Fertility preservation following pediatric cancer therapy programs has become a major avenue of infertility research. In vitro spermatogenesis (IVS) aims to generate sperm from banked prepubertal testicular tissues in a lab setting using specialized culture conditions. While successful using rodent tissues, progress with human tissues is limited by the scarcity of human prepubertal testicular tissues for research. This study posits that human induced pluripotent stem cells (hiPSCs) can model human prepubertal testicular tissue to facilitate the development of human IVS conditions. Testicular cells derived from hiPSCs are characterized for phenotype markers and profiled transcriptionally. HiPSC-derived testicular cells are bioprinted into core-shell constructs representative of testis cytoarchitecture and found to capture functional aspects of prepubertal testicular tissues within 7 days under xeno-free conditions. Moreover, hiPSC-derived Sertoli cells illustrate the capacity to mature under pubertal-like conditions. The utility of the model is tested by comparing 2 methods of supplementing retinoic acid (RA), the vitamin responsible for inducing spermatogenesis. The model reveals a significant gain in activity under microsphere-released RA compared to RA medium supplementation, indicating that the fragility of free RA in vitro may be a contributing factor to the molecular dysfunction observed in human IVS studies to date.© 2025 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.
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