SCIENTIFIC COMMITTEE - uwm.edu.pl · SCIENTIFIC COMMITTEE ... (IP-2016-06-9451), awarded by...

SCIENTIFIC COMMITTEE

Prof. dr hab. n. med. Krystyna Domańska-Janik

President of Cell Therapy Team of the Central Nervous System Diseases, Polish Academy of Sciences

Prof. dr hab. n. med. Wojciech Maksymowicz

Department of Neurosurgery, School of Medicine, University of Warmia and Mazury in Olsztyn

dr hab. n. med. Anna Sarnowska

Secretary of Cell Therapy Team of the Central Nervous System Diseases, Polish Academy of Sciences

Prof. dr hab. n. med. Leonora Bużańska

Cell Therapy Team of the Central Nervous System Diseases, Polish Academy of Sciences

dr n. med. Katarzyna Jezierska-Woźniak

Department of Neurosurgery, School of Medicine, University of Warmia and Mazury in Olsztyn

ORGANISING COMMITTEE

Katarzyna Jezierska-Woźniak

Wioleta Czelejewska

Sylwia Dargiewicz

Emilia Sinderewicz

Stem Cells: therapeutic outlook for central nervous system disorders Olsztyn, 7th June 2019

Dear Colleagues,

it is a great pleasure to welcome you in Olsztyn, Poland, on June 7th 2019 at the 6th

International Conference "Stem cells: therapeutic outlook for central nervous system

disorders". The conference aims to bring together academic scientists, researchers and

physicians to discuss the latest achievements in the field of stem cells application in the

progressive experimental and clinical therapies of neurodegenerative diseases. We believe

that planned lectures, presented by excellent speakers from Polish and European leading

research and medical centers, give us an opportunity to exchange the scientific experience,

new ideas and technologies.

Sincerely yours,

Scientific Committee


PROGRAMME 08:30 Registration

09:30 Welcome

Wojciech Maksymowicz

Session I

Chairman: Pavla Jendelova, Wojciech Maksymowicz

09:45-10:15 Dinko Mitrecic (University of Zagreb, Croatia)

Application of stem cells in neurovascular and neurodegenerative brain

diseases

10:15-10:45 Edgaras Stankevicius (Lithuanian University of Health Sciences, Kaunas, Lithuania)

Human induced pluripotent stem cell derived astrocyte based models in

neuropsychiatric disorders

10:45-11:15 Anna Sarnowska (Mossakowski Medical Research Centre PAS, Warsaw, Poland)

Methods for optimizing the derivation of MSC for clinical application

11:15-11:30 Konrad Żurawski (Macopharma)

Viral inactivation of human platelet lysate by gamma irradiation preserves its optimal

efficiency in the expansion of human bone marrow mesenchymal stromal cells

11:30-12:00 Coffee break

Session II

Chairman: Krystyna Domańska-Janik, Marcin Mycko

12:00-12:30 Pavla Jendelova (Czech Academy of Sciences, Praque, Czech Republic)

The mechanisms underlying the beneficial effect of stem cells in the animal

model of ALS

12:30-13:00 Sebastian Lewandowski (Karolinska Institutet, Stockholm, Sweden)

Perivascular fibroblasts contribute to ALS neurodegeneration


13:00- 13:30 Hans-Peter Hartung (Heinrich-Heine-Universität, Düsseldorf, Germany)

Multiple sclerosis: From immunomodulation to repair

13:30-13:45 Michał Konieczny (Merck)

The role of Imaging Flow Cytometry in modern cellular analysis

13:45-14:45 Lunch break

Session III

Chairman: Leonora Bużańska, Sebastian Lewandowski

14:45-15:15 Bogusław Machaliński (Pomeranian Medical University, Szczecin, Poland)

Novel evidence that neutrophines may play a significant role in the

neuroprotective stem cell-based therapies

15:15-15:45 Bogna Badyra (Jagiellonian University, Cracow, Poland)

