Derivation of Naïve Human Embryonic Stem Cells Using a CHK1 Inhibitor

Abstract

Embryonic development is a continuum in vivo. Transcriptional analysis can separate established human embryonic stem cells (hESC) into at least four distinct developmental pluripotent stages, two naïve and two primed, early and late relative to the intact epiblast. In this study we primarily show that exposure of frozen human blastocysts to an inhibitor of checkpoint kinase 1 (CHK1) upon thaw greatly enhances establishment of karyotypically normal late naïve hESC cultures. These late naïve cells are plastic and can be toggled back to early naïve and forward to early primed pluripotent stages. The early primed cells are transcriptionally equivalent to the post inner cell mass intermediate (PICMI) stage seen one day following transfer of human blastocysts into in vitro culture and are stable at an earlier stage than conventional primed hESC.

Keywords: CHK1 inhibitor; Epigenetic; Naïve; PICMI; Primed; hESC.

Fig. 1

Analysis of new hESC lines generated with CHK1 inhibition, A. Analysis of CHK inhibitors for survival of Elf1 naïve vs. primed cells. Viability following exposure to the CHK inhibitors was assessed by luminescence using CellTiter Glo for ATP content. B. Expression of naïve and primed markers. qRT-PCR was performed for naïve markers (DNMT3L, NLRP7, PVR and IL6ST) and primed markers (CD24, THY7, BG3) derived from published data [31, 32]. Expression of these genes is shown in newly generated hESC lines (Elm2, Elf3, Elf4 and Elf5), WIN1 [11], Elf1 [12] and HNES1 [13] cultured in ground naïve (5iLA) or late naïve (2iLIF; 4iL), and compared to primed TeSR. The fold changes of expression of markers in naïve conditions (ground or late) versus primed conditions is indicated for each cell line. Error bars indicate the SEM of 3 independent replicates. *** < 0.001. Two-way ANOVA was used to assess whether there are significance differences in means due to the culture conditons (i.e. mean of early naïve vs. mean of late primed and mean of late naïve vs. mean of late primed). C. Principal component analysis of RNA-seq from various studies [10, 11, 13, 14, 23, 30, 32, 34] and Elf3 and Elf4 (this study) reveal 4 main pluripotency stages. Batch effect adjustment using ComBat-seq was applied on the combined RNA-seq dataset. D. Cells cultured in 2iLIF ± PKCi or CHKi maintain the H19 imprint while 5iLA cells irreversibly erase the imprint as seen upon differentiation. Sanger sequencing traces of Elf1 genomic DNA for H19 containing allele-specific SNP (rs217727, indicated by an arrow) and H19 cDNAs of Elf1 cells cultured in various conditions: 5iLA, 2iLIF, 2iLIF + PKCi, 2iLIF + CHKi, T and cells pushed to undirected differentiation from 5iLA or T. Y = pyrimidine (either C or T nuclotide). Graphical summary of the data was generated using BioRender

Fig. 2

G-banded karyotype analysis of newly generated hESC lines, A. G-banded karyotype analysis was performed in naïve hESC (Elf1, Elm2, Elf3, Elf4, Elf5 cultured in 2iLIF and HNES1 cultured in 4iL) Elf5 (PKCi 1passage were established in 4iL and transferred to 2iLIF upon the second passage). Denotations of HNES1 passage number follows the convention of labeling from the Nichols lab from which they were obtained with a passage number change up from the second number upon each passage in 4iL. Elf1 number of passages in inhibitors is denoted as the number of passages in the particular inhibitor, e.g. “Elf1 with CHK1i (5 pass.) indicates Elf1 with a normal karyotype subsequently passaged 5 times in the presence of CHK1i. The final analysis of karyotype is printed below the cell line details. B. Table showing ploidy effects of culture in CHK1i or PKCi. Naïve hESC Elf1 [12], Elm2, Elf3, Elf4 and Elf5 were cultured in 2iLIF media supplemented with 0.5 mM CHK1i, 1 mM PKCi or 50 mM ZVAD for the indicated number of passages prior to G-banded karyotype analysis. Five metaphases for each culture were assessed