Abstract
Human pluripotent stem cells (hPSCs) are not only a promising tool to investigate differentiation to many cell types, including the germline, but are also a potential source of cells to use for regenerative medicine purposes in the future. However, current in vitro models to generate human primordial germ cell-like cells (hPGCLCs) have revealed high variability regarding differentiation efficiency depending on the hPSC lines used. Here, we investigated whether differences in X chromosome inactivation (XCI) in female hPSCs could contribute to the variability of hPGCLC differentiation efficiency during embryoid body (EB) formation. For this, we first characterized the XCI state in different hPSC lines by investigating the expression of XIST and H3K27me3, followed by differentiation and quantification of hPGCLCs. We observed that the XCI state did not influence the efficiency to differentiate to hPGCLCs; rather, hPSCs derived from cells isolated from urine showed an increased trend towards hPGCLCs differentiation compared to skin-derived hPSCs. In addition, we also characterized the XCI state in the generated hPGCLCs. Interestingly, we observed that independent of the XCI state of the hPSCs used, both hPGCLCs and soma cells in the EBs acquired XIST expression, indicative of an inactive X chromosome. In fact, culture conditions for EB formation seemed to promote XIST expression. Together, our results contribute to understanding how epigenetic properties of hPSCs influence differentiation and to optimize differentiation methods to obtain higher numbers of hPGCLCs, the first step to achieve human in vitro gametogenesis.
Highlights
Human pluripotent stem cells, such as human embryonic stem cellsand induced pluripotent stem cells, can be maintained in culture for long periods of time while preserving their pluripotency, self-renewal characteristics and the potential to differentiate to virtually all cell types of the body [1,2]
Female Human pluripotent stem cells (hPSCs) are subjected to greater repercussions of epigenetic instability due to the fact that they contain two X chromosomes and rely on a process known as X chromosome inactivation (XCI)
We investigated whether different XCI states in hPSCs were associated with the capacity to differentiate to primordial germ cell-like cells in vitro
Summary
Induced pluripotent stem cells (hiPSC), can be maintained in culture for long periods of time while preserving their pluripotency, self-renewal characteristics and the potential to differentiate to virtually all cell types of the body [1,2]. Female hPSCs cultured in standard conditions (FGF2 and Activin A) show characteristics of primed pluripotency [9]; their XCI state would be expected to be XaXi, which entails accumulation of XIST, EZH2 and H3K27me on the Xi. after plating human blastocysts for hESC derivation, the epithelizing epiblast (or PICMI) to be used as passage zero show XaXi [15]. Once derived and cultured over a prolonged period of time, female hPSCs gradually and inevitably start losing epigenetic marks, including XIST expression and H3K27me on the. DNA demethylation and remodelling of histone marks/variants, leading to the erasure of genomic imprints [25,26] and, in female hPGCs, the reactivation of the Xi, a process known as X chromosome reactivation (XCR) [26,27]. We investigated whether different XCI states in hPSCs were associated with the capacity to differentiate to primordial germ cell-like cells (hPGCLCs) in vitro
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