biostudies-literatureSartori-Maldonado RFinnish Cultural FoundationMagnus Ehrnrooth FundationBo and Kerstin Hjelt Diabetes FoundationThe Paulo FoundationHUS GroupFinnish Red Cross Blood ServiceChildhood Disease Research FundationSigne and Ane Gyllenberg FoundationDiabetes Research FoundationK Albin Johansson FoundationIda Mont FundationNovo Nordisk Fonden2535-2548https://www.ebi.ac.uk/biostudies/studies/S-EPMC11405178biostudies-literatureUnknown32(8)Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS^-/--pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic β cells. Our results suggest that supplMolecular therapy : the journal of the American Society of Gene TherapyThymidylate synthase disruption to limit cell proliferation in cell therapies.PMC11405178NNF22OC0078484NNF19OC0057286Balaz MSoppa IEurola SWartiovaara KJuutila JPuttonen HSaarimaki-Vire JSartori-Maldonado RMontaser HOtonkoski TfalseThymidylate synthase disruption to limit cell proliferation in cell therapies.Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS^-/--pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic β cells. Our results suggest that suppl2024-01-01T00:00:00Z2024 Aug2026-06-02T22:17:55.66Z2025-04-06T12:30:50.904ZS-EPMC114051783886745010.1016/j.ymthe.2024.06.014