<p>The present study was conducted in order to assess a role of the SH2 domain-containing adapter protein SHB in development and cell differentiation.</p><p>Embryonic stem (ES) cells overexpressing SHB and SHB with an inactive SH2 domain (R522K-SHB) were obtained. Microarray analysis in the SHB clone revealed altered expression of genes connected with neural cell function. The R522K-SHB clone exhibited altered expression of several transcription factors related to development. ES cells were differentiated by forming aggregates named embryoid bodies (EBs). The morphology of EBs was altered in the R522K-SHB clones, which showed fewer cavities. Expression of endodermal markers was decreased in the R522K-SHB EBs. </p><p>To further investigate the role of SHB in differentiation, murine ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-) were generated. SHB deficient clones increased the expression of mesendodermal and endodermal markers and decreased expression of two receptors, VEGFR2 and FGFR1, connected with blood vessel differentiation. Similarly, blood vessels showed an altered morphology in SHB+/- and SHB-/- EBs after VEGF stimulation. SHB-/- ES cells also formed fewer blood colonies than control ES cells.</p><p>Finally, the role of the SHB adapter protein in vivo was analyzed by generating a SHB deficient mouse (SHB-/-). SHB-/- animals are viable, fertile, but suffer from leukopenia and anemia. SHB-/- animals demonstrate an abnormal morphology of blood vessels in the liver and kidney. Breeding of SHB+/- animals revealed an abnormal segregation of the mutant allele with an increased number of SHB+/- animals and a decreased number of SHB-/- and SHB+/+animals. Backcross analysis of SHB+/- females with SHB+/+ males displayed an increased number of SHB+/- offspring already at the blastocyst level. Simultaneously, embryos from SHB+/- mothers show an increased malformation rate in comparison to embryos from SHB+/+ mothers.</p><p>In summary, the study suggests a role of SHB in reproduction and development and in mesodermal and endodermal specification. </p>

The present study was conducted in order to assess a role of the SH2 domain-containing adapter protein SHB in development and cell differentiation. Embryonic stem (ES) cells overexpressing SHB and SHB with an inactive SH2 domain (R522K-SHB) were obtained. Microarray analysis in the SHB clone revealed altered expression of genes connected with neural cell function. The R522K-SHB clone exhibited altered expression of several transcription factors related to development. ES cells were differentiated by forming aggregates named embryoid bodies (EBs). The morphology of EBs was altered in the R522K-SHB clones, which showed fewer cavities. Expression of endodermal markers was decreased in the R522K-SHB EBs. To further investigate the role of SHB in differentiation, murine ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-) were generated. SHB deficient clones increased the expression of mesendodermal and endodermal markers and decreased expression of two receptors, VEGFR2 and FGFR1, connected with blood vessel differentiation. Similarly, blood vessels showed an altered morphology in SHB+/- and SHB-/- EBs after VEGF stimulation. SHB-/- ES cells also formed fewer blood colonies than control ES cells. Finally, the role of the SHB adapter protein in vivo was analyzed by generating a SHB deficient mouse (SHB-/-). SHB-/- animals are viable, fertile, but suffer from leukopenia and anemia. SHB-/- animals demonstrate an abnormal morphology of blood vessels in the liver and kidney. Breeding of SHB+/- animals revealed an abnormal segregation of the mutant allele with an increased number of SHB+/- animals and a decreased number of SHB-/- and SHB+/+animals. Backcross analysis of SHB+/- females with SHB+/+ males displayed an increased number of SHB+/- offspring already at the blastocyst level. Simultaneously, embryos from SHB+/- mothers show an increased malformation rate in comparison to embryos from SHB+/+ mothers. In summary, the study suggests a role of SHB in reproduction and development and in mesodermal and endodermal specification.

