Direct comparison of distinct naive pluripotent states in human embryonic stem cells

Abstract

Until recently, human embryonic stem cells (hESCs) were shown to exist in a state of primed pluripotency, while mouse embryonic stem cells (mESCs) display a naive or primed pluripotent state. Here we show the rapid conversion of in-house-derived primed hESCs on mouse embryonic feeder layer (MEF) to a naive state within 5-6 days in naive conversion media (NCM-MEF), 6-10 days in naive human stem cell media (NHSM-MEF) and 14-20 days using the reverse-toggle protocol (RT-MEF). We further observe enhanced unbiased lineage-specific differentiation potential of naive hESCs converted in NCM-MEF, however, all naive hESCs fail to differentiate towards functional cell types. RNA-seq analysis reveals a divergent role of PI3K/AKT/mTORC signalling, specifically of the mTORC2 subunit, in the different naive hESCs. Overall, we demonstrate a direct evaluation of several naive culture conditions performed in the same laboratory, thereby contributing to an unbiased, more in-depth understanding of different naive hESCs.

Figure 1. Conversion of primed hESCs towards a naive state in different naive media combinations.

(a) Overview of small molecules and growth factors supplemented in NCM, NHSM and RT naive media. (b) Morphological analysis of hESC cultures in different naive media and their transition from primed pluripotency-specific flat morphology towards dome-shaped undifferentiated naive colonies. NCM and NHSM media comprised of several domed colonies whereas their frequency was lower in RT media. Scale bars, 200 μm.

Figure 2. Assessment of naive pluripotency in the newly formed naive hESCs from primed hESC line UGent11-70.

(a) Doubling time and single-cell clonogenicity assays for primed hESCs, NCM-, NHSM- and RT-naive hESCs. a* compared to a and b* compared to b, *P<0.05. (b) Depiction of chromosomal analysis for primed hESCs, NCM-, NHSM- and RT-naive hESCs via array comparative genome hybridization (aCGH). (c) Representative immunofluorescence image for pluripotency makers OCT4 and NANOG in NCM-, NHSM- and RT-naive hESCs; Scale bars, 200 μm. (d) qRT-PCR analysis for ectoderm, mesoderm and endoderm markers on spontaneously differentiated primed hESCs, NCM-, NHSM- and RT-naive hESCs as EBs for 14 days. (e) Gene expression analysis for naive-pluripotency specific genes on undifferentiated primed hESCs, NCM-, NHSM- and RT-naive hESCs. a* compared to a and b* compared to b, *P<0.05. The data are represented as mean±s.d.

Figure 3. Germ-layer specific differentiation of naive hESC line UGent11-70.

Gene expression analysis for lineage-specific genes upon directed differentiation of naive hESC line UGent11-70 towards (a) ectoderm, (b) mesoderm and (c) endoderm. All germ layer markers were analysed irrespective of the targeted lineage to determine the heterogeneity in differentiation between the different naive medium conditions as well as between primed and naive conditions. Dashed box represents the gene expression for markers specific for that lineage. The data are represented as mean±s.d.

Figure 4. Directed differentiation of primed and naive hESCs towards neuronal and cardiac lineages.

(a) Representative images of primed, NCM-, NHSM- and RT-MEF naive hESCs expressing β-TUBULIN when differentiated towards neuronal lineage. β-TUBULIN is expressed higher in neurons derived from primed and RT-MEF naive hESCs compared to NCM- and NHSM-MEF naive hESCs. (b) Representative images of primed, NCM-, NHSM- and RT-MEF naive hESCs expressing mature cardiac markers TROPONIN and MYOSIN when differentiated towards cardiomyocytes. Primed hESCs when differentiated towards cardiac lineage expressing markers TROPONIN and MYOSIN whereas all naive hESCs fail to express the mature markers. Scale bar, 200 μm.

Figure 5. Genome-wide expression analysis of parental primed hESC and converted naive hESC in different conversion conditions.

(a) PCA based on normalized transciptome data. (b) PCA based on rlog values of the differentially expressed genes (FC> 2, FDR<0.05) between primed hESC and all naive hESC. (c) Hierarchical clustering and heatmap of differentially expressed transcriptome data between parental primed and all naive hESCs.

Figure 6. Panel of genes of interest.

(a) Genes involved in PI3K/AKT/mTORC pathway. (b) Genes related to naive pluripotency upregulated in converted naive hESCS. (c) Genes related to lineage specification downregulated in converted naive hESCs. The data are represented as mean±s.e.m.