In vitro Ableitung und Maturierung von Oozyten aus embryonalen Stammzellen der Maus
Zusammenfassung der Projektergebnisse
We have established a standardized, serum-free differentiation protocol for the generation of mouse PGCs from ESCs and iPSCs to improve differentiation efficiencies and to gain compatibility with applications in the clinical setting. We developed a culture system that robustly and reproducibly supported PGC specification and commitment and produced structures mimicking follicles. We demonstrated the derivation of germ cells of different developmental follicular stages and granulosa cells that exhibited ultrastructural characteristics typical for metabolically active and steroid-producing cells. With the development of a human differentiation model we demonstrated the specification of PGCs from hESCs/hiPSCs in a serum-free, two-step differentiation regimen. The combinatorial action of ActA, BMP4, and bFGF induced PGC precursors, which enabled the subsequent robust induction of PGCLCs through the action of BMP4. Contrary to mouse PGCs, these PGCLCs expressed only nominal levels of PRDM14 /PRDM14, suggesting that different regulatory mechanisms underlie human and mouse PGC specification and development. To improve the isolation of putative PGCs from heterogeneous differentiation cultures, we identified novel specific germ cell marker genes that are not expressed in pluripotent SCs. Two new reporter-ESC lines enabled the isolation of homogeneous PGCLC fractions that exhibited reduced methylation marks of imprinted loci and genetic features of migratory PGCs. While germ cell commitment took place at satisfactory yields, the overall yield of gonocytes/oocyte-like cells remained unsatisfactory. We detected that lack of serum triggered a premature upregulation of the meiotic gene program in PGCLCs before their gonial maturation and before suppression of pluripotency-related genes like Oct4. In search of factors associated with Oct4 and its regulation in germ cells, particularly during meiosis, we investigated the role of GCNF during germ cell development. We observed that Gcnf expression is not critical for the embryonic segregation of the germ cell lineage or maintenance of PGCs during their migratory phase. However, Gcnf appeared to be essential for further development and maturation of PGCs into oocytes. Our data also suggest a possible function for Gcnf in the activation or initiation of meiosis by enhancing Stra8 expression and inhibiting the expression of pluripotency-associated genes. We did not yet accomplish meiotic maturation of SC-derived germ cells into functional gametes, but with the knowledge we gained during the funding period of this project, we got a step closer to reaching this goal.
Projektbezogene Publikationen (Auswahl)
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(2010) In vitro derivation of germ cells from embryonic stem cells. Front Biosci (Landmark Ed) 15: 46-56
Ko K, Hübner K, Müller-Keuker J, Schöler HR
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(2011) Identification of genes specific to mouse primordial germ cells through dynamic global gene expression. Hum Mol Genet 20: 115-25
Sabour D, Arauzo-Bravo MJ, Hübner K, Ko K, Greber B, Gentile L, Stehling M, Schöler HR
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(2011) Ultrastructural characterization of mouse embryonic stem cell-derived oocytes and granulosa cells. Stem Cells Dev 20: 2205-15
Psathaki OE, Hübner K, Sabour D, Sebastiano V, Wu G, Sugawa F, Wieacker P, Pennekamp P, Schöler HR
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(2013) In vitro differentiation of germ cells from stem cells. In Biology and Pathology of the Oocyte: Role in Fertility, Medicine and Nuclear Reprograming, Trounson A, Gosden R, Eichenlaub-Ritter U (eds) Cambridge University Press
Sugawa F, Hübner K, Schöler HR
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(2014) Germ cell nuclear factor regulates gametogenesis in developing gonads. PLoS One 9: e103985
Sabour D, Xu X, Chung AC, Le Menuet D, Ko K, Tapia N, Arauzo-Bravo MJ, Gentile L, Greber B, Hübner K, Sebastiano V, Wu G, Schöler HR, Cooney AJ
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(2015) Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile. EMBO J 34:1009-24
Sugawa F, Arauzo-Bravo MJ, Yoon J, Kim KP, Aramaki S, Wu G, Stehling M, Psathaki OE, Hübner K, Schöler HR