Murine neural precursor cells have been a well studied model for neural cell fate determination and stem cell function both in vivo and in primary culture. However, factors such as cell number, the presence of multiple cell populations and of niche intrinsic factors made it difficult to dissect the mechanisms regulating cortical development. To overcome this issue, we have developed a culture system where mouse embryonic stem cells (ES) are differentiated to cortical radial precursors through retinoic acid treatment of embryoid bodies. One day after plating in neural differentiation conditions, ~70% of cells in the culture are cortical radial precursors (RPs) as indicated by the definitive cortical marker Emx1, and over 8 days in culture, these RPs differentiate to pyramidal glutamatergic neurons of the cortex mimicking in vivo development. Astrocyte differentiation can be observed later as the culture progresses, which again mimics the typical timed genesis of cells in the cortex. The stem cell properties and cell fate of these RPs can be manipulated with growth factors in culture as they are in vivo. In particular, FGF2 promotes proliferation and survival, while ciliary neurotrophic factor (CNTF) induces precocious astrocyte formation. Thus, our ES-derived cortical RP cultures can serve as an alternate and complementary in vitro model to examine neural precursor biology during early development.

Murine neural precursor cells have been a well studied model for neural cell fate determination and stem cell function both in vivo and in primary culture. However, factors such as cell number, the presence of multiple cell populations and of niche intrinsic factors made it difficult to dissect the mechanisms regulating cortical development. To overcome this issue, we have developed a culture system where mouse embryonic stem cells (ES) are differentiated to cortical radial precursors through retinoic acid treatment of embryoid bodies. One day after plating in neural differentiation conditions, ~70% of cells in the culture are cortical radial precursors (RPs) as indicated by the definitive cortical marker Emx1, and over 8 days in culture, these RPs differentiate to pyramidal glutamatergic neurons of the cortex mimicking in vivo development. Astrocyte differentiation can be observed later as the culture progresses, which again mimics the typical timed genesis of cells in the cortex. The stem cell properties and cell fate of these RPs can be manipulated with growth factors in culture as they are in vivo. In particular, FGF2 promotes proliferation and survival, while ciliary neurotrophic factor (CNTF) induces precocious astrocyte formation. Thus, our ES-derived cortical RP cultures can serve as an alternate and complementary in vitro model to examine neural precursor biology during early development.

Pluripotent stem cells (PSCs: ESCs and iPSCs) provide an excellent model system for studying neural development and function. These cells also serve as a reliable source of cell replacement for the treatment of various neurodegenerative diseases and disorders. In view of these applications of PSCs, multiple protocols have been developed to direct their differentiation into neural lineage. However, many of these protocols are limiting in terms of (a) low efficiency of generation of neural cells after long-term culture, (b) requirement of exogenous factors to induce and enhance neural differentiation and (c) supplementation of PSC culture medium with serum. Therefore, in the present study, attempts were made to achieve enhanced efficiency of neural differentiation of PSCs in the absence of exogenous molecules by employing a defined culture medium containing knockout serum replacement (KSR). KSR-based culture system was tested with our in-house-derived EGFP-transgenic ‘GS-2’ ES-cell and ‘N9’ iPS-cell lines and the wild-type ‘D3’ ES-cell line. In KSR medium, PSC-derived EBs predominantly generated neural cells from their post-attachment outgrowths and the complexity of neural networks increased as the culture progressed. Molecular phenotyping of PSC-derived neural cells was performed based on the expression of neural markers both at the mRNA and protein levels. qPCR analysis revealed the expression of markers corresponding to multiple neural cell types, including glutamatergic, GABAergic, cholinergic, serotonergic and dopaminergic neurons, astrocytes and oligodendrocytes, at various time points during the culture. RNA expression studies were confirmed via immunocytochemical analysis of the expression of neural markers. On day 15 of culture, FACS quantitation revealed the efficient generation of NES+ neural progenitors (~16-18%), MAP2+ mature neurons (~12-26%) and GFAP+ astrocytes (~30-63%) from the three PSC lines. Functional assessment of the generated neurons was performed by electrophysiological analysis of passive (RMP) and active (threshold, amplitude, FWHM and outward and inward currents) membrane properties. In order to investigate the role of default pathway in neural differentiation of PSCs in KSR medium, various strategies were employed. GS-2 ES-cells were cultured in the presence of different serum-free supplements; predominant differentiation into neural lineage was achieved in the B27-supplemented medium. The supplementation of KSR medium with BMP4 failed to show any effect of neural differentiation of GS-2 ES-cells. Also, EBs were switched between KSR- and FBS-supplemented culture conditions on day 2 or day 5 of culture. These experiments indicated that KSR medium promoted the generation of neural cell fates at the expense of differentiation to non-neural lineages. Interestingly, differentiation of P19 EC-cells in KSR medium also resulted in the predominant neural differentiation. These experiments collectively suggested the importance of default pathway in neural differentiation of PSCs in KSR medium. Additionally, efforts were made to enrich PSC-derived neural cells and also to enhance the efficiency of neural differentiation of PSCs. The removal of central EB-core from its peripheral neural outgrowth via scooping resulted in the enrichment of neural cells by ~1.3-2.1 folds. Significant increases were observed in the percentages of GS-2 ES-cell-derived MAP2+ mature neurons and GFAP+ astrocytes. Also, FGF2 supplementation of KSR medium was tested as a strategy to achieve enhanced efficiency of neural differentiation. Preliminary studies suggested an increase in the percentage of NES+ neural progenitors in the presence of FGF2. Taken together, KSR-based culture system offers a simple, defined and efficient method to achieve neural differentiation of PSCs in short time duration in the absence of exogenous factors. KSR-based culture system can be employed to generate specific neural cell types, study molecular regulation of neural differentiation and in disease modeling. Also, it can be used to develop a platform for high-throughput screening of potential neurogenic molecules and for dissecting their mechanisms of action.

