A Comparative Study of Cell Culture Conditions during Conversion from Primed to Naive Human Pluripotent Stem Cells

The successful reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs) represented a turning point in the stem cell research field, owing to their ability to differentiate into any cell type with fewer ethical issues than human embryonic stem cells (hESCs). In mice, PSCs are thought to exist in a naive state, the cell culture equivalent of the immature pre-implantation embryo, whereas in humans, PSCs are in a primed state, which is a more committed pluripotent state than a naive state. Recent studies have focused on capturing a similar cell stage in human cells. Given their earlier developmental stage and therefore lack of cell-of-origin epigenetic memory, these cells would be better candidates for further re-differentiation, use in disease modeling, regenerative medicine and drug discovery. In this study, we used primed hiPSCs and hESCs to evaluate the successful establishment and maintenance of a naive cell stage using three different naive-conversion media, both in the feeder and feeder-free cells conditions. In addition, we compared the directed differentiation capacity of primed and naive cells into the three germ layers and characterized these different cell stages with commonly used pluripotent and lineage-specific markers. Our results show that, in general, naive culture NHSM medium (in both feeder and feeder-free systems) confers greater hiPSCs and hESCs viability and the highest naive pluripotency markers expression. This medium also allows better cell differentiation cells toward endoderm and mesoderm.This work was supported by the Health Department of the Basque Government (Grant 2019111068, 2019/4703, 2020111058, 2020333032, 2021333057 and 2021333012), Merck-Salud Founda- tion (FSALUD17/004), Economic Development and Infrastructures Department of the Basque Govern- ment (KK-2020/00068), EITB Maratoia (BIO21/COV/030), Project “PI18/01299” and “PI21/01187”, funded by Instituto de Salud Carlos III and co-funded by European Union (ERDF) “A way to make Europe”, “ICI21/00095” funded by Instituto de Salud Carlos III and co-funded by European Union (NextGenerationEU), “Plan de Recuperación Transformación y Resiliencia” Investigación Clínica Independiente 2021–Acción Estratégica Salud 2017–2020, RICORS: (RD21/00017/0024) Red Española de Terapias Avanzadas TERAV ISCIII. Funded by Instituto de Salud Carlos III (ISCIII) and co-funded by European Union (NextGenerationEU) “Plan de Recuperación Transformación y Resiliencia” Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) 2021–Acción Estratégica Salud 2017–2020. L.H. was supported by the Jesus Gangoiti Barrera Foundation and the Asociación Española contra el Cáncer (AECC) AECC16/501 and the Fundación Mutua Madrileña AP176182020. M.M-I was supported by Jesus Gangoiti Barrera Foundation. I.R was supported by Margarita Salas Grant “MARSA21/60” and the Jesus Gangoiti Barrera Foundation. M.I-F. was supported by Inocente Inocente Foundation FII18/003. J.R.P. has grant “RYC-2013-13450” funded by MCIN/AEI/10.13039/501100011033, by the European Social Fund “ESF investing in your future”