Targeting key signaling factors as away to control microglial activation and

induction of neuroinflammation

15:45-16:15 Krzysztof Selmaj (University of Warmia and Mazury, Olsztyn, Poland)

The role of microRNA in mesenchymal stem cell immunoregulatory function

and neurogenesis in experimental autoimmune encephalomyelitis

16:15-16:30 Joanna Zimniak (VWR)

VWR as a provider of solutions for all Life Science disciplines

16:30-16.45 Closing Remarks

Chemical reagents voucher drawing


Application of stem cells in neurovascular and neurodegenerative

brain diseases Dinko Mitrecic

Laboratory for Stem Cells, Croatian Institute for Brain Research, School of Medicine University

of Zagreb

Faced with a burden of brain diseases, modern medicine searches for new therapeutic strategies.

Regeneration of nervous tissue based on benefits linked to stem cells has attracted a significant

attention. Among numerous brain diseases, in this presentation we focus on two of them: while

stroke stands as the most significant and most common cause of a life-long disability, amyotrophic

lateral sclerosis (ALS) is a paradigm of a fatal progressive neurodegenerative disease defined by very

fast deterioration of motoric neurons and inevitable death of the patient within few years after

the first appearance of symptoms.

Here we present an overview of current state of the art in experimental application of stem cells

in stroke and amyotrophic lateral sclerosis. While for stroke our group uses a mouse middle carotid

artery occlusion model, ALS is successfully modeled by animal mutants of superoxide dismutase 1

(SOD1). Here we describe our experience with both direct intraparenchymal and intravascular

transplantation of mouse neural stem cells and our observation about rates of survival, migration

and differentiation of the transplanted cells. While stereotaxic intraparenchymal injection allows

to obtain a large concentration of cells in the targeted region, they survival is linked to formation

of a local niche. Intravascular transplantation is possible because of opened blood brain barrier

and cells which accumulate in the region affected by disease can survive for very long periods.

Based on our and similar experiences from preclinical studies, many clinical trials with stem cells have

been launched. Although characterized by rather heterogeneous conclusions in patients, possibly

linked to cell source, many measurable benefits have been observed. It is obvious that parallel

and coordinated work on both preclinical models and patient trials will help to gradually increase

therapeutic effects of stem cells transplanted to the brain tissue affected by these fatal diseases.

Funding: The work has been supported by project Orastem (IP-2016-06-9451), awarded by Croatian National

Foundation.


Human induced pluripotent stem cell derived astrocyte based models

in neuropsychiatric disorders Silvijus Abramavičius, Laura Martinkutė, Edgaras Stankevičius

Institute of Physiology and Pharmacology, Lithuanian University of Health Sciences, Kaunas, Lithuania

Astrocytes have a plethora of actions and play a major role in synapse maturation and function.

Pathological astrocyte changes (astrocytopathology) may result in various neuropsychiatric disorders

like schizophrenia, major depressive disorder and autism spectrum disorders. Even more interesting

is that human induced pluripotent stem cells (hiPSC) may be of vital importance in merging

the astrocyte pathophysiology, drug development and clinical practice. In this review we discuss basic

astrocyte physiology, pathological physiology of neuropsychiatric disorders with emphasis

on astrocytopathology, discuss the implications of hiPSC technology and recent developments

in the astrocyte centered modelling of neuropsychiatric disorders.


Methods for optimizing the derivation of MSC for clinical application Anna Sarnowska

Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

In recent years, we have seen the rapid development of stem-cell based therapy, mostly based

on mesenchymal stromal/stem cells (MSC) application. In few disorders their restorative potential and

ability to integration with injured tissue has been proved. However, for most indications, the use

of MSC remains within the framework of experimental therapies. To move regenerative medicine into

the realms of mainstream medicine, it should be clearly indicated what has been achieved so far

in regenerative medicine, explain to patients the possible therapeutic effect of MSC and treat most

stem cell applications as ongoing experiment.

Four decades of research on somatic cells regenerative potential ended with very ambiguous results.

