Characterisation of primate primordial germ cells in vitro
by Julia Kurlovich
Date of Examination:2023-10-05
Date of issue:2024-06-17
Advisor:Dr. Ufuk Günesdogan
Referee:Prof. Dr. Rüdiger Behr
Referee:Dr. Melina Schuh
Referee:Prof. Dr. Heidi Hahn
Referee:Dr. Lukas Cyganek
Referee:Prof. Dr. Bernd Wollnik
Persistent Address:
http://dx.doi.org/10.53846/goediss-10533
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Abstract
English
Primordial germ cells (PGCs) are embryonic precursors of sperm and eggs, responsible for passing on genetic and epigenetic information throughout the generations. For certain mammalian species, the development of PGCs can be reconstituted from pluripotent stem cells (PSCs) in vitro, opening up many new avenues to study germ cell biology. Human PGC-like cells (PGCLCs) can be derived by first differentiating human PSCs into precursors of mesendoderm (pre-ME) and then, using a cocktail of cytokines, into PGCLCs. In mice, both female and male gametogenesis have been reconstituted in vitro and the derived gamete-like cells have been used for in vitro fertilisation, confirming their functionality and resemblance to in vivo gametes. However, due to molecular differences, reconstitution of in vitro gametogenesis in primates has not yet been achieved. The ability to derive human iPSCs (hiPSCs) from a patient’s own cells allows to generate immunologically compatible differentiated iPSCs, paving the way for personalised therapy. The biomedical potential of hiPSCs could be extended by their differentiation into functional gametes in vitro, which holds great promise for the treatment of infertility. However, hiPSCs exhibit functional heterogeneity when differentiated into various lineages, i.e. their propensity to differentiate into a cell type of interest varies between cell lines. HiPSC differentiation bias is not fully understood, but previously it was suggested to be influenced by genetic and epigenetic background of the donor. These differences between hiPSC lines could be exploited during iPSC differentiation into PGCLCs to investigate the factors important for PGC development in humans. In addition, the reliable use of hiPSCs in research and medicine requires a thorough study of the factors influencing functional heterogeneity of hiPSCs. In this work we evaluated PGCLC differentiation potential of ten hiPSC lines derived from healthy donors. We showed that studied cell lines have a pronounced variable differentiation efficiency when induced into PGCLCs. We found that elevated expression levels of TFAP2C, UTF1 and NR5A2 genes in hiPSCs and pre-ME negatively correlate with their ability to give rise to PGCLCs. Moreover, the absence of repressive histone modification H3K27me3 correlates with the higher expression levels of these genes. Finally, we show that the differentiation potential of the same hiPSC line can vary depending on the induced cell type. Therefore, this work identifies the factors influencing PGCLC specification and highlights the role of epigenetic regulation in this context. Monitoring accurate in vitro differentiation of gametes from PGCLCs requires their subsequent use for in vitro fertilisation and comparison with embryonic germ cells, which is not possible in humans due to ethical reasons. Therefore, to establish an in vitro model system, which closely resembles humans, we studied PGC development in vivo and in vitro in the common marmoset Callithrix jacchus (cj), a New World monkey. Post-migratory marmoset PGCs (cjPGCs) in gonads at embryonic day 74 express the germ cell-related genes SOX17, AP2Ɣ, BLIMP1 as well as the pluripotency markers NANOG and OCT4A (POU5F1), which is reminiscent of human PGCs. We established transgene-free marmoset iPSC (cjiPSC) lines from fetal and postnatal fibroblasts. These cjiPSCs cultured in feeder-free conditions can be differentiated into pre-ME and subsequently into cjPGC-like cells (cjPGCLCs) with a transcriptome similar to human PGCLCs. Taken together, this study establishes an in vitro system to derive cjPGCLCs, which closely resemble human PGCLCs, reinforcing the role of the marmoset monkey as a model to study primate germ cell development. In conclusion, my doctorate work advances our knowledge of hiPSC functional heterogeneity and thus contributes to the field of stem cell-based reproductive medicine. In addition, my work elucidated molecular mechanisms of human PGCLC differentiation in vitro. Further, this study describes the derivation of marmoset PGCLCs in vitro, paving the way for the derivation of functional marmoset gametes in vitro in future studies.
Keywords: Primordial germ cells; Common marmoset monkey; Pluripotent stem cells; Embryonic development; In vitro gametogenesis
