In-vitro cellular reprogramming to model gonad development and its disorders

Abstract

During embryonic development, mutually antagonistic signaling cascades determine the fate of the bipotential gonad towards a testicular or ovarian identity. Errors in this process result in human Disorders of Sex Development (DSDs), where there is discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in-vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells towards gonadal progenitors. Transcriptomic analysis reveals that the in-vitro-derived murine gonadal cells are equivalent to E11.5 in-vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete AMH, migrate and form tubular structures. The cells derived from a 46,XY DSD female hiPSCs, carrying a NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR/Cas9-mediated correction of the variant rescued the phenotype. This is a robust tool to understand mechanisms of sex-determination and model DSDs.

Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression

“…This is in agreement with a recent study reporting that the COV434 cell line was mis-identified and actually originates from small cell carcinoma of the ovary, not a granulosa tumor ( Karnezis et al, 2021 ). Finally, some previous studies have reported growth-factor-based protocols to differentiate human pluripotent stem cells to ovarian granulosa-like cells ( Lipskind et al, 2018 ; Lan et al, 2013 ; Gonen et al, 2021 ). However, the efficiency of such protocols is low (4–12%, or not reported), the protocols require extended culture (multiple weeks vs. 5 days), and those studies did not demonstrate that the cells could support germ cell maturation.…”

Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression

“…Several studies have used mouse gonadal cells to support hPGCLC maturation 2,34 , but this process is much slower (~77 days vs. ~4 days in our protocol) and the mouse cells may not behave the same as human cells. Other studies have reported growth-factor based protocols to differentiate human pluripotent stem cells to ovarian granulosa-like cells [8][9][10] . However, the efficiency of such protocols is low (4-12%, or not reported) and those studies did not demonstrate that the cells could support germ cell maturation.…”

“…Therefore, these cells are a crucial component of any in vitro model of the human ovary. Although a few studies have attempted differentiating hiPSCs to granulosa-like cells by treatment with growth factors [8][9][10] , none has resulted in an efficient method, nor has any study evaluated their ability to support germ cell development. Therefore, we took a different approach based on the principle that cell identity can be manipulated through transcription factor (TF) overexpression 11 .…”

Sorting and Manipulation of Human PGC-LC Using PDPN and Hanging Drop Cultures

“…This complete mouse in vitro oogenesis highlights the importance of the matching between germ cells and their environment. Gonen et al [ 48 ] used a similar approach to generate gonadal somatic cell progenitors using hiPSC. This appears to be a promising tool compared to the use of endogenous ovarian somatic cells, whose use relies on poorly accessible biological material.…”