The Effects of Genetic and Epigenetic Variation on Human Pluripotent Stem Cell Differentiation

Human pluripotent stem cells (PSCs) are widely used for studying embryonic development, disease modelling, drug discovery and cell therapy development. Using human PSCs as a model has significantly contributed to our understanding of embryonic development and elucidating novel pathological mechanisms as well as developing new drugs. However, there are significant genetic and epigenetic variations among hPSCs which can potentially affect their utility and differentiation potential, and thus undermine their applicability for downstream applications. In addition, female hPSCs under conventional culture conditions are epigenetically unstable, which is a major concern for disease modeling. The heterogeneity among hPSCs may be inherited from source cell populations, introduced during reprograming or accumulated during culture. Regardless of the source of this heterogeneity and instability, if human PSCs are to be used in embryonic development and disease modeling studies or regenerative medicine, these variations need to be understood and perhaps adjusted for. Human PSCs can potentially differentiate into all cells of the body including hematopoietic cells, and thus hold great promise for hematopoietic stem cell transplantation or transfusion therapies. However, the heterogeneity among hPSCs can affect their hematopoietic differentiation potential. For example, the level of IGF2 expression in hiPSCs is correlated with their hematopoietic commitment capacity, and diversity in DNA methylation patterns of hiPSCs is associated with their hematopoietic maturation capacity. It has been reported that differentiation potential of iPSC lines is skewed in favor of their source cell lineage. For example, iPSC lines derived from blood cells have a greater ability to differentiate towards blood cell lineages compared to the lines that are derived from skin cells. In paper I, we evaluate the influence of donor and cell type on the epigenome and hematopoietic differentiation potential of iPSC lines derived from blood and fibroblast cells of multiple donors. We demonstrate that donor genetic background has a much greater influence on the epigenome and hematopoietic differentiation potential of iPSC lines. Due to the large impact of donor on differentiation potential, in paper II we propose the establishment of a broad spectrum HLA type-matched iPSC bank derived from a broad donor base of term amniotic fluid derived cells in connection to planned caesarean section deliveries. Term amniotic fluid cells, being from a neonatal source have likely accumulated fewer genetic mutations compared to adult sources and thus are a better cell source for iPSC generation and will reduce variations among hiPSC lines. It has been also suggested that erosion of the transcriptionally inactive X chromosome in female PSCs under conventional culture condition is correlated with poor differentiation propensity, including hematopoietic differentiation potential. In paper III, we evaluate the effects of epigenetic instability of female hiPSC on their hematopoietic differentiation potential. We demonstrate that extensive erosion of the inactive X chromosome has a negative impact on hematopoiesis, and suggest a role for the X-linked gene ZIC3 in mediating the hematopoietic differentiation defect via Wnt signaling. (Less)