Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing β-like cells

Abstract

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.

FIG. 1.

Pancreatic exocrine fractions dedifferentiate toward mesenchymal cells in culture. A: Phase contrast images of exocrine fractions when cultured in tissue culture dishes over a 14-day period. Scale bar = 50 µm. B: QRT-PCR analysis of pancreatic, epithelial, and mesenchymal markers in cultured exocrine fractions from day 2 up to passage 7 after plating in tissue culture dishes. Data are presented as mean ± SEM. Expression levels are relative to glyceraldehyde 3-phosphate dehydrogenase (n = 3). AML, amylase; a-SMA, α-smooth muscle actin; D, day; ECAD, E-cadherin; EPCAM, epithelial cell adhesion molecule; INS, insulin; P, passage; SNAI2, snail homolog 2; VIM, vimentin.

FIG. 2.

Dedifferentiation of pancreatic exocrine fractions is accompanied by EMT. Immunocytochemistry of plated pancreatic exocrine fractions was performed from day 2 to day 18 in culture. The pancreatic exocrine markers amylase (AML) and CK19, the epithelial marker E-cadherin (ECAD), and the mesenchymal marker vimentin (VIM) expressions were analyzed on days 2, 4, 10, and 18 of culture. Nuclei were counterstained with DAPI. Scale bar = 20 μm.

FIG. 3.

Genetic lineage tracing of acinar amylase-positive cells in pancreatic exocrine fractions. A: Schematic representation of the two viral vectors used for tracing amylase-positive cells. B: The dsRed-positive cells were monitored in culture for a 10-day period. The dsRed fluorescence (top row) and brightfield images (bottom row) were analyzed at days 4, 6, 8, and 10. Scale bar = 50 µm. C: Immunocytochemistry was performed on days 3, 7, and 10 on traced amylase-positive cells. The exocrine pancreatic markers amylase (AML) and CK19 were analyzed along with the mesenchymal marker vimentin (VIM) and the proliferation marker ki67. Nuclei were counterstained with DAPI. Scale bar = 20 µm.

FIG. 4.

Ectopic expression of pancreatic TFs reprograms exocrine pancreatic MSCs into glucagon-positive cells. A: Passaged human exocrine pancreatic fractions were plated in tissue culture dishes and subsequently transduced with different combinations of adenoviruses expressing the pancreatic TFs Pdx1 (P), MafA (M), Pax4 (Px), and Ngn3 (N), each with a multiplicity of infection of 100. After 7 days, QRT-PCR was performed and the data were expressed relative to glyceraldehyde 3-phosphate dehydrogenase and presented as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. INS, insulin; GLC, glucagon; SST, somatostatin; N/A, nontreated samples. B: Exocrine cells treated with different combinations of adenoviruses expressing P, M, and N in the presence or absence of Px were stained for the expression of glucagon, and cell nuclei were counterstained with DAPI. Scale bar = 20 µm.

FIG. 5.

SFM and chromatin-modifying reagents enhance reprogramming of exocrine pancreatic MSCs toward glucagon-producing cells. A: Representative phase contrast images of passaged exocrine pancreatic fractions cultured in serum-containing medium (SCM) or in SFM. Scale bar = 50 µm. B: Passaged exocrine pancreatic cells were cultured in SCM or SFM and transduced with adenoviruses expressing the 4TFs. After 24 h, 1 nmol/L betacellulin, 10 nmol/L exendin-4, and 10 mmol/L nicotinamide (BEN) were added to both SCM and SFM cultures. After 7 days, the cells were harvested and QRT-PCR was performed. The data are expressed relative to glyceraldehyde 3-phosphate dehydrogenase and presented as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. C: Passaged exocrine pancreatic cells were cultured in SFM supplemented with 1 μmol/L 5-Aza-2′deoxycytidine (A) and/or 1 mmol/L sodium butyrate (Bu), transduced with adenoviruses (4TF), and treated with BEN as indicated. QRT-PCR was performed and the data were expressed relative to glyceraldehyde 3-phosphate dehydrogenase and presented as mean ± SEM (n = 3). ***P < 0.001 relative to nontreated samples (N/A). D: Glucagon secretion in culture medium of N/A or reprogrammed (A + Bu + 4TFs + BEN) exocrine cells in the presence of basal (2.5 mmol/L) or after stimulation for 1 h with high (20 mmol/L) glucose. Glucagon levels were measured by ELISA and data represent the mean ± SEM (n = 3). *P < 0.05. E: Glucagon and C-peptide immunofluorescent staining in exocrine cells treated with 4TFs and BEN in the presence of 5-Aza-2′deoxycytidine and sodium butyrate. Scale bar = 20 µm. GLC, glucagon; INS, insulin; SST, somatostatin.

FIG. 6.

