From Fibroblasts to hPGCLCs: Germline Specification from hiPSCs for Fertility Regeneration

Abstract

Background and Objectives: Childhood cancer survivors face significant long-term health consequences, with infertility being one of the most prominent side effects of gonadotoxic cancer therapies. While current fertility preservation approaches for prepubertal boys rely on the cryopreservation of testicular tissue containing spermatogonial stem cells (SSCs), this strategy is not applicable to patients already lacking viable SSCs at the time of biopsy. For these patients, generating SSCs from human induced pluripotent stem cells (hiPSCs) represents the only viable option for achieving biological parenthood. This project aimed to establish a pipeline for generating hiPSCs from primary testicular somatic cells of childhood cancer patients and evaluate their competence for germline differentiation through human primordial germ cell-like cell (hPGCLC) specification protocols. Methods: Primary testicular somatic cells were isolated from fresh and cryopreserved testicular biopsies obtained from 16 prepubertal patients undergoing gonadotoxic cancer treatments as part of the NORDFERTIL fertility preservation project. Cells were expanded in culture and reprogrammed to hiPSCs using non-integrative RNA-based reprogramming protocols. Generated hiPSC lines were characterized by immunohistochemistry for pluripotency and trilineage (endoderm, mesoderm and ectoderm) differentiation capability. Germline competence was assessed through hPGCLC specification using two established protocols, with hPGCLCs identified by fluorescence-activated cell sorting (FACS) and immunohistochemistry. Results: Primary testicular somatic cells were successfully isolated and expanded from 15 of 16 patient samples. The mRNA-based reprogramming kit successfully generated hiPSCs from chemotherapy-exposed testicular somatic cells of a high-priority patient (TS1) with severe spermatogonial depletion. The resulting hiPSC-TS1-6 line demonstrated robust pluripotency characteristics, expressing OCT4 and SOX2, and successfully differentiated into the three embryonic germ layers. Importantly, hiPSC-TS1-6 retained competence for hPGCLC specification, generating 20-25% TNAP+/PDPN+ hPGCLCs, assessed by FACS, via peri-gastrulation precursors and resetting precursors, comparable to control dermal fibroblast-derived hiPSCs. Conclusions: This project demonstrates the feasibility of generating clinically compatible and compliant hiPSCs from primary testicular somatic cells of childhood cancer patients using non-integrative reprogramming methods. The successful specification of hPGCLCs from patient-derived hiPSCs represents a crucial step towards restoring the germline for patients lacking viable SSCs in their cryopreserved testicular tissue. To our knowledge, this is the first report of prepubertal testis-derived hiPSCs generated using a non-integrative method and their successful specification into hPGCLCs. These findings provide a proof-of-concept for hiPSC-based fertility restoration strategies and establish a foundation for future research aimed at complete in vitro gametogenesis from patient-specific stem cells. Clinical Significance: The development of hiPSC-based approaches for fertility restoration offers hope for childhood cancer survivors who cannot benefit from conventional fertility preservation methods. As advances in male germline differentiation from hiPSCs continue to progress towards SSC differentiation, patient-derived hiPSC biobanks will become increasingly valuable resources for personalized fertility treatments. This work contributes to the broader goal of improving the quality of life for childhood cancer survivors by addressing one of the most significant long-term consequences of their life-saving treatments.