Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells

Abstract

Pluripotent stem cells have been proposed as an unlimited source of pancreatic β cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional β cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ β-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.

Keywords: culture conditions; directed differentiation; pancreatic development; pancreatic progenitors; self-renewal; tissue stem cells; β cell differentiation.

Figure 1

Derivation of cPP Cell Lines from hESC and hiPSC (A) Pancreatic progenitors generated after 15 days of differentiation using the STEMdiff directed differentiation kit (PPd15 cells) were plated and expanded on a layer of 3T3-J2 feeder cells in medium supplemented with the indicated growth factors and signaling inhibitors. (B) Intracellular flow cytometric analysis for PDX1 and NKX6-1 at days 8, 10, and 15 of differentiation using H9 hESCs. (C) Phase-contrast images of cPP cells passaged as aggregates (left) and as single cells (right). Scale bar, 100 μm. (D) Gene expression measured by qRT-PCR using samples harvested from PPd15 cells and cPP cells at early (6–8), middle (11–13), and late (14–18) passages. Cells were derived from both AK6-13 hiPSC and H9 hESC. Gene expression in definitive endoderm (H9 hESCs after 4 days STEMdiff differentiation) is shown for comparison. Values are plotted on a log₂ scale and error bars represent the SE of three technical replicates. ND, not detected. (E) Immunofluorescence staining of cPP cells for key pancreatic transcription factors. Scale bar, 100 μm. (F) Intracellular flow cytometric analysis of cPP cells for PDX1. Gray dots represent control cells stained with isotype control antibodies.

Figure 2

Chromosome Counting and M-FISH Analysis Reveals cPP Cells Are Genetically Stable (A) Chromosome counting of cPP cells from diverse genetic backgrounds at different passage numbers. Values shown are the percentage of spreads with a given number of chromosomes, with the modal chromosome count for each cPP line highlighted. A modal (shared by >80% of cells) chromosome number of 46 is indicative of a normal karyotype and of karyotypic stability. Five out of six cPP cell lines analyzed exhibited a modal chromosome count of 46 after >6 passages, without evidence of fragments or dicentric chromosomes, and are considered karyotypically stable. In H9 pedigree #1, cells gradually acquired an additional isochromosome upon passaging. Traditional G-band karyotyping (data not shown) subsequently found this to be i(12) (p10)[20], an isochromosome commonly observed in hESC cultures. (B) Multicolor fluorescence in situ hybridization (M-FISH) enables the detection of chromosomal structural abnormalities at significantly higher resolution than chromosome counting alone. M-FISH of passage 20 AK6-13 cPP cells failed to detect aneuploidy, translocations or deletions in 19/20 spreads analyzed. A representative image of a single chromosome spread is shown.

Figure 3

Transcriptome Analysis of cPP Cells by RNA-Seq (A) Hierarchical clustering of Euclidean distances between transcriptomes of diverse adult and embryonic tissues shows that in vitro and in vivo pancreatic progenitors exhibit similar patterns of gene expression. Log₂-transformed gene count values were used to calculate Euclidian distances. For detailed information on the sources of data used here, see Table S1. (B) Heatmaps showing log₂-transformed gene expression levels of key endodermal and pancreatic markers by in vitro and in vivo pancreatic progenitors. Levels in brain are shown for comparison. (C) Genes specifically expressed by cPP, PPd15, and CS16-18 pancreatic progenitors. The coefficient of variance (CV) for each protein-coding gene across the 25 tissues shown in (A) was plotted against the corresponding Z score (see Supplemental Experimental Procedures). Specifically expressed genes are located in the upper right-hand quadrant (CV >1 and Z score >1) and include genes with well-characterized roles in early pancreatic development (labeled). The color scale denotes the number of genes. The Venn diagram shows overlap between genes specifically expressed by cPP, PPd15, and CS16-18 pancreatic progenitors. (D) Biological process Gene Ontology (GO) terms associated with all genes specifically expressed by cPP cells (above) or genes specifically expressed by cPP cells but not PPd15 or CS16-18 pancreatic progenitors (below). Only GO terms associated with >5 genes and/or an adjusted p value <0.01 are shown. (E) Heatmap of expression levels of genes associated with the enriched GO terms mitotic recombination, DNA strand elongation, telomere maintenance, and DNA packaging. Levels are shown for individual cPP and PPd15 populations derived from three different genetic backgrounds (H9, AK6-13, and HES3) relative to the maximum detected value across the 25 different tissues shown in (A). (F) Expression of selected telomerase pathway genes as measured by qRT-PCR in cPP and PPd15 cells. Error bars represent the SE of three technical replicates.