The Rho-kinase and TGF-β1 pathway inhibitors suppress dedifferentiation of cultured pancreatic exocrine cells. A: Unpassaged exocrine pancreatic cells were plated in tissue culture dishes. After 48 h to allow attachment, cells were untreated (N/A) or treated with 2 μmol/L ρ-kinase inhibitor Y27632 (Y) and 10 μmol/L TGF-β1 inhibitor SB431542 (S) individually or in combination, and the cells were incubated for another 5 days. Treated and N/A cells as well as baseline samples after 48 h in culture (day 0) were then harvested and RNA was extracted for QRT-PCR analysis for expression of pancreatic, epithelial, and mesenchymal markers. Data are expressed relative to glyceraldehyde 3-phosphate dehydrogenase and presented as mean ± SEM (n = 3). A one-way ANOVA was performed with Dunnet post hoc test comparing treatment groups with N/A. A t test was used to compare day 2 with Y + S. For all analyses, *P < 0.05, **P < 0.01. B: Immunocytochemistry for the pancreatic markers amylase (AML) and Pdx1, the epithelial marker E-cadherin (ECAD), and the mesenchymal marker vimentin (VIM) in cultured exocrine pancreatic cells after 10 days in the presence of Y27632 and SB431542 (Y+S). N/A cells also were analyzed for the same markers. Nuclei were counterstained with DAPI. Scale bar = 20 μm. EPCAM, epithelial cell adhesion molecule; GLC, glucagon; INS, insulin; SST, somatostatin.

FIG. 7.

The Rho-kinase and TGF-β1 pathway inhibitors enhance reprogramming toward insulin-producing cells. A: Unpassaged exocrine pancreatic cells were plated in tissue culture dishes. The cells were then cultured for 72 h in SFM containing Y27632 (Y), SB431542 (S), 5-Aza-2′deoxycytidine (A), and sodium butyrate (Bu). They were then transduced with adenoviruses expressing the 4TFs and cultured for 7 days in SFM containing 1 nmol/L betacellulin, 10 nmol/L exendin-4, and 10 mmol/L nicotinamide (BEN). Treated and untreated (N/A) cells then were harvested and RNA was extracted for QRT-PCR analysis. Data are represented as mean ± SEM and expressed relative to glyceraldehyde 3-phosphate dehydrogenase. *P < 0.05 or **P < 0.01 relative to N/A samples. B: Release of C-peptide to the medium in transdifferentiated (S+Y+A+Bu+4TFs+BEN) and N/A cells after incubation with 2.5 or 20 mmol/L of d-glucose for 1 h. The dashed line indicates the assay detection limit. Data are representative of triplicate experiments. ***P < 0.001. C: Insulin content of transdifferentiated (S+Y+A+Bu+4TFs+BEN) and N/A cells normalized to the DNA content of each sample. Data are representative of triplicate experiments. *P < 0.05. D: Immunostaining for insulin (INS) (panels a–c and g–i), C-peptide (C–PEP) (panels d–f), Pdx1 (panels g–i), and glucagon (GLC) (panels d–f) of transdifferentiated cells in culture. Nuclei were counterstained with DAPI. Data are representative of triplicate experiments. Inlets show a 2× higher magnification image of stained clusters. Scale bar = 50 µm. AML, amylase; E-CAD, E-cadherin; SST, somatostatin.

FIG. 8.

Reprogrammed insulin-producing cells prevent STZ-induced diabetes in vivo. A: Body weight and blood glucose levels were measured in NOD/SCID mice grafted with transdifferentiated cells or exocrine pancreatic cells or in nongrafted mice (Ctrl) over a 38-day period after surgery. A single dose (150 mg/kg) of STZ was administered 1 day before surgery. Transdifferentiated cells, n = 5; exocrine pancreatic cells, n = 3; control, n = 2. B: Serum C-peptide levels were measured in NOD/SCID mice grafted with transdifferentiated cells or exocrine pancreatic cells and in nongrafted mice (Ctrl) after a 4-h starvation period (fast) or with ad libitum feeding (fed) conditions. Transdifferentiated cells, n = 5; exocrine pancreatic cells, n = 3; control, n = 2. C: Immunostaining for insulin and glucagon of grafted kidneys after kidney removal. Yellow dashed lines indicate the border between the kidney (k) and the graft. The red circle in panel a indicates the difference in glucagon staining observed within the cluster. A 5× higher magnification of the cells inside this circle is shown in panel c; 10× higher magnifications of the cells inside (panel e) and outside (panel f) the circle are shown. Panel d shows a 5× higher magnification of insulin staining within the area marked by the red square in panel b. A 10× higher magnification of insulin-positive cells present in the center of the cluster is shown in panel g. Scale bar for panels a and b = 100 µm. Scale bar for panels c–g = 20 µm. D: Immunofluorescent staining for Pdx1 in kidneys grafted with transdifferentiated cells. Scale bar = 50 µm. A 5× higher magnification inlet is shown. Ctrl, control; Exoc Cells, exocrine pancreatic cells; GLC, glucagon; INS, insulin; Transdif Cells, transdifferentiated cells.