Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

Abstract

Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.

Graphical abstract

Figure 1

Cell Line Derivation from Dissociated Human Inner Cell Mass Cells (A) Day-6 blastocyst. (B) Trophoblast lysis. (C) Discarded trophoblast. (D) Isolated inner cell mass. (E) Decompacted ICM. (F) Dissociated ICM. (G) Primary stem cell clone grown from a single ICM cell. (H) Colony at passage 8. (I) qRT-PCR for pluripotency markers in HNES cells, conventional human PSCs (H9), and in vitro reset PSCs (Reset H9). Error bars indicate the SD of two independent reactions. (J) Immunofluorescence of pluripotency markers in HNES1 cells. (K) Immunofluorescence of KLF17 and NANOG in D6 human ICM cells. Scale bars: 25 μm.

Figure 2

Transcriptome and Methylome Analyses (A) Clustered expression data from HNES cells, and reset and conventional human PSCs for a panel of pluripotency and lineage markers selected by the International Stem Cell Initiative (Adewumi et al., 2007). Displayed are log₂ FPKM values (fragments per kilobase of exon per million reads mapped) scaled by the mean expression of each gene across samples. Published data are labeled with sample accession codes. (B) PCA of HNES cells, and reset and conventional PSCs with single-cell RNA-seq data from early human ICMs (Blakeley et al., 2015, Yan et al., 2013) and PSC explants. Embryo single-cell samples are those assigned an epiblast identity in the respective studies. (C) Pluripotency and lineage marker expression in human ICM, HNES cells, and reset and conventional PSC lines. (D) Proportion of whole-genome CpG methylation measured by bisulfite sequencing (BS-seq) analysis from three biological replicates. Error bars indicate the SD of three biological replicates. (E) Comparison of global methylation in HNES1 (male) and HNES3 (female) cells by averaging CpG methylation levels over 500-kb windows. (F) Comparisons of CpG methylation in HNES1 cells and primed derivatives, and reset H9 and ICM cells. (G) PCA of mean CpG island methylation. (H) CGI methylation in HNES1 and conventional PSCs.

Figure 3

Differentiation (A) Colonies of naive HNES1 cells in t2iLGöY and primed HNES1 cells after 12 passages in FGF/KSR. (B) qRT-PCR analysis of naive marker expression in naive HNES1 cells and derivatives after three passages in FGF/KSR. Error bars indicate the SD of two independent reactions. (C) qRT-PCR analysis of embryoid bodies formed from HNES1 and primed HNES1 cells. Error bars indicate the SD two independent reactions. (D) Immunofluorescence of embryoid body outgrowths: TuJ1, β-III tubulin; AFP, α-fetoprotein; SMA, α-smooth muscle actin (green); FOXA2 (red). Nuclei (DAPI; blue). Scale bars, 100 μm.