Chemically defined conditions for long-term maintenance of pancreatic progenitors derived from human induced pluripotent stem cells

Abstract

Large numbers of hormone-releasing cells, approximately 10⁹ endocrine cells, are required to treat type I diabetes patients by cell transplantation. The SOX9-positive pancreatic epithelium proliferates extensively during the early stages of pancreatic development. SOX9-positive pancreatic epithelium is thought to be an expandable cell source of β cells for transplantation therapy. In this study, we attempted to expand pancreatic progenitors (PPs: PDX1⁺/SOX9⁺) derived from four human iPSC lines in three-dimensional (3D) culture using a chemically defined medium and examined the potential of the derived PPs to differentiate into β-like cells. PPs from four human iPSC lines were maintained and effectively proliferated in a chemically defined medium containing epidermal growth factor and R-spondin-1, CHIR99021, fibroblast growth factor-7, and SB431542. PPs derived from one iPSC line can be expanded by more than 10⁴-fold in chemically defined medium containing two of the fives, epidermal growth factor and R-spondin-1. The expanded PPs were also stable following cryopreservation. After freezing and thawing, the PPs proliferated without a decrease in the rate. PPs obtained after 50 days of culture successfully differentiated into insulin-positive β-like cells, glucagon-positive α-like cells, and somatostatin-positive δ-like cells. The differentiation efficiency of expanded PPs was similar to that of PPs without expansion culture.

Figure 1

EGF and RSPO1 synergistically induced the proliferation of hiPSC-PPs in short-term culture. (a) Representative results of flow cytometry analyses for PDX1 and SOX9 (right panel) or PDX1 and Ki67 (left panel). hiPSCs 253G1 line were differentiated into PPs (Stage 4) and then stained with antibodies. (b) Phase-contrast images of cell aggregates of human iPSC (253G1 cell)-derived PPs. Cell aggregates were cultured for 6 days in the presence of EGF and/or RSPO1. (c) Fold-change in cell number within 12 days (mean ± standard deviation, n = 3, *p < 0.01, Tukey’s HSD test). On day 6, cell aggregates were subcultured. The results were expressed as fold-change relative to the initial cell numbers. (d) Representative results of flow cytometry analyses for PDX1 and SOX9 (upper panel) or PDX1 and Ki67 (bottom panel). Cell aggregates were cultured for 6 days and then stained with antibodies. (e) Summary of flow cytometry analyses (mean ± standard deviation, n = 3, **p < 0.01, *p < 0.05, Tukey’s HSD test). (f) Immunofluorescent micrographs of thin sections of cell aggregates cultured in microwells with or without EGF + RSPO1 for 6 days. Cells were immunologically stained with antibodies targeting pancreatic and cell proliferation markers. Cell nuclei were stained with Hoechst 33258. Inserts: high magnification images of single and double stained cells. Scale bars = 100 μm.

Figure 2

Long-term maintenance of PPs derived from hiPSCs (253G1 cells). (a–e) 253G1 cell-derived PPs were maintained in PP-GF for long-term analysis. Black circles indicate each passaging point. Cells were subcultured every 6 days. (a) Phase-contrast images of cell aggregates during long-term culture (passage 5). Top panel: image of cell aggregates cultured in microwell, bottom panel: image of cell aggregates retrieved from microwell. (b) Fold-change in cell number (n = 4). Circles indicate each passaging point. The results were expressed as fold-change relative to the initial cell numbers. (c) Representative results of flow cytometry analyses for PDX1 and SOX9 (upper panel) or PDX1 and Ki67 (bottom panel) though long-term culture. P0–P9: passage numbers. (d) Summary of flow cytometry analyses (mean ± standard deviation, n = 3, **p < 0.01, *p < 0.05, compared to P0, Tukey’s HSD test). (e) Fluorescent micrograph of thin sections of cell aggregates after long-term culture. Cells were stained with antibodies targeting pancreatic markers. Inserts: high-magnification images of single and double stained cells. Scale bars = 100 μm. (f) Summary of qPCR analyses. Expression levels were normalized to GAPDH expression (mean ± standard deviation, biological replicates, n = 3). PPs before expansion (P0) and after three (P3), six (P6), and nine passages (P9) were used for the study. *p < 0.05 compared to P0, Tukey’s HSD test.

Figure 3

Differentiation into endocrine cells. After long-term culture, PPs were differentiated into endocrine cells. (a) Representative results of flow cytometry analyses for pancreatic hormones. Left: results of before maturation culture, middle: results of after maturation culture, right: results of after maturation culture without expansion culture. Cells were stained with antibodies targeting endocrine markers. INS: insulin, GCG: glucagon, SST: somatostatin. (b) Fluorescent micrograph of thin sections of cell aggregates after maturation culture. Cells were stained with antibodies targeting endocrine markers. Inserts: high-magnification images of single and double stained cells. Scale bars = 100 μm. (c) Representative results of flow cytometry analyses for C-peptide and NKX6.1. After maturation culture, the cells were fixed and stained. (d) C-peptide secretion from differentiated cells in response to 2.5 or 22.5 mM glucose. With (P4: n = 1, P7: n = 1, P10: n = 2) or without (P0, n = 3) expansion culture, cells were differentiated into endocrine cells and exposed to glucose solution.

Figure 4

Expansion of PPs derived from various hiPSC lines. 253G1, RPChiPS771-2, and P11025 cell-derived PPs were cultured in improved PP-GM (EGF + RSPO1 + CHIR99021 + FGF7). (a) Fold-change in cell numbers (253G1: n = 2, 771-2: n = 3, P11025: n = 2). (b) Phase-contrast images of PPs after long-term culture (P9). (c) Fluorescent micrographs of thin sections of cell aggregates after long-term culture. Cells were stained with antibodies targeting PDX1/SOX9 or PDX1/Ki67. (d) Representative results of flow cytometry analyses for PDX1/SOX9 (upper panel) or PDX1/Ki67 (bottom panel) after long-term culture. Scale bars = 100 μm. (e,f) Maturation of various hiPSC-derived PPs into endocrine cells. PPs were expanded in the improved PP-GM (EGF + RSPO1 + CHIR99021 + FGF7) for long-term culture (P9) and differentiated into endocrine cells. (e) Fluorescent micrograph of cell aggregates after maturation culture. After maturation culture, cells were cultured on iMatrix-511-coated substrate for 2 days and stained with antibodies targeting endocrine markers. Cell nuclei were stained with Hoechst 33258. Scale bars = 100 μm. (f) Representative results of flow cytometry analyses for C-peptide and NKX6.1.