It has been shown how highly heterogeneous population are stromal cells, and even minor changes

in isolation or culture protocols can significantly affect the properties of isolated cells. The method

of cell isolation may substantially affect cells ability to differentiate toward neural direction

and presumably the neuroprotective properties.

Our results show significant differences in phenotype, proliferation rate, CFU-F formation and the

number of senescent cells between MSC populations obtained with two methods (mechanical

vs enzymatic) of isolation. Undifferentiated, SRTF expressing MSC, capable to time-locked

proliferation, migration and ultimately to neural differentiation were the most effective in various

therapeutic transplantation models. The strongest ability for neuroprotection was provided by freshly

isolated cells and the first cohort of migrating MSC cells (passage O). To keep the cells

in undifferentiated state, culture conditions e.g. 5% oxygen concentration were crucial.

Due to the appropriate methods of cell isolation and cultivation, we could obtain preMSC

subpopulation, which is the most closely related to neural crest derived stem cells and may possess

so needed restorative properties.


The mechanisms underlying the beneficial effect of stem cells in the

animal model of ALS Pavla Jendelová

Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic

A promising therapeutic strategy for ALS treatment is stem cell therapy. Human mesenchymal stem

cells (hMSC) could be an ideal option due to their immunomodulatory and anti-inflammatory

properties and the excretion of trophic factors and exosomes. Contrary, neural progenitors derived

from iPS cells (iPS-NPs) might replace dying motoneurons (MNs). However, the mechanisms

responsible for the beneficial effect are not fully understood. We therefore tested intrathecal repeated

application of hMSC into spinal canal and/or muscle injection and compared it with injection

of conditioned medium. We studied the effect of the applied therapies on apoptosis, necroptosis

and autophagy. We also intraspinally injected iPS-NPs to follow their fate in presymptomatic

and symptomatic ALS rats. All the animals were behaviorally tested (Grip strength test, BBB, rotarod),

and the tissue was analyzed immunohistochemically, and by western blot. Symptomatic SOD1 rats

treated with hMSC (into the spinal canal or in combination with intramuscular injection) had

a significantly increased lifespan, improved motor activity and reduced number of TUNEL positive

cells. Moreover, a combined hMSC delivery increased MN survival maintained neuromuscular junctions

and substantially reduced the levels of proteins involved in necroptosis (Rip1, MLKL,cl-casp8),

apoptosis (cl-casp 9) and autophagy (beclin 1). Furthermore, astrogliosis and elevated levels

of Connexin 43 were decreased after combined hMSC treatment. NP-iPS transplantation significantly

preserved MNs and restored their perineuronal nets, which slowed disease progression and extended

survival of all cell-treated animals. In the host spinal cord, transplanted cells adopted a glial

phenotype or remained as progenitors, many in contact with motoneurons. However, they did not

differentiate to MN phenotype. Our findings confirm that stem cell and progenitor therapy can modify

the progression of neurodegenerative pathology, mainly due to their multiple paracrine effect.

Supported by the Center of Reconstruction Neuroscience NEURORECON

(CZ.02.1.01/0.0/0.0/15_003/0000419) and LO1309.


Perivascular fibroblasts contribute to ALS neurodegeneration Sebastian Lewandowski

SciLifeLab, Royal Institute of Technology, Karolinska Institute, Solna, Sweden

Neurodegenerative diseases and dementia represent the biggest source of disability in the elderly

and despite their high incidence they still remain difficult to diagnose or predict. Amyotrophic lateral

sclerosis (ALS) patients show signs of decreased cerebral blood flow and glucose uptake, which

indicates poor survival prognosis. Although these clinical symptoms correlate with increased disease

severity, their cellular mechanisms remain largely unknown.

We examined the underlying mechanisms of vascular dysfunction in mouse models and ALS patients

using cell type specific transcriptomes and patient plasma proteomics. We found that genes specific

for perivascular fibroblast cells become active before onset of clinical symptoms

in the SOD1G93A mouse model with similar cell enrichment patterns present in sporadic ALS patients.

