Human pancreatic beta-like cells converted from fibroblasts

Abstract

Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes.

Figure 1. Conversion of human fibroblasts into definitive endodermal progenitor cells.

(a) Schematic illustrating our strategy to convert human fibroblasts (Fib) into definitive endodermal progenitor cells (cDE cells) by combining non-integrating episomal reprogramming plasmids with specific initiation and conversion conditions. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), CHIR 99021 (CHIR), 5′-N-ethylcarboxamidoadenosine (NECA), sodium butyrate (NaB), Parnate (Par) and RG108 (RG). (b) Bright-field images of control fibroblasts and a cDE colony at day 21. Scale bar, 20 μm. (c) Immunofluorescence staining of a representative cDE colony at day 21 for the endodermal progenitor markers SOX17 and FOXA2. Scale bar, 20 μm. (d) Small molecules sodium butyrate (NaB), Parnate (Par), RG108 (RG), CHIR99021 (CHIR) and 5′-N-ethylcarboxamidoadenosine (NECA) added to the basal condition further enhance endodermal reprogramming efficiency. Data represent the number of FOXA2-positive colonies scored at day 28 (mean values±s.e.m. of three experiments). Statistical significance calculated using two-tailed Student's t-test, compared with CHIR treatment. *P<0.05. (e) QPCR analyses of endodermal genes SOX17 and FOXA2, and endogenous pluripotent genes OCT4 (endoOCT4) and NANOG (endoNANOG) during the conversion process. Note that hESCs served as control. Mean value±s.e.m. are normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and relative to d28 cDE cultures and hESCs, respectively (n=3 experiments). Statistical significance calculated using two-tailed Student's t-test, compared with d28 cDE cultures and hESCs, respectively. *P<0.05, **P<0.01.

Figure 2. Specification, expansion and characterization of posterior foregut-like progenitor cells.

(a) Schematic representation of culture conditions for the amplification of cDE cells that resulted in further specification into expandable posterior foregut-like progenitor cells (cPF cells). (b) Improved culture conditions allow amplification of cDE/cPF cell colonies. Mean values±s.e.m. represent three experiments. Statistical significance calculated using two-tailed Student's t-test, compared with DMSO controls. *P<0.05, **P<0.01. (c) Immunofluorescence analysis of colonies after four passages in improved expansion media suggested specification towards cPF cells. Scale bar, 20 μm. (d) Illustration of cPF expansion strategy. (e) Growth curve of cPF cells. Data from three experiments are shown as average±s.e.m. (f) All four media supplements—EGF, bFGF, A83-01 and CHIR99021—are important for cPF cell self-renewal (n=3 experiments). Statistical significance calculated using two-tailed Student's t-test, compared with ALL supplementation. *P<0.05. (g) Bright-field image of established cPF cells showing epithelial colony morphology. Scale bar, 20 μm. (h) Immunofluorescence staining of SOX17, FOXA2, HNF4α, HNF6 and PDX1 in cPF cells at passage 15. Scale bar, 20 μm. (i) QPCR analysis demonstrates the enrichment of transcripts for SOX17, FOXA2, HNF1A, HNF1B, HNF4A, HNF6, SOX9 and PDX1, but not SOX1, BRY, OCT4 and NANOG in p15 cPF cells. Mean values±s.e.m. are normalized to GAPDH relative to control fibroblasts. (n=3 experiments). Statistical significance calculated using two-tailed Student's t-test, compared with fibroblast controls. *P<0.05, **P<0.01. (j) Immunofluorescence analysis of cPF cell grafts shows epithelial structures that express E-cadherin, HNF4α, PDX1, SOX9 and pan-cytokeratin. Human nuclear antigen (HuNu) demonstrates the human cell origin. Scale bar, 20 μm.

Figure 3. Differentiation of cPF cells into expandable pancreatic endodermal progenitor cells.

(a) Schematic strategy for the differentiation of cPF cells into pancreatic cPE cells. (b,c) Immunofluorescence staining of PE markers FOXA2, SOX9, HNF6, PDX1 and NKX6.1 in p1 cPE cells. Scale bar, 20 μm. (d) Flow cytometric analysis of PDX1 and NKX6.1 expression in p1 cPE cells. (e) Growth curve of cPE cells. Data from three experiments are shown as average±s.e.m. (f) Illustration for the expansion of cPE cells. (g) Immunofluorescence staining of PE markers PDX1 and NKX6.1 in p12 cPE cells. Scale bar, 20 μm. (h) qPCR analysis demonstrated the enrichment of NKX2.2, NKX6.1, PDX1, FOXA2, HNF4A, HNF6, HLXB9, PTF1A and NGN3, while SOX17 is downregulated in cPE cells. Mean values±s.e.m. are normalized to GAPDH and relative to cPF cells (n=3 experiments). (i) ELISA analysis reveals detectable levels of human C-peptide in serum of 67% of mice-bearing cPE cell grafts for 15 weeks 1 h after glucose challenge. Human C-peptide levels and percentage of mice exhibiting detectable levels of human C-peptide increases over time. Numbers on top of each bar indicate human C-peptide-positive mice out of total mice assayed. (j) ELISA analysis before and after glucose challenge of mice-bearing cPE cell grafts for 23–24 weeks demonstrates their functional response to glucose administration. Red line indicates values below or equal to detection limit of the ELISA assay. P value was calculated using a two-tailed Student's t-test. (k) Immunofluorescence analysis of 15-week-old cPE cell graft sections shows co-expression of insulin (INS) and the beta-cell transcription factors PDX1 and NKX6.1 but not the hormone glucagon (GCG). Scale bar, 20 μm.

