biostudies-literatureDuarte FMHORIZON EUROPE European Innovation Councile0331046https://www.ebi.ac.uk/biostudies/studies/S-EPMC12410764biostudies-literatureUnknown20(9)Neural crest stem cells (NCSCs) compose a highly migratory, multipotent, stem cell population arising from the neural plate border of the embryonic ectoderm. Investigating the development of NCSCs is critical in understanding both embryonic development and abnormal events that underlie neurocristopathies. Suggested seeding densities in in vitro human induced pluripotent stem cells (hiPSCs) differentiation protocols, varying between 10,000 cells/cm2 and 200,000 cells/cm2, demonstrate a lack of consensus on the optimal conditions to obtain NCSCs. Aiming to maximize the differentiation efficiency of hiPSCs towards the NCSCs lineage, we investigated the effect of the initial seeding density on NCSCs lineage commitment, both in fibroblast- and human peripheral blood mononuclear cell (PBMC)-deriPloS oneReaching a cell monolayer at the end of hiPSC differentiation enhances neural crest lineage commitment.PMC12410764860635Duarte FMMathijssen IMJvan Neck JWvan de Peppel JfalseReaching a cell monolayer at the end of hiPSC differentiation enhances neural crest lineage commitment.Neural crest stem cells (NCSCs) compose a highly migratory, multipotent, stem cell population arising from the neural plate border of the embryonic ectoderm. Investigating the development of NCSCs is critical in understanding both embryonic development and abnormal events that underlie neurocristopathies. Suggested seeding densities in in vitro human induced pluripotent stem cells (hiPSCs) differentiation protocols, varying between 10,000 cells/cm2 and 200,000 cells/cm2, demonstrate a lack of consensus on the optimal conditions to obtain NCSCs. Aiming to maximize the differentiation efficiency of hiPSCs towards the NCSCs lineage, we investigated the effect of the initial seeding density on NCSCs lineage commitment, both in fibroblast- and human peripheral blood mononuclear cell (PBMC)-deri2025-01-01T00:00:00Z20252026-05-29T22:38:08.607Z2026-04-08T06:17:10.304ZS-EPMC124107644090674910.1371/journal.pone.0331046