JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):123
Poster Presentation
29^th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263757

P-111. Generation of human female germline cells from hiPSCs stimulated by granulosa cells

Sarah Abreu Coxir¹, Raquel de Lima Leite Soares Alvarenga², Camilla Fernandes dos Santos¹, Samyra Maria dos Santos Nassif Lacerda¹

¹ Universidade Federal de Minas Gerais - Belo Horizonte - MG - Brasil
² Cegonha Medicina Reprodutiva - Belo Horizonte - MG – Brasil

Objective: This study aimed to evaluate the potential of conditioned medium derived from human granulosa cells (GCs) to induce the in vitro differentiation of human primordial germ cell-like cells (hPGCLCs) from induced pluripotent stem cells (hiPSCs).
Methods: The GC-conditioned medium was obtained from 48-hour cultures of cells collected from patients undergoing assisted reproductive treatments at a private reproductive medicine clinic. Prior to hPGCLC induction, hiPSCs were first differentiated into incipient mesoderm-like cells (iMeLCs) to optimize germline lineage specification. Subsequently, iMeLCs were cultured for six days in 96-well V-bottom plates under three experimental conditions: 1) Conditioned Medium Group: iMeLCs cultured in GK15 medium supplemented with 50% GC-conditioned medium. 2) Standard Group: iMeLCs cultured in GK15 medium supplemented with growth factors as previously described by Yamashiro et al. (2020). 3) Control Group: iMeLCs cultured in GK15 medium without any supplementation. The resulting cells were evaluated for the expression of early hPGCLC markers (BLIMP1, AP2-γ, SOX17) and migratory/post-migratory markers (NANOS3, DDX4, DAZL, DPPA3) by immunofluorescence and qPCR, as well as spheroid morphology.
Results: Gene expression analyses revealed higher levels of BLIMP1, AP2-γ, SOX17, NANOS3, DDX4, and DPPA3 in all three media compared to hiPSCs. For DPPA3, only the GC-conditioned media showed a statistically significant increase compared to hiPSCs (p=0.0115). For AP2-γ, a significant increase was observed only in the standard medium (p=0.0002). When comparing all groups relative to hiPSCs, the greatest differences were observed for early hPGCLC genes (BLIMP1, AP2-γ, SOX17, NANOS3), whereas late hPGCLC genes (DDX4, DAZL, DPPA3) showed smaller differences. These findings indicate that all tested media are capable of upregulating genes associated with human PGC specification. Immunofluorescence analyses showed strong and comparable staining for BLIMP1, AP2-γ, and SOX17 in spheroids cultured in GC-conditioned media and in the standard medium, but little to no staining in the control group. Morphological assessment revealed that spheroids cultured in the standard medium were smaller, less compact, and had more irregular edges compared to those in GC-conditioned media. Spheroids in the control medium displayed variable size, lower compactness, and partial disintegration during handling.
Conclusion: Our results highlight the promising potential of GC-conditioned medium for in vitro induction of hPGCLCs. This medium promoted strong expression of mRNA and protein markers of newly specified PGCs and generated a higher number of induced cells compared to the standard protocol. These findings suggest that GC-conditioned medium may offer a more efficient and biologically relevant approach for maintaining cell proliferation and viability during germ cell induction, with significant potential for developing cost-effective protocols for generating human female gametes in vitro.