8%

TOTAL 832 / 1462 1210 2396 469 / 48938.7%

04/14/2008Reproductive Genetics Institute [email protected]




- Stem cells

Stem Cells

- clonogenic, self-renewing

progenitor cells that generate one

or more specialised cell types

A. Embryonic stem cells and germline stem cells

- pluripotential and immortal

B. Organ or tissue specific stem cells

- multipotential

Stem Cells


CNS PNS Hema. Liver Skin Mesen. etc

Primitive germ cell(germline stem cell)

Embryonic stem cell (ES cell)

Embryo

Embryo(gonads)

Pluripotent stem cell

Culture

BlastocystTotipotent stem cell

Pluripotent somatic stem cell

Multipotent stem cells

Differentiation of Stem Cells


SOURCES OF STEM CELLS

ADULT TISSUE

UMBILICAL CORD

INNER CELL MASS

NERVOUS, HAEMATOPOIETIC,EPITHELIAL, INTESTINAL

EMBRYONIC STEM CELLS

(ES)

- SPECIFIC CELL TYPE- TRANSDIFFERENTIATION

HIGH PROLIFERATION

VERY HIGH DIFERENTIATON

POTENTIAL


STEM CELLS

CRUCIAL FCTORS

NUMBER OF CELLS

PROLIFERATION POTENTIAL

ES

UMBILICAL CORD TISSUES

HIGH

MEDIUM

LOW

HIGH

LOW

THERAPEUTIC APPLICATIONS

MEDIUM


Applications for Embryonic Stem Cells and their Derivatives

• ES cells for research and discovery

• Progenitor cells for drug screening

• Progenitor cells for toxicology

• Gene products (proteins), growth and

differentiating factors, cell surface

molecules for pharmaceutical use in

regenerative medicine


Embryonic Stem Cell Derivatives Applications for Cell and Tissue Therapy

• Vehicles for the delivery of gene therapies

- correcting genetic disease

- new immunization strategies

- targeting cancers


Making ES Cells and their Derivatives Compatible for Transplantation

• Make ES cells from all the necessary HLA subtypes –

subject to advice from transplant divisions about the

number necessary

• Remove the cell surface expression of the major

histocompatibility antigens from ES cells and their

derivatives

• Make customized ES cells for each and every patient –

therapeutic cloningS.I.S.ME.R.

VISION 2000

Embryonic Stem Cells Formed by Nuclear Transfer of Adult Cells (Therapeutic Cloning)

• ES cells contribute to all tissues in an apparently normal way

- in chimeras

- in vitro

- in teratomas

• No indication of defects seen in cloned foetuses/offspring

- helper cell effects

- transdifferentiation phenomenon

• May be an extremely efficient way to produce transplantation

compatible cells and tissues for patientsS.I.S.ME.R.

VISION 2000

Cells eg skin

Problems:

Inefficient - may need large numbers (50 to several hundreds) of eggs

Technically demanding - need to be available in many or all hospitals

Source of eggs: self, mother, relative, egg bank

Therapeutic Cloning

Compatible transplants

ES cells

Nuclear transfer


Patient Specific Stem Cells

Nucleartransfer

Blastocyst

Unaffectedneurones

How Why

Drug Screening

Diseasephenotype

Patients with diseasesof undetermined cause, eg. Glioblastoma

Alzheimer’s DiseaseParkinson’s DiseaseMotor Neurone Disease


Inject stem cells to correct Rag2 disease

Cultured skin cells

Differentiate into functional hematopoietic lineage

Rag2 Mouse (severe combined immuno

deficiency)

Nuclear transfer

ES Cells

DNA repair

Insert HoxB4 Homeobox gene

Skin cell

Mouse in a Bubble

Genetic Correction

Cell fusion

or

Cure of Genetic Diseases Using Stem Cells

S.I.S.ME.R.VISION 2000(Rideout et al. Cell 2002)

Master bank of HLA typed ES cells

Allogenic Cell Therapy

Neural stem cells

Propogate cells to master ES cell bank

MHC gene knockouts

Induce tolerance

Immune suppression

Non-transferable embryos in frozen storage

Select null HLA expression

Direct

Neuronal precursors TH+ dopaminergic neurone

> 200,000 HLA types

Brain transplantation

differentiation


Induction of tolerance across MHC histocompatibility barriers by pretransfer of ES cell- like cells/haematopoietic cells.

