Naïve human iPSCs obtained by culturing the ICGi022-A cell line with primed pluripotency in HENSM medium efficiently differentiate into endothelial derivatives

Naïve human pluripotent stem cells (PSCs) are a promising new tool in biomedical research. They provide access to the early embryonic development programmes and offer breakthrough solutions in regenerative medicine. However, the current inability to obtain long-term cultures of genetically and epigenetically stable naïve human PSC lines poses a challenge to their effective application in biomedicine. The recently proposed HENSM culture medium is claimed to enable the obtaining and long-term maintenance of naïve PSC lines. In this study, the potential of the HENSM medium for obtaining stable naïve human PSC lines was investigated. We successfully reset the primed induced pluripotent stem cell (iPSC) line ICGi022-A (K7-4Lf), derived from a healthy donor, to a naïve state using the HENSM medium. Naïve iPSCs grow in the form of dome-shaped colonies, both with and without a feeder layer of cells. The resulting cells retained expression of the key pluripotency factors and activated the naïve PSC transcriptional programme, including expression of endogenous retroviral elements, early epiblast marker genes and genes associated with totipotency. The naïve iPSC line was capable of differentiating into derivatives of the three primary germ layers, as well as producing trophoblast derivatives. Culturing naïve iPSCs in low-adhesion conditions resulted in the spontaneous formation of three-dimensional structures (blastoids) resembling early human blastocysts. The X chromosome, which was in an eroded inactive state in the original cell line, was reactivated in the naïve cells, but returned to its normal inactive state when the naïve cells were re-primed. Notably, naïve iPSCs demonstrated limited ability to directly differentiate into endothelial cells. However, their competence to give rise to mature endothelial derivatives was restored upon returning to the primed state, achieving comparable efficiency to the original primed iPSCs. Thus, the resulting naïve iPSC line has significant potential for studying the early stages of embryogenesis and for other biomedical applications, including disease modelling. However, the naïve ICGi022-A line proved to be karyotypically unstable during long-term cultivation using HENSM medium. As there is a risk of karyotypic aberrations during the maintenance of naïve PSCs, further improvement of the culture conditions is necessary to obtain reliable, karyotypically stable lines of naïve pluripotent cells.