We show that perivascular fibroblasts are located between vascular basement membrane layers

and express specific proteins to the extracellular matrix. Using plasma proteomics we found that

increase in perivascular fibroblast specific proteins associates disease progression dynamics

and survival in ALS patients.

Our work brings evidence that blood vessel function contributes to ALS neurodegeneration.

We suggest that perivascular fibroblasts contribute to the mechanisms of vascular injury in ALS and

can serve as prognostic biomarkers in clinical setting. Cerebral blood flow is decreased in a broader

range on neurodegenerative conditions and vascular injury proteins may ultimately become candidate

biomarkers for other neurodegenerative diseases including Alzheimers dementia.


Multiple sclerosis: From immunomodulation to repair Hans-Peter Hartung

Department of Neurology and Center of Neurology and Neuropsychiatry, Heinrich-Heine-Universität,

Düsseldorf, Germany

The past 2 decades have witnessed enormous progress in the treatment of multiple sclerosis. This

commonest immune-mediated inflammatory disease of the central nervous system is the most

frequent cause of lasting neurological disability in young adults. It initially runs a relapsing course

which eventually transitions to a progressive stage. MS is considered to be autoimmune in origin with

genetic and environmental factors synergizing to produce the characteristic pathology: multifocal

inflammation, demyelination and axonal damage. The relapsing phase most likely is driven

by aberrant immune responses to myelin antigens whereas with advancing disease neuronal

degeneration takes precedence. A number of immunomodulatory agents are available that show

modest to high efficacy: platform injectables, oral medications and monoclonal antibodies. Only very

recently a new drug was approved in the US to treat active secondary progressive MS. Clearly

the rare unmet needs – both in terms of achieving disease control during the relapsing stage

and slowing progression. To address these needs other than immunomodulatory strategies those that

aim at restoring function and structure are warranted. First successes have been achieved with drugs

that foster remyelenation. Other approaches use stem cells of different sources.

I will discuss these strategies and perspectives for improving the outcome of MS.


Novel evidence that neutrophines may play a significant role in the

neuroprotective stem cell-based therapies Bogusław Machaliński

Department of General Pathology, Pomeranian Medical University in Szczecin, Szczecin, Poland

Several studies have reported that neuroprotective effects could be supported by adult stem cell

transplantation to the site of the damaged tissue. Many hopes for the cure for neurodegenerative

diseases, are placed in growth factors that show neuroprotective abilities and are known to promote

the survival of neurons (e.g., NGF, BDNF, and others). Neurotrophic factors/neurotrophins (NTs) act

via different classes of receptors, leading to the subsequent activation of various signaling pathways

in the target cells. Those pathways may modulate the development and maintenance of the nervous

system via promoting survival, migration, proliferation, differentiation and death

of neurons. However, their plasma delivery in pure protein form is rather ineffective, often

due to their poor pharmacokinetic profiles and inability to cross the blood–brain barrier in substantial

amounts. Therefore, different stem and progenitor cell therapy approaches step forward, as stem

cells are avid growth factors 'producers'. Human intrathecal transplantation of different stem cell

populations, including bone marrow (BM)-derived hematopoietic stem and progenitor cells, have been

previously attempted in patients with ALS, as well as in other neurological disorders including retinal

degeneration. Accordingly, improved administration of exogenous NTs and consequent

neuroprotection has been considered as a potential novel treatment for neurodegenerative diseases.

Our measurements revealed the NT-4-dendrimer nanoparticles can be used for continuous

neurotrophic factor delivery enhancing its distribution into mouse vitreous as well as damaged retina.

Understanding of polyvalent neurotrophins interactions with dendrimer nanoparticles might be useful

to obtain well-ordered protein layer, targeting future development of drug delivery systems especially

for neuroprotection of damaged retinal neurons.