An epithelial progenitor stem cells population (positive for glycoprotein MTS24) has been identified that is competent and sufficient to fully reconstitute the thymus.

Transplantation of Cells Derived from Human Embryonic Stem Cells

(Gill et al Nature Immunology June 17 2002). S.I.S.ME.R.VISION 2000

Delivering stem cell therapy:Avoiding immune rejectionby reconstructing the immune system

The thymus is the site where immune systemcells learn the difference between self and non-self Richard Boyd and colleagues have shownthat rare progenitor cells can rebuild a thymus


LONG-TERM ALLOGENEIC GRAFT ACCEPTANCE USING EMBRYONIC STEM CELL-LIKE CELLS


(Fandrich et al. Nature Medicine 8: 171 2002)

IVFFemale infertility

Idiopathic infertility

ICSI

Manyeggs

Fertility drugs given towomen to make many

mature eggs

2-cell

4-cell

8-cell

Embryos

Biopsy forgenetic diagnosis

few eggs

Immature eggs recoveredin natural cycle

(no fertility drugs)

Use in regenerative medicine(eg. diabetes, Parkinson’s Disease,stroke, respiratory disease, cardiacdamage, quadriplegia)

Transfer to patient

Trophectoderm biopsy fordevelopmental competence

Transfer to patient

Blastocyst

Inner cell mass

Reproductive Medicine Therapeutic Medicine S.I.S.ME.R.VISION 2000

EU hESC line Registry (hESCReg)

• Primary objective: provide information about all human ESC lines derived and used in Europe which are available to the scientific community.

• Specific Support Action funded by VI FP European Commission (1.048.000 €, 2007 - 2010)

Coordinated by: Joeri Borstlap (BCRT - Technical Coord.)

Anna Veiga (CRMB - Scientific Coord.)


S.I.S.ME.R.

VISION 2000

RGI’s Repository of Human Embryonic Stem CellsNORMAL (258 lines) AUTOSOMAL DOMINANT CONDITIONS (34)

CHROMOSOMAL ABNORMALITIES (14 lines) BRCA2 (2 lines – of which 1 also has MEN1)

46X +mar 47,XX,+12 FSHD (7 lines)

46,XX,der(4)t(4;13) 47,XY,+12 Huntington Disease (7 lines)

46,XX t(14;17) 47,XX,+13 Marfan Syndrome

46,XX iso (17q) 47,XX,+14 Myotonic Dystrophy (2 lines)

47,XY+der(21)t(2,21) 47,XX,+21 Neurofibromatosis, type 1 (7 lines)

69,XXX 47,XXX Popliteal Pterygium Syndrome

47,XXY (2 lines) Torsion Dystonia, DYT1 (2 lines)

X-LINKED CONDITIONS

Tuberous Sclerosis, TSC1 (2 lines)

Treacher Collins-Franschetti syndrome (3 lines)

AUTOSOMAL RECESSIVE CONDITIONS (24)

Adrenoleukodystrophy Alpha Thalassemia (1 lines)

Becker Muscular Dystrophy Beta Thalassemia (9 lines)

Duchenne Muscular Dystrophy (4 lines) Cystic Fibrosis (8 lines)

Emery-Driefuss Muscular Dystrophy (2 lines) Fanconi Anemia A

Fragile X syndrome (2 lines) Sandhoff Disease (3 lines)

Ocular Albinism, X-linked (2 lines) Spinal Muscular Atrophy (2 lines)

Reproductive Genetics InstituteAs of 10/2008





- Stem cells

CONCLUSIONS:

CB Stem CB Stem CellsCells

CB Stem CB Stem CellsCells

Direct Direct treatmenttreatment

In vitroIn vitro ExpansionExpansion

Low Low quantityquantity

High High quantityquantity

Complete engraftmentComplete engraftmentLocatelli, unpubblishedLocatelli, unpubblished


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CONTRIBUTIONS OF ART TECHNOLOGY IN NEW THERAPEUTIC APPROACHES S.I.S.ME.R. Reproductive Medicine Unit...

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