No description available.

Unbegrentzte Selbsterneuerungkapazität und Pluripotenz sind charakteristische Merkmale von embryonalen Stammzellen (ES-Zellen). Dennoch sind die molekularen und zellulären Mechanismen, die für das Schicksal der ES-Zellen zuständig sind, noch nicht genau definiert. Um regulierende Faktoren des undifferenzierten Zustands von ES-Zellen zu identifizieren, wurden undifferenzierte ES Zellen, "Embryoid Bodies", spontan differenzierte und mit Retinsäure differenzierte ES Zellen mittels Microarray-Analysen verglichen. Neben bereits etablierten Pluripotenz-Markern, wurde Sprouty4 als eines der am stärksten degerulierten Transkripte unter diesen Bedingungen identifiziert. Sprouty4 ist als Inhibitor des ERK (Extracellular signal-regulated protein kinase)-Signalweges bekannt, aber seine Rolle in ES-Zellen wurde noch nicht definiert. Mittels Genexpression und Western BlotAnalysen konnte gezeigt werden, dass Sprouty4 in undifferenzierten ES-Zellen stark exprimiert ist und im Verlauf der Differenzierung schnell herunterreguliert wird. In vivo war Sprouty4 auf die innere Zellmasse (ICM) der Mausblastozyste beschränkt. Außerdem wurde gezeigt, dass der Sprouty4 Promotor durch direkte Bindung der PluripotenzMarkern Nanog, Klf4 und Stat3 reguliert wird. ES-Zellen, die Sprouty4 konstitutiv exprimieren, waren resistent gegen Differenzierung durch Zugabe von Retinsäure oder Bildung von Embryoid Bodies. Hingegen führte die Expression einer dominant-negativen Mutante von Sprouty4 zu einer erhörten Sensitivierung von ES-Zellen gegenüber der Differenzierung und zur Bildung extraembryonaler Gewebe begleitet von Endoreduplikation. Zusammenfassend konnten unsere Ergebnisse zeigen, dass die enge Regulation des ERK-Signalweges und warscheinlich anderer Signalwege durch Sprouty4 notwendig ist, um die Balance zwichen Pluripotenz und Differenzierung embryonaler Stammzellen zu kontrollieren. / A hallmark feature of embryonic stem (ES) cells is the ability to self-renew indefinitely while maintaining pluripotency. However, the molecular and cellular mechanisms underlying ES cell fate are poorly understood. To identify signaling pathway molecules that maintain the uncommitted state of ES cells, a microarray analysis was performed comparing undifferentiated ES cells, mature embryoid bodies, spontaneously differentiated and retinoic acid-induced differentiated ES cells. Among several well-validated pluripotency markers, Sprouty4 was identified as one of the most highly deregulated transcripts under these conditions. Sprouty4 is known as an inhibitor of the extracellular signal-regulated protein kinase (ERK/MAPK) pathway however its role in ES cells has not yet been defined. Gene expression and western-blot analyses have shown that Sprouty4 is highly expressed in ES cells and strongly downregulated upon differentiation whilst in vivo, Sprouty4 is confined to the founder population of ES cells, the inner cell mass of mouse blastocysts. Moreover, the Sprouty4 promoter was found to be regulated via the direct binding of the intrinsic pluripotency-associated factors Nanog, Klf4 and Stat3. ES cells engineered to constitutively express a wild-type version of Sprouty4 were found to be resistant to differentiation induced by retinoic acid or embryoid bodies formation. Conversely, expression of a dominant negative Sprouty4 mutant activating the ERK/MAPK pathway in a sustained manner sensitized ES cells to differentiation and triggered endoreduplication leading to the formation of extraembryonic tissue. Taken together, these results highlight the essential role of Sprouty4 in the tight regulation of the ERK/MAPK pathway- and probably others- for the balance between pluripotency and lineage commitment in mouse embryonic stem cells.