Targeting key signaling factors as a way to control microglial

activation and induction of neuroinflammation Bogna Badyra, Anna Tejchman, Marcin Majka

Department of Transplantation, Medical College Jagiellonian University, Krakow Poland

Neuroinflammation is co-occurring phenomenon during pathological processes in the nervous system.

Key player in this process is microglia. As moderate activation of microglia is beneficial, excessive one

however, leads to more severe degeneration of tissue and inhibition of its endogenous regeneration.

One way to prevent this situation is to modulate or inhibit microglia activation.

Aim of this study was to use gene silencing technique to influence microglial activation. By targeting

key proteins - NF-κB, MyD-88 and TRIF, we intended to decrease inflammatory signaling network.

Gene silencing was optimized on stable murine microglia BV-2 cell line. Before stimulation

with lipopolysaccharide (LPS), cells were transfected with designed siRNA sequences. Efficacy

of transfection was assessed by evaluating expression of NF-κB, MyD-88, TRIF as well as IL-1β, IL-6,

TNF-α, TREM1, TREM2 at mRNA and protein level. Optimized sequences of siRNA were then used

on primary microglia.

Our results showed that siRNA can successfully inhibit activation of microglia in vitro after stimulation

with LPS. Significant decrease was observed in expression of signaling proteins. However, depending

on targeted factor, different decrease patterns were observed for IL-1β, IL-6 and TNF-α. Thus,

mixture of siRNA was combined to achieve most successful effect.

Our results provide a new method to successfully limit microglia activation with siRNA technique. This

approach will be further used in vivo, in our models of Parkinson’s disease and hypoxia-ischemia

encephalopathy, in which severe inflammation is observed.

Acknowledgements: The project was supported by the research grant from the Jagiellonian University

Medical College: 2015/17/B/NZ5/00294, K/DSC/003575.


The role of microRNA in mesenchymal stem cell immunoregulatory

function and neurogenesis in experimental autoimmune

encephalomyelitis Krzysztof Selmaj

Department of Neurology, Faculty of Medicine, University of Warmia and Mazury, Olsztyn, Poland

Transfer of bone marrow mesenchymal stem cells (BMSC) induced amelioration of experimental

autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The primary

mechanism of BMSC-induced EAE inhibition was linked with immunoregulation and this effect was

diminished with neuronal differentiation of BMSC. MicroRNA (miRNA) are single-stranded non-coding

RNA molecules, 21-23 nucleotides in length that regulate the expression of genes encoding proteins

and are involved in multiple basic biological processes. Several studies indicates that miRNA play

a role in stem cells (SC) self-renewal, differentiation and tissue repair. We analyzed the expression

profile of miRNAs in BMSCs in mice as a correlate of their immunoregulatory potential in EAE.

We found that several miRNAs were significantly differentially expressed (11 downregulated

and 19 upregulated) in neuronal BMSCs that had lost immunoregulatory activity in mice with EAE.

Inhibition of miR-146a with a complementary antagomir restored the immunoregulatory activity

of neuronal BMSCs. We mapped miR-146a to its multiple predicted target mRNA transcripts

and found that miR-146a was predicted to block prostaglandin E2 (PGE2) synthase (ptges-2) which

resulted in decreased secretion of PGE2. In our earlier studies we have found that BMSC secreted

large amounts of PGE2 which correlated with higher efficacy to EAE inhibition. In addition, high levels

of PGE2 secretion correlated with high expression of indoleamine-2,3-dioxygenase (IDO),

a recognized tryptophan-dependent immunosuppression mechanism. Thus, the current findings

on the differentially expressed miRNAs in BMSCs contribute to better explanation of functional

relation between PGE2 and IDO and induction of BMSC-induced immunoregulatory function

in EAE.Our data support the conclusion that epigenetic mechanisms dependent on miRNAs are

involved in BMSC-induced inhibition of inflammation in demyelination conditions, like MS, and provide

a mechanistic link between BMSC-derived miRNAs and IDO-dependent immunoregulation.


