Genomic integrity in mouse embryonic and induced pluripotent stem cells can be compromised by factors such as extended time in culture and cellular reprogramming. Surprising, only a few studies have thus far examined the accumulation of chromosomal imbalances in mouse pluripotent populations upon prolonged propagation in vitro. It is presumed that specific recurring genetic changes can confer selective growth advantage and resistance to apoptosis and/or differentiation to the affected cells, although the genes that drive these processes remain elusive. The presence of these changes in published studies can confound the analysis of the data and hinder the reproducibility of the results. At the transcriptional level, aneuploidy manifests as large chromosomal regions of aberrant gene expression. This thesis presents a method to identify these regions in large-scale datasets and interrogate for recurrent patterns. The present analysis shows that over half of the 315 mouse pluripotent samples examined carry whole or partial-chromosome spanning clusters of aberrant transcription. Furthermore, there are common gene expression changes across samples with any type of predicted aneuploidy and samples with chromosome-specific aberrations. These transcriptional signatures have been used to train classification models which can predict aneuploid samples with over 90% accuracy. This is an important step towards the development of a low-cost and reliable transcriptional validation assay for the presence of aneuploidy.

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.

Pluripotent stem cells (PSCs) are specialized cells, which have remarkable ability to maintain in an undifferentiated state and are capable of undergoing differentiation to three germ-layer lineage cell types, under differentiation-enabling conditions. PSCs include embryonic stem (ES)-cells, embryonal carcinoma (EC)-cells and embryonic germ (EG)-cells. ES-cells are derived from the inner cell mass (ICM) of day 3.5 blastocysts (mouse). On the other hand, EC- and EG-cells have different source of origin and exhibit some differences in terms of their differentiation abilities and culture requirements. These PSCs act as an ideal in-vitro model system to study early mammalian development and cell differentiation and, they could potentially be used for experimental cell-based therapy for a number of diseases. However, one of the problems encountered is the immune rejection of transplanted cells. For this, immune-matched induced pluripotent stem (iPS)-cells have been derived from somatic cells, by forced expression of a few stemness genes. Although, human PSCs lines are being experimented, their cell-therapeutic potential is still far from being thoroughly tested due to lack of our understanding regarding 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 PSCs 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: GU-3 line (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. Recently, we have derived a ‘GS-2’ ES-cell line from the GU-3 mouse line (Singh et al., 2012). Additionally, we envisaged the need for developing an iPS-cell line from the GU-3 mouse and then use them for studying cell differentiation. Thus, aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked iPS-cell line from a genetically non-permissive FVB/N mouse strain, characterize the established iPS-cell line and achieve differentiation of various cell types from EGFP-expressing iPS-cell line; (2) to study differentiation phenomenon, in particular to cardiac lineage, using select-cardiogenesis modulators and (3) to assess the gene-expression profiles and signaling system associated with cardiomyocyte differentiation of PSCs. This thesis is divided into four chapters with the 1st chapter being a review of literature followed by three data chapters. In the chapter I of the thesis, a comprehensive up-to¬date review of literature is provided pertaining to PSCs, their classification, derivation strategies especially for reprogramming of somatic cells for iPSC generation, their differentiation potential and characterization, particularly to cardiac lineage. Various molecular regulators involved in cardiac differentiation of PSCs with emphasis on epigenetic regulation involving DNA methylation and signaling pathways involved are described in detail. Subsequently, various approaches used for enhanced cardiac differentiation of PSCs and the therapeutic potential of PSC-derived differentiated cell types to treat disease(s) are discussed. Chapter-II describes the successful establishment of a permanent iPS-cell line (named ‘N9’ iPS-cell line) from the non-permissive FVB/N EGFP-transgenic GU-3 ‘green’ mouse. This chapter provides results pertaining to detailed derivation strategy and characterization of the ‘N9’ iPS-cell line which includes colony morphology, expansion (proliferation) efficiency, alkaline phosphatase staining, pluripotent markers’ expression analysis by qPCR and immunostaining approaches and karyotyping analysis. Further, in order to thoroughly assess the differentiation competence of the ‘N9’ iPS¬cell line, assessment of in-vitro and in-vivo differentiation potential of the ‘N9’ iPS-cell line by embryoid body (EB) formation and teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives were performed, followed by the generation of chimeric blastocysts by aggregation method. This established N9 iPS-cell line could potentially offer a suitable model system to study cardiac differentiation along with other established PSC lines such as the GS-2 and D3 ES-cell lines and the P19 EC-cell line. Following the establishment of the system to study cardiac differentiation of PSC lines, efforts were made to understand the biology of cardiac differentiation of PSCs (wild¬type and EGFP-transgenic PSC lines and P19 EC-cell line) using small molecules as modulators. Data pertaining to this is described in Chapter-III. The possible involvement of epigenetic regulation of cardiogenesis for example, DNA methylation changes in cardiogenesis-associated genes is studied using 5-aza cytidine as one of the chromatin modifiers. In order to understand the cardiac differentiation phenomenon, as a consequence of using 5-aza cytidine in cell culture, it was important to investigate its ability to induce/mediate cardiac differentiation. This involved an assessment by quantitating the cardiac beating phenotype and correlating this with enhanced cardiac-gene expression profiles. Further, DNA methylation regulation of cardiogenesis¬associated genes is described using various DNA methylation analysis techniques. Moreover, the possible involvement of other signaling members in mediating the cardiac differentiation is also studied using the P19 EC-cells. Results pertaining to the above findings are described in detail in the Chapter-III. Chapter-IV is focused on various efforts made towards investigating the ability of ascorbic acid to enhance cardiac differentiation of mouse ES-cells (GS-2 and D3 lines). Ascorbic acid has been implicated to be influencing cardiogenesis and it is reported to enhance differentiation of various cell types under certain culture conditions. Results pertaining to enhancement of cardiac differentiation of PSCs using ascorbic acid are presented in this chapter. This included assessment by quantitating cardiac beating phenotype and its correlation with enhanced cardiogenesis-associated gene expression profiles. Besides, estimation on the sorted cardiomyocyte population, derived from PSCs was also made using mature-cardiac marker. The possible underlying signaling mechanism involved was also studied in detail, using specific inhibitors for pERK (U0126), integrin signaling (pFAK; PP2) and collagen synthesis (DHP), in order to ascertain their involvement in ascorbic acid-mediated cardiac differentiation of mouse ES-cells. Subsequent to the three data chapters (II-IV), separate sections are provided for ‘Summary and Conclusion’ and for ‘Bibliography’, cited in the thesis. The overall scope of the study has been to understand the basic biology of cardiac differentiation from PSCs (EC-cells, iPS-cells and transgenic and wild-type ES-cells) and to assess, by using certain small molecules, whether PSCs could be coaxed to enhance the differentiation to a particular cell type (cardiac). The data contained in this thesis addresses the above theme.