Les cellules souches pluripotentes sont capables de donner naissance à tous les types cellulaires constituant un organisme, ce qui leur confère un fort intérêt thérapeutique. A partir de l’embryon de souris on peut en dériver deux types : les cellules souches embryonnaires (ES) au stade blastocyste et les cellules souches d’épiblaste (EpiSC) au stade œuf cylindre. Ces deux types de cellules partagent leurs propriétés pluripotentes mais se distinguent par de nombreux aspects comme leurs conditions de culture et les gènes qu’elles expriment. Nous avons montré que la reprogrammation par clonage par transfert de noyau permet d’obtenir des EpiSC présentant un méthylome et un transcriptome similaires à ceux des EpiSC issues d’embryons après fécondation. Nous avons également caractérisé le profil de méthylation de l’ADN des EpiSC, et montré une tendance à l’hyperméthylation des promoteurs des EpiSC par-rapport aux cellules ES et à l’épiblaste. De plus, l’absence de méthylation empêche la conversion des cellules ES en EpiSC. Les EpiSC semblent donc dépendre fortement de la méthylation de l’ADN pour réguler l’expression de leurs gènes, ce qui les distingue des cellules ES. / Pluripotent stem cells are of great therapeutic interest because of their capability to give rise to all the cells composing an organism. We can derive two types of these stem cells from the mouse embryo: embryonic stem cells (ESCs) from the blastocyst and epiblast stem cells (EpiSCs) from the egg cylinder stage. These two cell types share their pluripotent properties but are distinct on several features, like their culture conditions and gene expression. We showed that reprogramming using cloning by nuclear transfer allows the obtention of EpiSCs with a methylome and a transcriptome similar to those of EpiSCs derived from embryo after fertilisation. We also characterised the DNA methylation pattern of EpiSCs and showed their tendency to present a hypermethylation at their promoters compared to ESCs and epiblast. We also observed that the absence of DNA methylation blocks the conversion of ESCs into EpiSCs. As a conclusion, it seems that EpiSCs are strongly dependant of DNA methylation to regulate gene expression, which distinguishes them from ESCs.

Emerging trends in cell-therapy based tissue repair have focused on the renewable source of adult stem cells including human bone marrow-derived mesenchymal stromal cells (hMSCs). Due to immunomodulatory properties as well as a potential to differentiate into cells characteristic of all three germ layers, hMSCs provide a source of immature cells for utilization in cell-therapy based treatments. Marrow isolated adult multilineage inducible (MIAMI) cells are a homogeneous sub-population of hMSCs which maintain self-renewal potential during ex vivo expansion, in addition to efficiently undergoing trans-differentiation into neuron-like cells in vitro. Even though hMSCs have the potential to be used for neural tissue repair, the molecular mechanisms by which they are stimulated to become neuron-like cells have not been fully characterized. Therefore the work described herein focuses on the molecular mechanisms by which MIAMI cells undergo NT-3 dependent neuronal commitment. MIAMI cells express both the full length (FL-) and tyrosine kinase deficient (TKd-) isoforms of the NTRK3 receptor, the primary NT-3 receptor, at the protein level. NT-3 stimulation of MIAMI cells during neuronal commitment induced the phosphorylation of FL-NTRK3, degradation of TKd-NTRK3, downstream activation of the Mek1/2-Erk1/2 signaling cascade, and subsequent up-regulation of a limited number of pro-neuronal genes. These findings were verified using chemical inhibitors to block NTRK autophosphorylation (K252a) and Erk1/2 activation (U0126). TKd-NTRK3 is hypothesized to activate Rac1 upon NT-3 stimulation. Rac1 was found to suppress NT-3 stimulated Erk1/2 phosphorylation, as well as downstream gene expression, as determined using a Rac1 chemical inhibitor. Further characterization confirmed that Rac1b is the predominant Rac1 isoform in MIAMI cells. Rac1b siRNA mediated knock-down resulted in increased expression of the pro-neuronal genes NGN2, MAP2, NFH and NFL during NT-3 stimulation via regulation of Mek1/2-Erk1/2. Rac1b is also involved in NT-3 stimulated cell proliferation, as well as repression of CCND1 and CCNB1 mRNA expression. In an attempt to enhance neuronal differentiation of MIAMI cells, EGF and bFGF were used to pretreat MIAMI cells prior to NT-3 stimulated neuronal commitment. EGF/bFGF pretreatment increased NTRK3 and NTRK1 protein levels along with NT-3 stimulated Erk1/2 phosphorylation. In addition, bFGF versus EGF/bFGF pretreatment restricted the expression of the pro-neuronal transcription factors Ngn2 and Prox1 versus the neural stem cells self-renewal transcription factor Musashi-1, respectively. The culmination of this work provides a model for the NT-3 induced neuronal commitment of MIAMI cells in vitro, as well as insight into the neurogenic potential of MSCs for future applications in cell-therapy based tissue repair.