Figure 4

A Layer of 3T3-Feeder Cells and Exogenous Signaling Molecules Are Required for the Maintenance and Expansion of cPP Cells (A) Phase-contrast images of H9 and AK6-13 cPP cells after 7 days culture in complete medium on 3T3-feeder cells plated at densities of 5 × 10⁴, 2.5 × 10⁴, and 1.25 × 10⁴ cells/cm². Scale bar, 100 μm. (B) Gene expression measured by qRT-PCR for samples harvested from cultures in (A) for endocrine (NGN3 and NKX2-2), ductal (KRT19 and CA2), and acinar (CPA1 and AMY2B) marker genes. Error bars represent the SE of three technical replicates. (C) Phase-contrast images of cPP cells cultured for 6 days in complete medium with individual components omitted. Scale bar, 100 μm. (D) PDX1 and SOX9 expression measured by qRT-PCR for samples harvested in (C). Error bars represent the SE of three technical replicates. (E) Microbioreactor array (MBA) screening of factors required to propagate PDX1+SOX9+ cPP cells. Effects of reducing or removing selected factors (EGF, RA, DAPT) from complete medium containing all factors at the following levels: EGF (50 ng/mL), RA (3 μM), DAPT (1 μM), SB431542 (10 μM), and FGF10 (50 ng/mL). Top panels: effects on total nuclei per chamber, and PDX1 and SOX9 mean nuclear intensity. Lower panels: effects on the total number of PDX1+SOX9+ cells per chamber and percentage of PDX1+SOX9+ cells. Data represent the mean of ten chambers within a column treated with the given condition ± the SE. (F) Heatmap showing RNA-seq expression levels of components of signaling pathways that regulate cPP proliferation: EGF (EGFR), FGF10 (FGFR1-4, 6 and FGFRL2), RA (RARA, RARB, RARG, RXRA, RXRB, and RXRG), SB431542 (ACVR1B [ALK4], TGFBR1 [ALK5], and ACVR1C [ALK7]), and DAPT (NOTCH1-4 and its ligands DLL1,3,4 and JAG1,2). Levels are shown relative to those observed across all 25 tissues shown in Figure 3A.

Figure 5

Testing cPP Potency In Vitro and In Vivo (A) Feeder-depleted passage 15 H9 cPP cells were replated on Matrigel and exposed to the indicated factors that promote multilineage differentiation toward the endocrine, duct, and acinar lineages. (B) Endocrine, exocrine, and ductal gene expression analysis in (A) after 3, 6, and 12 days. Values are shown relative to levels in undifferentiated cPP cells (day 0). Error bars represent the SE of three technical replicates. (C) Directed differentiation of passage 10 AK6-13 cPP cells to insulin+ β-like cells using a modified version of Russ et al. (2015). (D) Phase-contrast image of differentiating spheres undergoing branching morphogenesis after 4 days. Scale bar, 100 μm. (E) Intracellular flow cytometric analysis of day 4 cells shows approximately 70% reactivate NKX6-1 and maintain PDX1. (F) PDX1 and NKX6-1 immunostaining on day 4. Scale bar, 100 μm. (G) On day 9, the majority of cells are NKX2-2+ with a proportion of these transiently NGN3+. Scale bar, 100 μm. (H) Phase contrast image of day 16 spheres. Scale bar, 100 μm. (I) Approximately 20% of cells are C-peptide+ on day 16. (J) Day 16 C-peptide+ cells do not coexpress glucagon. Scale bar, 100 μm. (K) Gene expression measured by qRT-PCR of cPP cells on days 4, 9, and 16 harvested from the differentiation protocol in (C). Levels are shown relative to those in undifferentiated cPP cells and human islets for comparison. Error bars represent the SE of three technical replicates. (L) Immunostaining of transplanted cPP cells for markers of endocrine (C-peptide and glucagon), duct (keratin-19), and acinar (trypsin) lineages. Scale bar, 100 μm.