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Correlating the function and phenotype of pluripotent stem cells
thesisposted on 08.02.2017, 04:53 by Ho, Shi Hui Mirabelle
The unique properties of self-renewal and multilineage differentiation enables pluripotent stem cells (PSCs) such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to be well-positioned for a range of applications. However, outcomes of these potential applications are contingent on gaining a more in-depth understanding of their biology, as well as, safety of these cells. To date the comparative safety of hESCs and iPSCs have yet to be adequately addressed. Safety of their differentiated derivatives also necessitates the development of technology which permits the reliable elimination of pluripotent cells. Current literature describing the biology of hESCs and iPSCs has utilized heterogeneous cell populations. To derive a more precise definition of what constitutes a pluripotent stem cell, defined sub-fractions were obtained via fluorescence activated cell sorting (FACS) based on the expression of surface markers CD9 (TG30) and GCTM-2. The TG30-GCTM-2 double sort yields four immunologically defined sub-fractions arbitrarily termed TG30Neg-GCTM-2Neg (P4) to TG30High-GCTM-2High (P7). This double sorting strategy was rigorously interrogated by a series of assays, including microarray analysis, colony-forming assay and teratoma assay to be a robust strategy to enrich for pluripotent cells. Having established the validity of the TG30-GCTM-2 double-sort, the equivalency of iPSCs and hESCs was then assessed by the objective comparison of corresponding sub-fractions. In agreement with current literature, initial flow cytometric and microarray analysis of both cell types displayed highly similar profiles. However, our studies subsequently demonstrated for the first time the existence of functional differences between hESCs and iPSCs. Colony-forming and teratoma formation assays, as well as, immunostaining results revealed that all four sub-fractions of iPSCs were able to give rise to stem cell colonies. In contrast, stem cell colonies were notably absent in P4 and P5 hESCs and only observed in P6 and P7 hESCs. Furthermore, flow cytometric analysis revealed spontaneous reversion of differentiated derivatives of the P4 sub-fraction of iPSCs within seven days. Gene expression studies using Q-PCR noted that iPSCs possess a marked dysregulation of a selected number of pluripotency genes. A logical progression from the findings of these initial studies was to determine the possible mechanism(s) responsible for the observed reversion phenomenon. Utilizing an additional three iPSC lines generated using different approaches and gene cocktails, it was found that whilst persistence of viral transgenes could have increased colony-forming efficiency, in isolation this factor proved to be insufficient to cause spontaneous reversion. The reversion phenomenon was also noted to be independent of the concentration of FGF-2 used to culture the two cell types. Finally, we determined that the presence of residual OCT-4+ cells in the P4 sub-fraction could not account for the reversion phenomenon. Collectively, whilst these studies have yet to definitively elucidate the exact mechanism(s) responsible for the spontaneous reversion of differentiated derivatives of iPSCs, we have at least ruled out some plausible mechanism(s). A useful tool which lends itself to the understanding the biology of PSCs, as well as, the mechanism(s) responsible for reversion and reprogramming, is a reporter cell line linked to a pluripotency gene. Such a reporter cell line would also enable the enrichment and/or elimination of pluripotent cells, with the latter addressing safety concerns regarding the clinical use of PSCs. In this regard, two pluripotency genes - GALANIN and GDF-3 were identified based on results obtained from microarray and Q-PCR analysis. GDF-3/GALANIN promoter constructs were used to create the resultant reporter cell line using a lentiviral vector approach. The GALANIN reporter cell line which appeared to be constitutively active regardless of the culture condition used was excluded from further studies. The GDF-3 reporter cell lines were then characterized using colony-forming assay, immunostaining, EB formation and karyotyping. However, karyotyping results revealed that both the GDF-3 and GALANIN cell lines possess gross chromosomal abnormalities, an outcome possibly attributed to the random insertion of viral vectors into the cells’ genome. Whilst both the GDF-3 and GALANIN reporter cell lines may not be utilized as reporter cell lines as initially desired, these constructs provide a framework for future creation of reporter cell lines.