Differentiated human stem cells resemble fetal, not adult, β cells

Abstract

Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells.

Keywords: MARIS; beta cells; differentiation; transcriptional profiling.

Fig. 1.

RNA profiling of sorted hPSC-derived insulin-expressing cells. (A) FACS plot of stage-6 H1-derived cells sorted for insulin-APC. (B) Quantitative RT-PCR of unsorted and insulin-sorted stage-6 hPSC-derived cells for pancreatic hormone genes INS (insulin), GCG (glucagon), SST (somatostatin), PPY(pancreatic polypeptide), and GHRL (ghrelin) suggests significant enrichment of mRNA specific for pancreatic hormones in the insulin-APC sorted population (*P < 0.05, **P < 0.01). (C) Three human pluripotent stem cell lines, HUES8, H1, and iPS-17b, were differentiated to stage 6 and sorted for INS⁺ cells. RNA was isolated from undifferentiated cells, stage-6 cells, and sorted INS⁺ cells for all three cell lines. Global gene expression for each sample was analyzed using the Illumina microarray platform. Hierarchical clustering identified three major groups of samples. Lengths in the dendrogram represent correlation value. Approximately unbiased (AU) P values are displayed. INS⁺ cells from different cell lines form a statistically significant cluster. (D) r² values based on microarray data across all genes are shown. The average r² value between stage-0 cells, 0.94, is similar to the average r² value between sorted INS⁺ cells, 0.93. ins, insulin⁺ MARIS-sorted stage-6 differentiated, pluripotent stem cells; S0, unsorted, undifferentiated pluripotent stem cells; S6, unsorted stage-6 differentiated pluripotent stem cells.

Fig. 2.

Human β-cell maturation. (A) FACS plots of human adult islets and human fetal pancreata sorted for INS⁺ cells (APC⁺). (B) Differentially expressed transcription factors between adult and fetal β cells. (C) Relative expression of UCN3 in mouse and human fetal and adult β cells. Expression normalized to fetal levels in each species. (D) Top five most significant (Benjamini q value) Gene Ontology biological processes relatively enriched in either adult or fetal β cells.

Fig. 3.

hPSC-derived insulin-expressing cells resemble human fetal β cells. (A) Glucose stimulated insulin secretion of dispersed cells. In contrast to adult β cells, fetal β cells and hPSC-INS⁺ cells both seem functionally immature, as indicated by increased basal glucose secretion and lack of glucose stimulation. (B) Hierarchical clustering based on microarray global gene expression across all genes indicated that hPSC-INS⁺ cells cluster closely with human fetal and not adult β cells. Numbers in parentheses indicate biological replicates. Lengths in the dendrogram represent correlation distances. (C) r² values based on microarray data across all genes are shown. Each row and column represents one sample. r² values between biological replicates of adult β-cell samples (Adult_ins) are on average 0.89 ± 0.04. r² values between sorted hPSC-derived insulin⁺ stage-6 cells and sorted fetal β cells are 0.88 ± 0.02. The biological variation between adult β cells is not statistically smaller than the variation between fetal β cells and hPSC-INS⁺ stage-6 cells (P = 0.49). This indicates a high degree of similarity between hPSC-INS⁺ cells and human fetal β cells. HUES8_ins, H1_ins, and iPS_ins are hPSC-derived MARIS-sorted stage-6 INS⁺ cells; Fetal_ins are MARIS-sorted INS⁺ cells from week-16 human fetal pancreata; Adult_ins are MARIS-sorted INS⁺ cells from islet preparations of adult human pancreata.

Fig. 4.

Differential expression between human β cells and hPSC-derived insulin-expressing cells. (A) The list of 152 pancreatic lineage genes is colored for genes that are differentially overexpressed in adult β cells (red) or hPSC-INS⁺ cells (blue). Differential gene expression was calculated based on microarray data between human adult β cells and hPSC-INS⁺ cells (greater than threefold change, P < 0.05) and confirmed by RNA-seq between HUES8-INS⁺ cells and adult β cells (greater than threefold change). Asterisk indicates genes that are also differentially expressed between fetal and adult β cells. (B) Immunofluorescence. hPSC-INS⁺ cells with PDX1, NKX6-1, and MAFA. (Scale bars, 100 μm.) (C) Relative gene expression of selected differentially expressed genes normalized to expression in hES-INS⁺ cells. ARX, FOXA1, IRX2, MNX1, NKX6-1, and PDF are expressed at similar levels between fetal and adult β cells but differentially expressed in hES-INS⁺ cells. PAX4, CHGB, KCNK1, and KCNK3 are expressed at similar levels between hES-INS⁺ cells and fetal β cells but differentially expressed in adult β cells. (D) Top 10 most significant (Benjamini q value) gene ontology biological processes enriched hPSC-INS⁺ cells over adult β cells. (E) A list of 42 transcription factors that were differentially expressed based on microarray data between human adult β cells and hPSC-derived INS⁺ cells (greater than threefold change, P < 0.05) and confirmed by RNA-seq between HUES8-INS⁺ cells and adult β cells (greater than threefold change).