Viral inactivation of human platelet lysate by gamma irradiation

preserves its optimal efficiency in the expansion of human bone

marrow mesenchymal stromal cells Konrad Żurawski

Macopharma

Human platelet lysate (hPL) represents a powerful xeno-free alternative to fetal bovine serum (FBS)

for human mesenchymal stem cell (hMSC) expansion. However, the characterization and the batch-

to-batch standardization of such products still remain a challenge. In addition, the general chapter

5.2.12 of the European Pharmacopeia requires the addition of a step of viral inactivation during

the production process of such raw material of biological origin used for cell-based and gene therapy

medicinal products. We will report the extensive characterization and document the robust

standardization of our different clinical grade hPL products (MultiPL'), including growth factors (GF)

contents, multiplex assay and biochemical and proteomic analysis. Data of comparison of hMSCs

cultured with either FBS or MultiPL' will be presented (expansion, morphology, membrane marker

expression, potential of differentiation and immunosuppressive properties). We will highlight some

key characteristics of hMSC cultured in hPL. Importantly, we will also show that the use

of standardized hPL improves the standardization of biological features of hMSCs. Finally, the efficacy

of the gamma irradiation to inactivate a broad range of viruses in MultiPL' will be documented.

The impact of the gamma irradiation on MultiPL' and the biological features of hMSCs cultured

in MultiPL'i will be described.


The role of Imaging Flow Cytometry in modern cellular analysis Michał Konieczny

Merck

Imaging Flow Cytometry (IFC) combines the speed, sensitivity, and phenotyping abilities of flow

cytometry with the detailed imagery and functional insights of microscopy. This unique combination

enables a broad range of applications that would be impossible using either technique alone.

By collecting large numbers of digital images per sample and providing a numerical representation

of image-based features, the Amnis ImageStreamX Mk II combines the per cell information content

provided by standard microscopy with the statistical significance afforded by large sample sizes

common to standard flow cytometry. With the ImageStreamX Mk II System, fluorescence intensity

measurements are acquired as with a conventional flow cytometer; however, the best applications

for the ImageStreamX Mk II take advantage of the system's imaging abilities to locate and quantitate

the distribution of signals on in or between cells.


VWR as a provider of solutions for all Life Science disciplines Joanna Zimniak

VWR

VWR, part of Avantor provides global access to a huge portfolio of trusted, quality brands, as well as

products from thousands of other well recognized manufacturers.

Our VWR range includes a broad selection of choice products for proteomics, genomics, cell culture,

microbiology and more, all designed to help scientists explore new frontiers. We provide access

to a reliable supply of exceptional quality reagents, consumables and instruments offering the choices

for all life science disciplines!

We would like to present our solutions for all Life Science disciplines especially including our brand

portfolio.


Tailoring of fibrin hydrogel for Mesenchymal Stem Cells culture Paskudzka Monika1, Lech Wioletta2, Bużańska Leonora2, Zychowicz Marzena2

1.Faculty of Pharmacy with the Laboratory Medicine Division, Medical University of Warsaw 2. Department of Stem Cell Bioengineering, Mossakowski Medical Research Centre Polish Academy

