Ligand-based directed differentiation to produce granulosa-like cells expressing steroidogenic enzyme genes

The ovarian granulosa cells are responsible for producing hormones and supporting oocytes through maturation and meiotic resumption. There is a need to generate granulosa-like cells (GLCs) from human induced pluripotent stem cells (hiPSCs) to better model human gonadal development and to test the effects of exogenous or pharmaceutical compounds on the ovary. Here we report a rapid ligand-based protocol for differentiating hiPSCs into cells that express markers of the transient developmental lineages and steroidogenic pathway genes. Single-cell RNA-sequencing (scRNA-seq) analysis identified canonical granulosa cell genes were expressed in a subset of cells and identified new genes of interest that were significantly associated with computationally modeled pseudotime. HSD17B1 was expressed in resulting GLCs but at low levels, suggesting an immature granulosa cell phenotype. The GLCs were produced using a simple culture method that could be augmented for granulosa cell functions such as sustaining oocyte growth. Producing GLCs through protocols such as this one is a first step toward designing large-scale ovarian endocrinology assays and developing personalized cell-based fertility and hormone restoration technologies in the future. This rapid protocol produced cells that express steroidogenic enzyme genes etoc blurb. Kubo and colleagues present a 5-day rapid protocol to generate immature granulosa-like cells from hiPSCs. Cells differentiated with inhibition of DKK1, a WNT signaling target gene, expressed gonadal ridge markers and FOXL2 transcripts and protein. Additionally, steroidogenic enzyme genes were expressed. A small population of differentiated cells were identified as expressing early-stage granulosa cell genes by single-cell RNA-seq.