Figure 4. Maturation of cPE cells into insulin-producing, glucose-responsive pancreatic beta-like cells in vitro.

(a) Schematic representation of protocol 1 differentiation strategy employed to mature cPE cells into pancreatic beta-like cells (cPB cells) in vitro. (b) Immunofluorescence staining of PDX1 and C-peptide expression in beta-like cells generated with basal pancreatic differentiation condition in vitro. Scale bar, 20 μm. Basal pancreatic differentiation media contains A83-01 (A83), Nicotinamide (NIC), Forskolin (FSK), Dexamethasone (DEX) and Exendin-4 (Ex-4). (c) Several small molecules, Compound-E (C–E), Vitamin C (Vc) and BayK-8644 (BayK) further increase the percentage of C-peptide-positive cells. Note that combined treatment of all molecules results in an additive effect, further increasing the percentage of C-peptide-positive cells (n=3 experiments). Statistical significance calculated using two-tailed Student's t-test, compared with DMSO controls. **P<0.01. (d) Immunofluorescence analysis of converted pancreatic beta-like cells (cPB cells) generated with the improved pancreatic maturation conditions. Many of the insulin (INS) positive cells co-express key beta-cell transcription factors including, PDX1, NKX6.1, NKX2.2 and NEUROD1, but only rarely co-express endocrine progenitor marker NGN3 and the endocrine hormones, glucagon (GCG) and somatostatin (SST). Scale bar, 20 μm. (e) In vitro, glucose-stimulated insulin secretion (GSIS) assays (n=7 cell cultures of 4 experiments) demonstrated that cPB cells release insulin in response to physiological levels of glucose. Depolarization by higher KCl concentration further increased insulin secretion. Note that insulin release was measured by human-specific C-peptide ELISA assay. Red line indicates values below or equal to detection limit of the ELISA assay. P value was calculated using a two-tailed Student's t-test.

Figure 5. Improved maturation of cPE cells into insulin-producing, glucose-responsive cPB cells in vitro.

(a) Schematic representation of the improved approach (protocol 2) employed to mature cPE cells into cPB cells in vitro. The improved protocol 2 consists of two candidate factors, Vitamin C (Vc) and BayK-8644 (BayK), identified by our chemical screen in conjugation with recently published protocols. (b) Addition of Vitamin C (Vc) and BayK-8644 (BayK) increases mRNA levels of INSULIN (INS) gene in differentiated cPB cultures at day 21. n=3 experiments. Statistical significance calculated using two-tailed Student's t-test, compared with DMSO controls. **P<0.01. (c) Immunofluorescence analysis of cPB cells generated with the improved pancreatic maturation conditions. Many of the C-peptide (C-pep)-positive cells co-express key beta-cell transcription factors, PDX1 and NKX6.1, but only rarely co-express other endocrine hormones, glucagon (GCG) and somatostatin (SST). Scale bar, 50 μm. (d) Flow cytometric analysis of cPB cells for human C-peptide (C-pep), glucagon (GCG) and somatostatin (SST). (e) Quantification of flow-based analysis of the percentage of single- and double-positive cells for C-pep and GCG or SST. n=3 experiments. (f) In vitro, glucose-stimulated insulin secretion (GSIS) assay (n=7 cell cultures of 3 experiments) demonstrate that cPB cells release insulin in response to physiological levels of glucose. Depolarization by higher KCl concentration further increased insulin secretion. Note that insulin release was measured by human-specific C-peptide ELISA assay. P value was calculated using a two-tailed Student's t-test. (g) Schematic of the lentiviral reporter construct employed to infect cPE cultures before differentiation into cPB cells. (h) Insulin-expressing cPB cells at day 21 were sorted based on mCherry expression. qPCR analysis of sorted cPB cells in comparison to primary human islets shows comparable expression levels of key beta-cell genes, including INS, PDX1, NKX6.1, NKX2.2, NEUROD1, PAX6, RFX6, MAFA, GCK, PCSK1, KIR6.2, SUR1, UCN3 and SLC30A8. P value was calculated using a two-tailed Student's t-test. *P<0.05, **P<0.01.

Figure 6. Transplanted cPB cells remain functional and protect mice from chemically induced diabetes.

(a) Schematic representation of the transplantation of cPB into immunodeficient mice. (b) ELISA analysis of serum from fasted and glucose-challenged mice 2 months post transplantation with either fibroblasts (Fib) or cPB are shown. cPB graft-bearing mice exhibit significant higher levels of circulating human C-peptide in serum after a glucose bolus, indicating that transplanted cPB cells remain functional in vivo. Mice transplanted with Fib controls do not exhibit circulating human C-peptide. n=12 mice for cPB and n=5 mice for Fib. P value was calculated using a two-tailed Student's t-test. (c) Immunofluorescence analysis of 2-month-old cPB cell grafts shows co-expression of C-peptide (C-pep) and the beta-cell transcription factors PDX1 and NKX6.1 but not the hormones glucagon (GCG) and somatostatin (SST). Scale bar, 50 μm. Data shown are representative of two mice. (d) Fed blood glucose levels of mice-bearing cPB grafts with circulating human C-peptide levels above 200 pM after glucose stimulation and control fibroblasts are shown. Mice were treated with the mouse-specific beta-cell toxin streptozotocine (STZ) to ablate endogenous beta cells. Uni-lateral nephrectomy of cPB graft-bearing mice 5 weeks after STZ treatment resulted in a rapid rise in blood glucose levels, directly demonstrating euglycemic control due to cPB grafts after STZ treatment in these mice. n=6 mice for cPB and n=6 mice for Fib. P value was calculated using a two-tailed Student's t-test. *P<0.05.