of Sciences

Our group has shown before that specific microenvironment (culturing in low oxygen level as well as three dimensional scaffold structure) influence the parameters of MSC derived from Wharton Jelly (WJ-MSCs), including proliferation, secretory profile and differentiation. Here we perform the basic MSC characterization depending on culture conditions and test whether biofunctionalization of fibrin hydrogels with extracellular matrix proteins can influence the biological processes of WJ-MSCs. WJ-MSC were isolated from human umbilical cord and cultured in standard (21%) and low (5%) O2 level. The cells were investigated for their main characteristics: clonogenicity, mesodermal differentiation, senescence and presence of mesenchymal and neural markers. Different concentrations of fibrin and thrombin and ECM proteins were investigated to optimize properties of scaffolds for WJ-MSC culture. Culturing of WJ-MSC in 5%O2 as compared to 21% O2 in 2D, revealed their higher clonogenic potential and proliferation rate, lower senescence while sustaining of mesodermal and neural differentiation potential. Moreover, coverage of culture surface with ECM proteins (laminin and fibronectin) as well as fibrin allows for WJ-MSC enhanced cell number , with higher proliferation rate observed in 5%O2 conditions. In 3D culture optimal combination of fibrinogen and thrombin concentration to improve WJ-MSC proliferation rate were investigated. We have shown that the concentration of fibrinogen applied in the scaffold influence cell behavior and migratory capabilities while not affected cell number. Despite the relatively high number of living cells during the culture time, the proliferation of WJ-MSC inside the 3D fibrin hydrogel as well as ECM-enriched scaffold was sustained during 7 days of culture. Oxygen level and ECM proteins improved WJ-MSC proliferation in 2D culture while in 3D scaffold cell number was sustained during culture time. The applied thrombin and fibrinogen concentration influence the porosity of scaffold structure. The ECM incorporation have no effect on proliferation of cells in fibrin hydrogels and presence of laminin or fibronectin keep cells in the scaffolds and could promote differentiation of cells, however it still needs to be further investigated.

The work was supported by statutory funds to MMRC.


3D cultured Wharton Jelly Mesenchymal Stem Cells exhibit features of

neurospheres Agnieszka Kamińska1, Katarzyna Jezierska-Woźniak2, Wojciech Maksymowicz3,

Krystyna Domańska-Janik4, Anna Sarnowska1, 4

1 Translative Platform for Regenerative Medicine, Mossakowski Research Centre, Polish Academy of Science, Warsaw, Poland; 2 Department of Neurosurgery, Laboratory of Regenerative Medicine, School of Medicine, Collegium Medicum-University of Warmia and Mazury in Olsztyn, Olsztyn, Poland; 3 Department of Neurosurgery, School of Medicine, Collegium Medicum-University of Warmia and Mazury in Olsztyn, Olsztyn, Poland; 4 Department of Stem Cell Bioengineering, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland;

Our group has shown before that specific microenvironment (culturing in low oxygen level Spheroids as a 3D cell culture system are referred as floating aggregates of cell with visible differences across the structure. One of spheroid example is the neurosphere formed by neural stem cells (NSC). Neurospheres are induced in serum-free medium containing mitogens – epithelial growth factors (EGF) and basal fibroblast growth factor (bFGF). By applying similar culture conditions, there can be obtained spheroids from other cells, like mesenchymal stem cells (MSC). Although, the neurosphere derivation from MSC is difficult and the protocols are still under optimization, cells cultured in this system exhibit better potential for neural differentiation and pluripotency. Here, we describe initial results of establishing the methods of cell culture MSC derived from WJ-MSC (WJ-MSC), detection of viability of spheroids and early neural marker immunostaining to confirm neurosphere properties. Spheroids were obtained from primary cultured WJ-MSC. WJ-MSC cells were seeded on antiadhesive surface. For first 3 days cells were cultured in medium DMEM/F12 containing EGF. Then, medium was replace for Neurobasal Medium with EGF and bFGF. Cells were cultured as long as possible. Every 3 and 5div spheres were stained with mix of 4 µM ethidium homodimer-1 (Eth-D1) and 2 µM AM calcein to confirm the viability of cells in spheroids. In 4-5 div some spheres were seeded on adhesive surface and cultured for 5-6 days in standard medium for WJ-MSC. Spheres were fixed, embed in OCT medium and cut on cryostat. Cryostat sections were immunostained for the presence of neural markers and pluripotency marker. To make comparison, we also obtained neurospheres from NSC. WJ-MSC spheres were morphologically similar to NSC spheres: similar shape, longevity and size were observed. WJ-MSC spheres exhibited the expression of early neural markers such as Nestin but also markers for precursors such as β-III-Tubulin, GFAP and A2B5. 3D WJ-MSC resembles NSC neurospheres in some features.


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Date post:	23-May-2020
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