Tissue of Origin, but Not XCI State, Influences Germ Cell Differentiation from Human Pluripotent Stem Cells

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.

Controlled X‐chromosome dynamics defines meiotic potential of female mouse in vitro germ cells

“…Two recent studies used human in vitro PGCLC differentiation systems to investigate if the X‐inactivation state of human pluripotent stem cells would affect their propensity to differentiate towards the germ cell lineage. While the first study did not find a significant effect (Chang et al , 2021 ), the second study reported that human iPSCs with eroded X‐inactivation showed a lower efficiency in forming PGCLCs when compared to cells with a higher degree of X‐inactivation (Yokobayashi et al , 2021 ). This suggests that faithful X‐dosage control might be an important feature of both mouse and human germ cell development.…”

Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay

“…In previous work using EB-based differentiation, we identified lines F20 and M72 as inefficient hPGCLC-generating lines (Chang et al ., 2021). We observed the same in BME overlay differentiation, with F20 yielding 15% and M72 0.3% respectively (Figure S1D).…”

“…The most widely used directed-differentiation protocols from hPSCs to hPGCLCs include aggregation into EBs and treatment with high concentration of BMP4. However, while these EB-based methods were instrumental in understanding hPGCLC formation (Irie et al, 2015; Sasaki et al, 2015), they are characterized by low efficiency and high variability per hPSC line (Chang et al, 2021; Chen et al, 2017). Reported hPGCLC yields ranged from 5%-60%, with efficiencies above 30% being uncommon, and with many hPSCs showing hPGCLC-differentiation yields below 10%.…”

The compleX balancing act of controlling X-chromosome dosage and how it impacts mammalian germline development

“…At the moment human in vitro germ cell development is less far advanced than the mouse model system [ 152 ] and only early meiotic stages have been achieved so far [ 153 ]. Nevertheless, in two recent studies, it has been tested whether the original X-chromosome status of the pluripotent stem cells used as the starting material would affect human PGCLC-specification efficiency [ 154 , 155 ]. While the first study did not observe any significant impact, but rather that cell type of origin might affect PGCLC-differentiation efficiency [ 154 ], the second reported that stem cells with an eroded XCI state showed reduced germ cell competence when compared with cells with more complete XCI [ 155 ].…”

“…Nevertheless, in two recent studies, it has been tested whether the original X-chromosome status of the pluripotent stem cells used as the starting material would affect human PGCLC-specification efficiency [ 154 , 155 ]. While the first study did not observe any significant impact, but rather that cell type of origin might affect PGCLC-differentiation efficiency [ 154 ], the second reported that stem cells with an eroded XCI state showed reduced germ cell competence when compared with cells with more complete XCI [ 155 ]. In the study with the most advanced in vitro development into oogonia-like cells, progressive demethylation of X-linked gene promoters and partial XCR has been observed [ 153 ].…”