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’.

Proteomic techniques were used to analyze the protein expression profile of the early-stage differentiation of pig embryonic stem cells (ES cells). The pig ES cells were induced to develop to neuronal cells by all-trans retinoic acid (ATRA) in vitro by Tainan Livestock Research Institute. The ES cells were cultured with ATRA and collected at time intervals of 0, 1, 2, 4, 8 and 10 days. The cell lysates were analyzed by two-dimensional electrophoresis, and the differentially expressed proteins are identified by MALDI-TOF. Our data shows that the expression profile of pig ES cells is similar to other mammalian models but with some differences. Preliminary pig ES cells 2D database was set up. Six spots each with up or down-regulation in neurogenesis were identified by MS. These proteins may become the good markers of pig ES cells into neural cells by RA. Among those proteins, vimentin, prohibitin and annexin A10 were up-regulated, zinc finger protein 482 (ZNF482), fyn-related kinase (FRK) and annexin A1 were down-regulated during differentiation of pig ES cells to neural cells. Addtionally, we ultilized RT-PCR technique to investigate mRNA expression during neurogenesis, vimentin and prohibitin was up-regulated, anxa1(annexin A1) was slightly down-regulated, neuroD1 and neurogenin 2 were high expression on day 10, beta-catenin was high expression on day 8 to 10.

Renal tissue engineering and regenerative medicine represent a significant clinical objective because of the very limited prospect of cure after classical kidney treatment. Thus, approaches to isolate, manipulate and reintegrate structures or stimulating the selfregenerative potential of renal tissue are of special interest. Such new strategies go back to knowledge and further outcome of developmental biological research. An understanding of extracellular matrix (ECM) structure and composition forms thereby a particularly significant aspect in comprehending the complex dynamics of tissue regeneration. Consequently the reconstruction of these structures offers beneficial options for advanced cell and tissue culture technology and tissue engineering. In an effort to investigate the influence of natural extracellular structures and components on embryonic stem cell and renal embryonic tissue, methodologies which allow the easy application of exogenous signals on tissue in vitro on the one hand and the straight forward evaluation of decellularization methods on the other hand, were developed. Both systems can be used to investigate and modulate behaviour of biological systems and represent novel interesting tools for tissue engineering. The novel technique for culturing tissue in vitro allows the growing of embryonic renal explants in very low volumes of medium and optimized observability, which makes it predestined for testing additives. In particular, this novel culture set up provides an ideal opportunity to investigate renal development and structure formation. Further studies indicated that the set is universally applicable on all kinds of (embryonic) tissue. Following hereon, more than 20 different ECM components were tested for their impact on kidney development under 116 different culture conditions, including different concentrations and being either bound to the substrate or dissolved in the culture medium. This allowed to study the role of ECM constituents on renal structure formation. In ongoing projects, kidney rudiments are exposed to aligned matrix fibrils and hydrogels with first promising results. The insights gained thereof gave rise to a basis for the rational application of exogenous signals in regenerative kidney therapies. Additionally new strategies for decellularization of whole murine adult kidneys were explored by applying different chemical agents. The obtained whole matrices were analysed for their degree of decellularization and their residual content and composition. In a new straight forward approach, a dependency of ECM decellularization efficiency to the different agents used for decellularization could be shown. Moreover the capability of the ECM isolated from whole adult kidneys to direct stem cell differentiation towards renal cell linage phenotypes was proved. The data obtained within this thesis give an innovative impetus to the design of biomaterial scaffolds with defined and distinct properties, offering exciting options for tissue engineering and regenerative kidney therapies by exogenous cues.