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.

Tissue engineering strategies represent exciting potential therapies to repair cartilage injuries; however, difficulty regenerating the complex extracellular matrix (ECM) organization of native cartilage remains a significant challenge. Cartilaginous ECM molecules, specifically chondroitin sulfate (CS) glycosaminoglycan, may possess the ability to promote and direct MSC differentiation down a chondrogenic lineage. CS may interact with the stem cell microenvironment through its highly negative charge, generation of osmotic pressure, and sequestration of growth factors; however, the role of CS in directing differentiation down a chondrogenic lineage remains unclear. The overall goal of this dissertation was to develop versatile biomaterial platforms to control CS presentation to mesenchymal stem cells (MSCs) in order to improve understanding of the interactions with CS that promote chondrogenic differentiation. To investigate chondrogenic response to a diverse set of CS materials, progenitor cells were cultured in the presence of CS proteoglycans and CS chains in a variety of 2D and 3D material systems. Surfaces were coated with aggrecan proteoglycan to alter cell morphology, CS-based nano- and microspheres were developed as small particle carriers for growth factor delivery, and desulfated chondroitin hydrogels were synthesized to examine electrostatic interactions with growth factors and the role of sulfation in the chondrogenic differentiation of MSCs. Together these studies provided valuable insight into the unique ability of CS-based materials to control cellular microenvironments via morphological and material cues to promote chondrogenic differentiation in the development of tissue engineering strategies for cartilage regeneration and repair.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.

Heart failure is the leading cause of death worldwide. In 2013, the American Heart Association estimated that one American will die of cardiovascular disease every 39 seconds. While heart transplantation is the most viable treatment option, the limited availability of donor hearts has necessitated the search for treatment alternatives such as the use of adult stem cells for cardiac repair and regeneration. Following myocardial infarction (MI), the inflammatory cardiac microenvironment, limited survival of stem/progenitor cells, myocardial scarring and fibrosis affect cardiac regeneration. This dissertation examines adult stem cell based approaches for cardiac regeneration by studying the effect of i) H₂O₂- mediated oxidative stress on mesenchymal stem cells, ii) Notch1 activation in cardiac progenitor cells using a self-assembling peptide hydrogel containing the Notch1 ligand mimic RJ in vitro and functional consequences in a rat model of MI. Through these approaches, the central hypothesis that modulation of stem cell response using cues such as oxidative stress and activation of Notch1 signaling can improve functional outcome following myocardial infarction has been studied.

No description available.

