Modeling human germ cell development with pluripotent stem cells and characterizing the putative oogonial stem cells

The current understanding of human germ cell development is limited and is at most parts extrapolated from studies of mice or other model organisms. Particularly, the specification events of human germ cell lineage are largely unknown, which is at least in part due to inaccessibility of early stages of human development. In addition, debate on the existence of oogonial stem cells (OSCs) in adult ovaries further indicate that more research is needed in order to elucidate human germ cell development.

Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer a promising in vitro strategy to study fundamental questions regarding human germ cell development and to model genetically caused infertility. In this thesis, we used human PSCs to elucidate the genetic requirements of human germ cell development. Specifically, we assessed the potential of human iPSCs to differentiate to pre- meiotic and post-meiotic germ cells, studied the function of germ cell specific NANOS3 and Deleted in azoospermia like (DAZL) proteins via over expression in hESCs, and optimized clinically relevant iPSC reprogramming conditions for the derivation of patient specific iPSCs from fibroblasts of infertile Klinefelter syndrome (KS) patients. Furthermore, we studied the existence of OSCs by isolating and characterizing cells from adult human and mouse ovaries.

We found that human iPSCs derived from fetal- and adult somatic cells could differentiate to pre-meiotic germ cells with similar or higher efficiency relative to hESCs. Furthermore, in response to the over expression of intrinsic meiotic regulators, we observed that like hESCs, iPSCs formed meiotic and post-meiotic haploid cells. In addition, we identified several candidate genes for NANOS3 and DAZL mediated functions by over expression in hESCs. We found that human NANOS3 may have a conserved role in suppression of differentiation and inhibition of apoptosis, and DAZL in induction of germ cell differentiation and cell cycle arrest. Furthermore, we identified a possible new role of DAZL in inhibition of cell migration. For optimizing clinically relevant iPSC reprogramming conditions, we achieved efficient reprogramming using xeno-free and chemically defined laminin-521 and NutriStem medium, and derived KS patient specific iPSCs. Finally, we contradicted the previous study of OSCs by showing that the reported DEAD (Asp-Glu-Ala-Asp)-box polypeptide 4 (DDX4) antibody –based isolation of OSCs did not select for DDX4 expressing cells from adult human or mouse ovaries, and the isolated cells were not functional OSCs.

In conclusion, human PSCs have an enormous potential to elucidate the complex mechanisms of human germ cell development, and may ultimately contribute to new strategies for the diagnosis and treatment of infertility in years to come. In addition, we found no evidence for the existence of OSCs in adult human ovaries, thus, supporting the central dogma of fixed ovarian reserve.

List of scientific papers

I. Sarita Panula, Jose V. Medrano, Kehkooi Kee, Rosita Bergström, Ha Nam Nguyen, Blake Byers, Kitchener D. Wilson, Joseph C. Wu, Carlos Simon, Outi Hovatta and Renee A. Reijo Pera. Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells. Human Molecular Genetics, 2011 Feb 15;20(4):752-62.
https://doi.org/10.1093/hmg/ddq520

II. Sarita Panula, Ahmed Reda, Cyril Ramathal, Meena Sukhwani, Jan-Bernd Stukenborg, Kazutoshi Takahashi, Daniel Edsgärd, Michiko Nakamura, Olle Söder, Kyle E. Orwig, Shinya Yamanaka, Renee A. Reijo Pera and Outi Hovatta. Forced expression of germ cell specific genes in human embryonic stem cells. [Manuscript]

III. Sarita Panula, Jonathan Arias Fuenzalida, Pauliina Damdimopoulou, Outi Hovatta. A xeno-free and chemically defined culture system for efficient derivation of integration-free patient specific iPSCs. [Manuscript]

IV. Hua Zhang, Sarita Panula, Sophie Petropoulos, Daniel Edsgärd, Kiran Busayavalasa, Lian Liu, Xin Li, Sanjiv Risal, Yan Shen, Jingchen Shao, Meng Liu, Susann Li, Dongdong Zhang, Xiaoxi Zhang, Romana Raphaela Gerner, Mona Sheikhi, Pauliina Damdimopoulou, Rickard Sandberg, Iyadh Douagi, Jan-Åke Gustafsson, Lin Liu, Fredrik Lanner, Outi Hovatta, and Kui Liu. No DDX4-expressing functional oogonial stem cells in adult human and mouse ovaries. Nature Medicine. [Accepted]
https://doi.org/10.1038/nm.3775