In vivo reprogramming of adult pancreatic exocrine cells to beta-cells

Abstract

One goal of regenerative medicine is to instructively convert adult cells into other cell types for tissue repair and regeneration. Although isolated examples of adult cell reprogramming are known, there is no general understanding of how to turn one cell type into another in a controlled manner. Here, using a strategy of re-expressing key developmental regulators in vivo, we identify a specific combination of three transcription factors (Ngn3 (also known as Neurog3) Pdx1 and Mafa) that reprograms differentiated pancreatic exocrine cells in adult mice into cells that closely resemble beta-cells. The induced beta-cells are indistinguishable from endogenous islet beta-cells in size, shape and ultrastructure. They express genes essential for beta-cell function and can ameliorate hyperglycaemia by remodelling local vasculature and secreting insulin. This study provides an example of cellular reprogramming using defined factors in an adult organ and suggests a general paradigm for directing cell reprogramming without reversion to a pluripotent stem cell state.

Figure 1.. A combination of three transcription factors induces insulin⁺ cells in adult mouse pancreas in vivo.

a, Schematic diagram of experimental strategy. Adenoviruses encoding bicistronic transcription factor (T.F.) and nuclear GFP (nGFP) via an IRES element (I) were injected into the pancreas of an adult mouse (Rag−/−). b, Wild type (WT) pancreas is predominantly exocrine tissue with insulin⁺ β-cells in islet (outlined). Nuclei stained blue with DAPI. c, One month after infection with a combination of Ngn3, Pdx1, and MafA viruses (pAd-M3), numerous insulin⁺ cells appear outside islets. d, e, Quantification of induction one month after infection. M9, M6: mixture of 9 and 6 different viruses, respectively. Data presented as mean ± s.d. n=3 animals. ~1,000 nGFP⁺ cells/animal. Asterisk, P < 0.05; two asterisks, p < 0.01; three asterisks, p < 0.001.

Figure 2.. Induced new β-cells originate from differentiated exocrine cells.

a, 10 days after nGFP viral infection, most infected cells are Amylase⁺ (Amy) mature exocrine cells, not duct cells (Ck19⁺). b, Quantification of nGFP infected cell types. Data presented as mean ± s.d. n=3 animals. ~1,000 nGFP⁺ cells/animal. c, Mature exocrine cells are labeled with βgalactosidase (βgal) in double heterozygous Cpa1CreER^T2;R26R adults with tamoxifen injection, reprogramming is subsequently induced by infection with pAd-M3. d-f, 10 days after infection, many βgal⁺insulin⁺ cells (arrows) are present. e and f are red (insulin)/green(GFP) and blue(βgal) channels of d, respectively.

Figure 3.. Endogenous and induced β-cells are indistinguishable in morphology and ultrastructure.

a, b, Islet β-cells (a, arrowheads) and induced β-cells (b, arrowheads) are similar in size and shape but distinctly different from exocrine cells (a, b, arrows). Ecadherin staining was used to visualize cell boundaries. c, Size comparison of exocrine cells (white bar), islet β-cells (gray bar), and induced β-cells (black bar). Data presented as mean ± s.d. n=3 animals. >100 cells/animal. Three asterisks: p < 0.001. d, Electron micrograph of a β-cell (outlined) in an islet. e, Example of an induced β-cell situated between two exocrine cells. Endogenous and induced β-cells contain small insulin granules (In) and lack zymogen granules (Zy) of exocrine cells and extensive endoplasmic reticulum (ER). Nuc: nucleus. Scale bar: 2um.

Figure 4.. Molecular marker characterization of induced β-cells.

a-f, One month after infection with pAd-M3, most Insulin⁺ induced β-cells coexpress endocrine genes: glucose transporter 2 (Glut2, a), glucokinase (GCK, b), prohormone convertase 1/3 (PC1/3, c), and β-cell transcription factors NeuroD (d), Nkx2.2 (e), and Nkx6.1 (f). Arrowheads indicate examples of cells that express marker genes but not insulin. g-i, Induced β-cells do not express Amylase (g), glucagon (h), or somatostatin/pancreatic polypeptide (j, SomPP). cPPt: c-peptide.

Figure 5.. Induced new β-cells remodel vasculature and ameliorate hyperglycemia.

a-c, New β-cells synthesize vascular endothelial growth factor (VEGF) (a), and induce local angiogenic remodeling (b). Note the close proximity of blood vessels (PECAM⁺) with the reprogrammed β-cells (b) versus control infected cells (c). d, Improvement of fasting blood glucose level in diabetic mice after injection with pAd-M3 (diamond), compared with controls with nGFP virus (square). Triangle: non-diabetic controls. STZ: streptozotocin. Arrows indicate timing of injection. n= 6-8 animals. e, non-fasting serum insulin levels 6 weeks after injection. n= 6-8 animals. f, average insulin⁺ β-cell number per section 8 weeks after injection. n= 3 animals. Both islet and reprogrammed β-cells were counted for the pAd-M3 samples. One asterisk, p < 0.05; two asterisks, p < 0.01; three asterisks, p < 0.001. Data presented as mean ± s.d.