Immunohistochemical characterization of pancreatic duodenal homeobox protein-1, neurogenin-3 and insulin protein expressions in islet-mesenchymal cell in vitro interactions from injured adult pancreatic tissues: a morphochronological evaluation

Document Type: Original Article


Islet and MSK Research Group, Anatomy and Histology, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Western Cape, South Africa


Objective(s): The use of a co-culture of islets with mesenchymal stromal cells (MSCs) is a promising therapy in islet transplantation to revert hyperglycaemia, but the resulting insulin-producing cells (IPCs) express low levels of pancreas endocrine developmental genes. This study aims to investigate the morphochronology of a co-culture of islets with MSCs from injured adult pancreata, and characterize pancreatic duodenal homeobox protein-1 (Pdx1), neurogenin-3 (Ngn3) and insulin protein expressions to establish the fate of their interaction.
Materials and Methods: Islets and MSCs were isolated from sham operated control (SOC) and duct-ligated (PPDL) pancreata. Islets from SOC or PPDL tissues were cultured with or without MSCs in RPMI1640, supplemented by 1% Penicillin-Streptomycin, and maintained at 37 °C±1 °C at 95% relative humidity and 95% /5% air/CO2. Pdx1, Ngn3 and insulin expressions were determined by immunohistochemistry and islet morphochronological changes were assessed.
Results: Pdx1 was expressed in all islet-cell cultures with or without MSCs. Pdx1+ islet cells were significantly increased in the presence of MSCs compared to the islet culture without MSCs. Similarly, Ngn3 was highly expressed in all cultures with MSCs from both SOC and PPDL tissues and the expression was prolonged in cultures using PPDL tissues before it was down-regulated, thereby, extending the period of Ngn3+ cell expansion and differentiation into mature functional islets.
Conclusion: In vitro, MSCs maintain a pool of Ngn3+ that contributes to insulin production from mature beta cells but the activation of insulin production from non-beta cells may not be induced by direct signals from MSCs.


Main Subjects

1. Chintinne M, Stangé G, Denys B, Ling Z, Pipeleers D. Beta cell count instead of beta cell mass to assess and localize growth in beta cell population following pancreatic duct ligation in mice. PLoS One 2012;7:e43959.
2. Xu X, D’Hoker J, Stange G, Bonne S, Leu N De, Xiao X, et al. β cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell 2008;132:197–207.
3. Tchokonte-Nana V. Cellular mechanisms involved in the recapitulation of endocrine development in the duct ligated pancreas. Stellenbosch: University of Stellenbosch; 2011.
4. Inada A, Nienaber C, Katsuta H, Fujitani Y, Levine J, Morita R, et al. Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth. Proc Natl Acad Sci U S A 2008;105:19915–19919.
5. Murtaugh LC, Kopinke D. Pancreatic stem cells. In Cambridge (MA): L. Charles Murtaugh and Daniel Kopinke; 2008. (StemBook).
6. Page BJ, du Toit DF, Muller CJ, Mattysen J, Lyners R, Arends E. Autogenous transplantation of a duct ligated pancreas: a functional and histological study. J Pancreas 2004;5:71–80.
7. Solar M, Cardalda C, Houbracken I, Martín M, Maestro MA, Medts N De, et al. Pancreatic exocrine duct cells give rise to insulin-producing β cells during embryogenesis but not after birth. Dev Cell 2009;17:849–60.
8. Wang RN, Klöppel G, Bouwens L. Duct-to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 1995;38:1405–1411.
9. Kopp JL, Dubois CL, Schaffer AE, Hao E, Shih HP, Seymour PA, et al. Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas. Development 2011 ;138:653–665.
10. Cano DA, Rulifson IC, Heiser PW, Swigart LB, Pelengaris S, German M, et al. Regulated beta-cell regeneration in the adult mouse pancreas. Diabetes. 2008 ;57:958–966.
11. Pagliuca FW, Melton DA. How to make a functional beta-cell. Development 2013 ;140:2472–2483.
12. Gittes GK, Galante PE, Hanahan D, Rutter WJ, Debase HT. Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors. Development 1996 ;122:439–447.
13. Pictet RL, Clark WR, Williams RH, Rutter WJ. An ultrastructural analysis of the developing embryonic pancreas. Dev Biol 1972;29:436–467.
14. Kramer B, Andrew A, Rawdon BB, Becker P. The effect of pancreatic mesenchyme on the differentiation of endocrine cells from gastric endoderm. Development 1987 ;100:661–671.
15.  Kedinger M, Simon-Assmann PM, Lacroix B, Marxer A, Hauri HP, Haffen K. Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells. Dev Biol 1986;113:474–483.
16. Manda JK. An in vitro study of mesenchyme--islet cell interactions in islet neogenesis: A model for tissue replacement therapy in diabetes mellitus. Stellenbosch: Stellenbosch University; 2017.
17. Manda JK, Page BJ, Tchokonte-Nana V. Mesenchymal cells are required for epithelial duct cell-to-beta cell maturation and function in an injured adult pancreas in the rat. Acta Histochem 2017 1;119:689–695.
18. Yu K, Fischbach S, Xiao X. Beta Cell Regeneration in Adult Mice: Controversy Over the Involvement of Stem Cells. Curr Stem Cell Res Ther 2016;11:542–546.
19. Hogan BLM. Morphogenesis. Cell 1999;96:225–233.
20. Trott J, Tan EK, Ong S, Titmarsh DM, Denil SLIJ, Giam M, et al. Long-Term Culture of Self-renewing pancreatic progenitors derived from human pluripotent stem cells. Stem cell reports 2017;8:1675–1688.
21. Puri S, Hebrok M. Cellular plasticity within the pancreas-lessons learned from development. Dev Cell 2010;18:342–356.
22. Tchokonte-Nana V, Manda JK. Early islets and mesenchyme from an injured adult pancreas improve syngeneic engraftments and islet graft function in diabetic rats. Acta Histochem 2018;120:356-362.
23. Schauwer C De, Meyer E, Cornillie P, Vliegher S De, van de Walle GR, Hoogewijs M, et al. Optimization of the isolation, culture, and characterization of equine umbilical cord blood mesenchymal stromal cells. Tissue Eng Part C Methods 2011;17:1061–1070.
24. Ohlsson H, Karlsson K, Edlund T. IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J 1993;12:4251–4259.
25. Waeber G, Thompson N, Nicod P, Bonny C. Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol Endocrinol 1996;10:1327–1334.
26. Watada H, Kajimoto Y, Miyagawa J, Hanafusa T, Hamaguchi K, Matsuoka T, et al. PDX-1 induces insulin and glucokinase gene expressions in alphaTC1 clone 6 cells in the presence of betacellulin. Diabetes 1996;45:1826–1831.
27. Pedersen JK, Nelson SB, Jorgensen MC, Henseleit KD, Fujitani Y, Wright CVE, et al. Endodermal expression of Nkx6 genes depends differentially on Pdx1. Dev Biol 2005;288:487–501.
28. Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 2002;129:2447–2457.
29. Gros L, Montoliu L, Riu E, Lebrigand L, Bosch F. Regulated production of mature insulin by non-beta-cells. Hum Gene Ther 1997;8:2249–2259.