Immortalized Primate Granulosa Cell Lines and Pluripotent Stem Cell-Derived Early Germ Cells: Towards Ovarian Follicles In Vitro
by Shabnam Ghazanfari Sarabi
Date of Examination:2025-09-26
Date of issue:2025-10-24
Advisor:Prof. Dr. Rüdiger Behr
Referee:Prof. Dr. Rüdiger Behr
Referee:Prof. Dr. Stefan Pöhlmann
Persistent Address:
http://dx.doi.org/10.53846/goediss-11589
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Abstract
English
Access to primate ovarian tissue and mature oocytes is extremely limited, yet these resources are crucial for advancing both infertility treatment and fundamental research in reproductive biology. Conventional sources are scarce and subject to ethical and practical constraints. New in vitro strategies, particularly those using stem cells and immortalized somatic cell lines, are increasingly viewed as promising alternatives for generating functional gametes. In this study, we establish and integrate two complementary in vitro models to reconstruct the somatic and germ cell (GC) components of the primate ovarian follicle. First, we generated eight clonal immortalized granulosa cell (IGrC) lines from the common marmoset (Callithrix jacchus) to overcome the limited lifespan and donor variability of primary granulosa cells (GrCs). Our new IGrC lines retain key GrC identity markers (such as FOXL2 co-expressed with SOX9) and form characteristic intercellular junctions and hybrid epithelial-mesenchymal cytoskeleton of natural follicular GrCs. Notably, all clones demonstrated robust, long-term proliferation with bypass of senescence, while preserving lineage-specific features, including steroidogenic capacity in select clones. Functional assays revealed heterogeneity between the clones: a subset of IGrC lines maintained aromatase expression and hormone production (estradiol (E2) and progesterone (P4)) when stimulated by increased intracellular cAMP. At the same time, other lines show diminished steroidogenesis concurrent with a loss of gonadotropin receptor expression. Transcriptomic profiling confirmed that immortalization induces partial dedifferentiation by downregulating differentiated granulosa functions (e.g., FSHR, STAR, and INHBA/B) while upregulating cell-cycle, survival, and developmental regulators. IGrC clones clustered closely with primary cells, underscoring preserved somatic cell identity. In the second part, we directed human induced pluripotent stem cells (hiPSCs) toward a GC fate by BMP priming and overexpression of the germline-specific RNA-binding proteins DAZL and BOLL. This approach yielded primordial germ cell-like cells (PGCLCs) that organized into cystic structures and expressed early germline markers (BLIMP1, TFAP2C, SOX17, NANOS3), demonstrating successful PGC specification. Notably, the pluripotency state exerted a significant influence on differentiation outcomes. This was shown by the naïve-state hiPSC line demonstrating superior induction of GC fate and partial activation of meiotic gene expression when compared to primed-state hiPSC lines. However, according to the transcriptome data, all differentiations remained arrested at a pre-meiotic, oogenic stage. Due to the absence of KIT/KITLG and GDF9/WNT4 signaling axes as somatic support signals, induced GCs failed to progress into fully grown oocytes. This may have led to the downregulation of folliculogenic factors. Even with three-dimensional culture to provide a more sterically permissive microenvironment, germ-somatic coordination remained incomplete, leading to stalled follicle-like assemblies. Together, this work delivers a sustainable primate GrCs model and contributes to the advancement of human in vitro gametogenesis (IVG). Our findings highlight the necessity of re-establishing cross-talk between somatic and GCs for complete folliculogenesis and provide insights for future ovarian bioengineering and fertility preservation efforts.
Keywords: Ovarian folliculogenesis; Granulosa cells; Cell immortalization; Common marmoset (Callithrix jacchus); Germ cell differentiation; In vitro gametogenesis (IVG)
