A Human Gonadal Cell Model From Induced Pluripotent Stem Cells

Abstract

Sertoli cells are main players in the male gonads development and their study may shed light on 46,XY disorders of sex development (DSD). Mature primary Sertoli cells are incapable of proliferating in prolonged in vitro cultures and the available Sertoli cell models have several limitations since they derive from mouse or human cancer tissues. We differentiated human fibroblasts (HFs)-derived induced pluripotent stem cells into Sertoli-like cells (SLC) and, in order to characterize this new Sertoli cell model, we performed gene expression analyses by NextGeneration Sequencing techniques. This approach revealed that our putative SLC have reduced expression of pluripotency markers and expressed Sertoli cell markers such as SRY-Related HMG-Box 9 (SOX9), vimentin (VIM), and claudin-11 (CLDN-11). More in detail, the transcriptional profile analysis suggested that these cells are in an early stage of Sertoli cells maturation. Harnessing the power of induced pluripotent stem cells, we were able to generate SLC that show genetic and functional similarities to human Sertoli cells (HSerCs). SLC could become an excellent source of patient-specific Sertoli cells that could be of paramount benefit for both basic research and personalized medicine in sex development and reproductive medicine.

An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development

“…Our protocol uses a stepwise directed differentiation approach to drive iPSCs to bipotential gonad lineages after 7–10 days, followed by the induction of testis cells, in particular, Sertoli-like cells, by days 12–15. This approach allows us to closely follow the iPSCs as they differentiate through the PS, IM/LPM, bipotential gonad, and early testicular lineages, something not evident in earlier Sertoli cell induction protocols ( Bucay et al., 2009 ; Rodríguez Gutiérrez et al., 2018 ; Shlush et al., 2017 ). Furthermore, as our protocol uses growth factor-mediated differentiation, there is no requirement for feeder cells, co-culture, or lentiviral transduction.…”

“…Such an approach has the potential to provide an in vitro model of the human gonad. To date, several studies have used pluripotent cells to induce gonad-like cells: bipotential gonads ( Sepponen et al., 2017 ), Sertoli or Leydig cells ( Bucay et al., 2009 ; Buganim et al., 2012 ; Chen et al., 2019 ; Rodríguez Gutiérrez et al., 2018 ; Jadhav and Jameson 2011 ; Kjartansdóttir et al., 2015 ; Yang et al., 2015 ; Yang et al., 2017 ; Yazawa et al., 2006 ), and germ cell lineages ( Hayashi et al, 2011 ; Irie et al, 2015 ; Hikabe et al, 2016 ; Shlush et al., 2017 ; Yamashiro et al., 2018 ; Gell et al., 2020 ). Some have overexpressed transcription factors to directly reprogram mouse or human fibroblasts into Sertoli and Leydig-like cells ( Buganim et al., 2012 ; Jadhav and Jameson 2011 ; Yang et al., 2015 , 2017 ; Yazawa et al., 2006 ).…”

“…The ectopic expression of the transcription factors GATA4 and NR5A1 appears to be sufficient to reprogram human fibroblasts into Sertoli-like cells ( Liang et al., 2019 ). Other protocols have used co-cultures of human and mouse cells ( Rodríguez Gutiérrez et al., 2018 ; Shlush et al., 2017 ; Yamashiro et al., 2018 ) or co-cultures of human iPSCs with NT2D1 cells to differentiate Sertoli-like cells ( Rodríguez Gutiérrez et al., 2018 ). Finally, bone morphogenetic protein (BMP) and WNT signaling drives bipotential gonad marker expression in human ESCs without the requirement for co-culture or transfection/transduction ( Sepponen et al., 2017 ).…”

Oligogenic Origin of Differences of Sex Development in Humans

“…Due to the complexity of sex development itself, a proper cell model has been searched for, for many years, to properly mimic patients' phenotypes and study DSD-related candidate genes and role of candidate variants. Nowadays, new techniques based on cell reprograming and in vitro guided differentiation (from differentiated cells to pluripotent stem cells to redifferentiated cells again) open the field to adequate models for DSD in the near future to predict the role of candidate variants [57][58][59].…”

Applying Single-Cell Analysis to Gonadogenesis and DSDs (Disorders/Differences of Sex Development)

“…Organoids can be derived from induced pluripotent stem cells (iPS cells) and can be used to model normal and abnormal development. For the gonad, iPS cells have been used to generate Sertoli-like cells, and also Leydig and germ cells [ 182 , 183 , 184 , 185 ]. Single-cell RNA-seq could be used to chart the differentiation of these cell types, identifying important genes activated during formation of the testis.…”