A Comparative Study Of Cell Culture Conditions During Conversion From Primed To Naive Human Pluripotent Stem Cells

50 p.Since the successful reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs), stem cells have opened up a large field of research to generate new therapies due to their ability to differentiate into any cell type. Embryonic stem cells (ESC) are pluripotent cells which give rise to all somatic cell types in the embryo. Their self-renewal ability and plasticity allows for in vitro generation of many distinct cell types, raising new challenges for regenerative medicine and therapies. The “naive” state of cell pluripotency is the result of cells that come from the preimplantation epiblast in vivo. This state was observed in mouse embryonic stem cells and was characterized by a high proliferation and differentiation capacity as well as a global DNA hypomethylation. Human embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos and correspond to a later stage called “primed” stage of embryonic development. The conversion of this “primed” human embryonic stem cells (hESCs) to a “naive” state is desirable as their characteristics would facilitate many techniques such as gene editing and a more efficient differentiation. The main objective of this conversion is to facilitate the application of cell therapies to be able to use them in clinical treatments to model different diseases, such as human primary immunodeficiencies related to NK cell defects. In the present study, the main objective is to compare and evaluate different culture conditions for conversion from primed to naive state of an hES cell line called ES-2. The different culture conditions are based on different conversion media (Gafni, Fine and T2iLGö) in both feeder and feeder-free cells conditions

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”