Human mesenchymal stem cells (MSC) have attracted a great deal of interest for their potential use in regenerative medicine and suppression of the inflammation. Nevertheless, all known therapy protocols require large amounts of MSCs, which can be obtained only by in vitro expansion. One of the most important methodological problems is associated with the use of animal-derived components in the cell culture medium. The main aim of the current research was to elucidate the influence of different serum substitutes on the proliferation, differentiation, expression of cell surface markers, and total protein expression of mesenchymal stem cells derived from human adipose tissue. In addition we were aiming to determine the features of mesenchymal stem cell populations from an exfoliated deciduous tooth (SHED) and their response to the multifunctional proinflammatory protein alpha1-antitrypsin. Our results indicate that adipose tissue derived MSCs cultivated in the presence of fetal calf serum and allogeneic human serum display similar properties, while synthetic serum substitute induces increase in growth and differentiation potential of MSCs. Moreover, our results indicate, that synthetic serum substitute also activates transcription of genes related to adipogenic and osteogenic differentiation and diminishes expression of cell surface marker CD146. In the present study, we used a proteomic approach that allowed us to compare protein expression signatures between primary cell... [to full text] / Žmogaus suaugusio organizmo mezenchiminės kamieninės ląstelės (MKL) pradėtos sėkmingai naudoti pažeistų audinių regeneracijai ir uždegiminio proceso slopinimui. Šiuolaikiniams terapijos protokolams yra reikalingi dideli ląstelių kiekiai, todėl prieš naudojimą yra būtina jas padauginti in vitro. Tačiau naudojant skirtingus MKL išskyrimo, in vitro kultivavimo ir padauginimo protokolus, yra sunku lyginti mokslinių tyrimų duomenis ir klinikinių tyrimų rezultatus. Nevienodos kultivavimo sąlygos gali įtakoti MKL funkcines savybes (augimo greitį, gebėjimą diferencijuotis, migracinį aktyvumą ir kt.). Šiame disertaciniame darbe tyrėme auginimo terpių, praturtintų skirtingais serumais (gyvulinės kilmės serumai, žmogaus alogeninis serumas, sintetinis serumas), poveikį žmogaus riebalinio audinio MKL. Taip pat charakterizavome MKL išskirtas iš žmogaus pieninių dantų pulpos ir palyginome šių ląstelių kultūros bei jų dukterinių klonų savybės. Kompleksiškai tyrėme uždegimo antiproteinazės alfa1-antitripsino (AAT) poveikį šioms ląstelėms. Nustatėme, kad, lyginant su kitais tyrime naudotais serumais, sintetinis serumo pakaitalas geriausiai skatino MKL augimą bei gebėjimą diferencijuotis adipogenine ir osteogenine kryptimis. Taip pat, skirtingai nuo kitų naudotų gyvulinės kilmės ir žmogaus serumų, sintetinis serumo pakaitalas veikė kaip adipogeninės ir osteogeninės diferenciacijos indukcijai svarbių genų PPARγ ir Msx2 transkripcijos aktyvatorius. Be to, sintetinis serumo pakaitalas slopino... [toliau žr. visą tekstą]

No description available.

The central properties of stem cells are the pluripotency and the capacity of self-renewal. Hematopoietic stem cells (HSCs) posses such common features that allows them to generate all the cells of the hematopoietic compartments, maintaining in the same time the HSC pool. We develop approaches focused on ex vivo HSC expansion through activation by exogenous HOXB4 (human HSCs) or Notch/Dll-4 ligand (murine HSCs). Two independent transcriptomic analyses surprisingly converged toward an increased expression of two genes never identified sofar as crucial for HSC functions: Pumilio1 (Pum1) and Pumilio2 (Pum2). Pum1 and Pum2 are posttranscriptional regulators belonging to the Pumilio-FBF (PUF) family of RNA-binding proteins. Although it was established that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells in Invertebrates, so far nothing is known about the role of Pum1 and Pum2 in human and murine HSCs.For these reasons, we have investigated the roles and mechanisms of action of Pum1 and Pum2 in murine and human HSCs through shRNA strategy. Pum1 and Pum2 knockdown (KD) in murine HSCs led to a decreased HSC expansion and clonogenic potential ex vivo, associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase. KD of both Pum1 and Pum2 enhanced these effects, suggesting a cooperative effect. Expansion and clonogenic potential of KD Pum1 HSCs were rescued by enforced expression of Pum1 (insensitive to our shRNA), thus validating the specificity of our shRNA. Enforced expression of Pum1 could not rescue the functions of Pum2 KD HSCs, highlighting the non-redundant role of these proteins. Furthermore, when Pum1 or Pum2 KD HSCs were inoculated into lethally irradiated mice to follow the long-term hematopoietic potential, only rare bone marrow cells derived from Pum1 and Pum2 KD HSCs were evidenced after 4 months, contrary to control HSCs. Identical results were obtained with human Pum1 or Pum2 KD HSCs.In conclusion, our results demonstrate the involvement of Pumilio factors in stemness maintenance, expansion and survival of murine and human HSCs. Identification of Pumilio factors and their targets as new regulators of HSCs expansion will allow consider them as new tools for therapeutic perspectives.