Abstract: The field of naive human pluripotent stem cells (hPSC) has seen a surge in research activity over the years, particularly their use for generation of blastoids, a powerful 3D model to study human embryogenesis in vitro. A major hurdle today is the lack of a standardized protocol for deriving naive hPSC without the use of mouse embryonic fibroblasts (MEF) or transgene overexpression. In this study, we cultured hPSC on laminin 521 (Biolamina LN521), a key cell adhesion protein in the inner cell mass of human blastocysts, to mimic their natural niche. We established this novel approach of culturing cells on LN521 during epigenetic resetting through histone deacetylation inhibition following the PXGL protocol (Austin Smith Lab). We used four in-house derived primed hPSC lines to generate feeder-free naive hPSC. After resetting, the cells were cultured in PXGL conditions on LN521 and passaged regularly into a single cell suspension. Two female hPSC lines (VUB14 and VUB26) produced hPSC with naive characteristics. The cells showed expression of naive pluripotency markers KLF17, NANOG and SUSD2 through RT-qPCR while 78.0% of VUB14 cells were SUSD2+/CD75+ and 82.1% VUB26 cells were SUSD2+/CD75+ using flow cytometry at passage 20. Both VUB14 and VUB26 showed presence of KLF17 through immunostaining and differentiated successfully towards primitive endoderm showing GATA4+/NANOG- cells. We also tested genome stability of the reset lines through CNV assays for chromosomal aberrations common in hPSC cultures and found a 20q11.21 duplication for VUB14 and a 12p duplication for VUB26. We hereby report a novel and standardized protocol for the derivation and sustained culture of naive hPSC importantly on a cell-free matrix, without the need for MEFs. Future experiments include resetting more cell lines towards naïve pluripotency on a LN521 matrix as well as investigating their potential to generate blastoids in comparison to MEF derived naïve hPSC.
Funding Source: This research is funded by FWO11H9825N.
