Pluripotent Cell Models for Gonadal Research

Abstract

Sex development is a complex process involving many genes and hormones. Defects in this process lead to Differences of Sex Development (DSD), a group of heterogeneous conditions not as rare as previously thought. Part of the obstacles in proper management of these patients is due to an incomplete understanding of the genetics programs and molecular pathways involved in sex development and DSD. Several challenges delay progress and the lack of a proper model system for the single patient severely hinders advances in understanding these diseases. The revolutionary techniques of cellular reprogramming and guided in vitro differentiation allow us now to exploit the versatility of induced pluripotent stem cells to create alternatives models for DSD, ideally on a patient-specific personalized basis.

Keywords: DSD; cell model; cellular reprogramming; germ cells; granulosa cells; iPSCs; induced pluripotent stem cells; patient-specific model; sertoli cell.

Figure 1

Human sex determination and differentiation: Gonadal somatic supporters, Sertoli cells and granulosa cells, play a central role in sexual development, orchestrating the differentiation of the other gonadal cells and secreting SOX9 and forkhead box L2 (FOXL2). Both factors counteract each other in a delicate equilibrium and its expression must be maintained through different stages of development to keep the final sexual faith into males or females respectively.

Figure 2

Cellular reprograming and guided differentiation of hiPSCs: The scheme represents the four stages of the iPSCs-derived gonadal cell model generation for DSD patients. Isolation of fibroblasts, peripheral blood mononuclear cells or even urinary precursors directly from the patient have been considered as easy to obtain and patient-friendly cell sources. Up to date, three cellular reprogramming techniques are commonly used to generate hiPSCs from diverse cell sources: viral transduction of Yamanaka and Thomson reprograming factors; episomal transduction adding a EBNA1 factor and silencing expression of the TP53 gene; and synthetized RNA. Guided differentiation by expression specific factors can force transformation of cell into Sertoli-like (BMP4, Activin A, PDG2 and FGF9) Granulosa-like (Estradiol, AMH, FSH, inhibin α and inhibin β) or germ-like cells (DAZL, DAZ, BOULE, GDF9 and BMP15). Addition of both those factors into the culture medium and forced overexpression proved to induce differentiation of hiPSCs. Gonadal cell-like models directly derived from DSD patients suppose unique tools to perform patient-specific in vitro studies and open the door for more advances in cell therapy and personalized medicine for DSD patients. hiPSCs: human induced pluripotent stem cells, PBMCS: peripheral blood mononuclear cells, UPS: urinary progenitors.

Figure 3

Known factors involved in guided differentiation of hiPSCs into gonadal cells: Expression of growth factors can guide the differentiation of embryoids bodies into Sertoli, Granulosa or germ cell-like cells. Bone morphogenic protein 4 (BMP4), Activin A, prostaglandin 2 (PDG2) and fibroblast growth factor 9 (FGF9) and retinoid acid (RA) successfully differentiated into Sertoli-like cells are able to endogenously express SOX9, anti-Mullerian hormone (AMH), claudin-11 (CLDN11) and other Sertoli markers [37,98]. Granulosa-like cell differentiation has shown to be stimulated by estradiol, AMH, follicle stimulating hormone (FSH), inhibin α and inhibin β [109,110,111]. BMPs proteins together with GDNF, DAZL and BOULE are known to initiate differentiation into a meiotic primordial germ cell-like (PGCL) phenotype [55,102,103,104]. While complete spermatogenesis in human cells was not achieved yet, PGCs differentiated with SOX17, GDF9 and BMP15 factors showed the ability to induce folliculogenesis [108].