A novel xeno-organoid approach: exploring the crosstalk between human iPSC-derived PGC-like and rat testicular cells

Abstract

Specification of germ cell-like cells from induced pluripotent stem cells (iPSCs) has become a clinically relevant tool for research. Research on initial embryonic processes is often limited by the access to fetal tissue, and in humans the molecular events resulting in primordial germ cell (PGC) specification and sex determination remain to be elucidated. A deeper understanding of the underlying processes is crucial to describe pathomechanisms leading to impaired reproductive function. Several protocols have been established for the specification of human PSC towards early PGC-like cells (PGCLC), currently representing the best model to mimic early human germline developmental processes in vitro. Further sex determination towards the male lineage depends on somatic gonadal cells providing the necessary molecular cues. By establishing a culture system characterised by the re-organisation of somatic cells from postnatal rat testes into cord-like structures and optimising efficient PGCLC specification protocols, we facilitated the co-culture of human germ cell-like cells within a surrogate testicular microenvironment. Specified conditions allowed the survival of rat somatic testicular and human PGCLCs for 14 days. Human cells maintained the characteristic expression of octamer-binding transcription factor 4, SRY-box transcription factor 17, and transcription factor AP-2 gamma, and were recovered from the xeno-organoids by cell sorting. This novel xeno-organoid approach will allow the in vitro exploration of early sex determination of human PGCLCs.

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

“…However, the only previously reported method that allows human primordial germ cell-like cells (hPGCLCs) to develop to later DAZL-positive stages in vitro is to aggregate them with mouse fetal ovarian ( Yamashiro et al, 2018 ) or testicular ( Hwang et al, 2020 ; Kobayashi et al, 2022 ) cells, which mimic the environment of the early gonad. Another study tested aggregation of hPGCLCs with rat neonatal testis cells ( Mall et al, 2020 ), but only showed that the hPGCLCs could survive and did not assess maturation.…”

Germline stem cells in human

“… 318 , 319 hPGCLCs are also co-cultured with somatic cells from postnatal rat testes. 320 These somatic cells provide an appropriate niche for hPGCLCs to mature into oogonia or prospermatogonia similar to those of hPGC differentiation in vivo, respectively. Nevertheless, differentiation in vitro from hPGCLCs to mature gametes (ova and spermatozoa) and function testing remain to be explored further.…”

Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations

“…Previous studies focused on the generation of TOs have reported the resuspension of testicular cells directly in Matrigel or agarose [ 6 , 31 , 50 , 51 , 52 ]. In these studies, the time reported for TO formation varies between 2 and 9 days depending on the substrate or culture method and the number of cells used [ 30 , 31 , 32 , 33 , 40 ].…”