ArticleHuman reproduction open2026
Human iPSCs derived from cryopreserved testicular somatic cells enable germline regeneration in childhood cancer survivors.
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Abstract
study questionCan cryopreserved primary testicular somatic cells from childhood cancer survivors with severely decreased fertility potential be reprogrammed into human-induced pluripotent stem cells (hiPSCs) competent for efficient specification into early human germ cells? SUMMARY ANSWER: Primary testicular somatic cells from cryopreserved testicular samples with severely compromised spermatogonial pools can be reprogrammed into hiPSCs using a non-genome-integrating, feeder-free approach and subsequently differentiated into human primordial germ cell-like cells (hPGCLCs) with high efficiency. WHAT IS KNOWN ALREADY: Infertility is one of the most concerning long-term side effects of cancer therapy in prepubertal boys, yet it remains largely unaddressed. Worldwide, biobanks storing cryopreserved immature testicular tissue are expanding to support the development of fertility preservation strategies, including future tissue and cell transplantation approaches. However, whether these samples can also serve as starting material to generate hiPSCs and subsequently differentiate into STUDY DESIGN SIZE DURATION: Two biological replicates of cryopreserved prepubertal testicular tissue were used to obtain primary somatic cells, which were reprogrammed into hiPSCs and subsequently differentiated into hPGCLCs. This experimental pipeline had a duration of approximately four months. PARTICIPANTS/MATERIALS SETTING
methodsCryopreserved testicular tissue samples were obtained from two prepubertal cancer patients (6.2 and 6.3 years old) with depleted spermatogonial pools (spermatogonia count per round tubular cross-section of 0.04 and 0.02, and age-standardized MAIN RESULTS AND THE ROLE OF CHANCE: Here, we present the first successful generation of hiPSCs from cryopreserved testicular somatic cells of childhood cancer patients with a severely depleted germ cell pool. We accomplished this using a non-genome-integrating mRNA-based reprogramming approach. We further demonstrated the specification of hPGCLCs from these patient-derived hiPSCs, effectively regenerating their germline. This provides proof-of-concept for a stem cell-based fertility regeneration strategy in childhood cancer survivors with non-functional or absent germ cells. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This proof-of-concept study was limited by a small sample size due to the restricted access to cryopreserved human prepubertal testicular tissues. Although multiple hiPSC clones were generated per individual, only one clone per patient was used for downstream analyses, precluding systematic assessment of intra-individual clonal variability, which remains an important aspect for future studies. While G-banding karyotyping was sufficient to validate genomic integrity in this proof‑of‑concept study, more comprehensive genetic and epigenetic analyses should be prioritized in future hiPSC and hPGCLC validation before any clinical application is considered. WIDER IMPLICATIONS OF THE
findingsChildhood cancer patient-derived hiPSCs represent a powerful platform to investigate and address a broad range of long-term, cancer therapy-related complications. These hiPSCs and their derived germ cells may be used not only to develop
fundingT.M. was supported by the Erasmus+ program, as part of the projects WORK4ALL 2023 and WORK4ALL 2024 (2023-1-PT01-KA131-HED-000121324 and 2024-1-PT01-KA131-HED-000214636). L.N.A. was supported by a Marie Skłodowska-Curie Actions Individual Fellowship (101278886: GERMFIT) from the European Commission. Y.B. was supported by the Scientific Fund Willy Gepts. K.J. was supported by the Foundation for Pediatric Research, the Finnish Cancer Society, the Swedish Childhood Cancer Foundation (KP2020-0012), and the Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00335, 2021-00079, and 2023-00380). J.-B.S. was supported by the Swedish Childhood Cancer Fund (PR2019-0123; PR2022-0115; TJ2020-0023) and the Swedish Research Council (2018-03094; 2021-02107). J.P.A.-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council, the Birgitta and Carl-Axel Rydbeck Research Grant for Paediatric Research 2024 (2024-00208), and the Scientific Fund Willy Gepts. DISCLOSURES: All authors declare no conflicts of interest.
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