Mouse Pancreas Stem/Progenitor Cells Get Augmented by Streptozotocin and Regenerate Diabetic Pancreas After Partial Pancreatectomy

  • Subhan Ali Mohammad
  • Siddhanath Metkari
  • Deepa BhartiyaEmail author


Existence of stem cells in adult pancreas remains contentious. Single cells suspensions obtained by collagenase and trypsin digestion separately from adult mouse pancreas and pancreatic islets were spun at 1000 rpm (250 g) to collect the cells. At this speed the stem/ progenitor cells remained buoyant and were further enriched by spinning the supernatant at 3000 rpm (1000 g). Two distinct populations of stem cells were detected including pluripotent, very small (2–6 μm) embryonic-like stem cells (VSELs) that expressed nuclear OCT-4A and pluripotent transcripts (Oct-4A, Sox2, Nanog, Stella) and slightly bigger progenitors, pancreatic stem cells (PSCs) that expressed cytoplasmic OCT-4B and PDX-1. Streptozotocin treated diabetic pancreas showed an increase in numbers of VSELs (2–6 μm, 7AAD-, LIN-CD45-SCA1+ cells) and up-regulation of transcripts specific for stem/ progenitor cells. Diabetic mice were further subjected to partial pancreatectomy to study involvement of VSELs/ PSCs during regeneration. VSELs/ PSCs were mobilized in large numbers, were observed in the lumen of blood vessels and PCNA expression suggested their proliferation. Initially, new acini assembled to regenerate the exocrine pancreas and later by Day 30, neogenesis of islets was observed in the vicinity of the blood vessels and pancreatic ducts by the differentiation of endogenous VSELs/ PSCs which may be targeted to regenerate diabetic pancreas in clinical settings.


Pancreas Stem cells VSELs Diabetes Regeneration PDX-1 OCT-4 



Thanks to Ms. Pushpa Singh and Ms. Ankita Kaushik for their help. Authors acknowledge the help provided by Drs Ramesh Bhonde (Pune) and Shahir Gaikwad (Department of Surgery, Bombay Veterinary College, Mumbai) towards surgical manipulations of mouse pancreas. Sincere thanks to Dr. Aleem Khan (Deccan College of Medical Sciences, Hyderabad) for his help to extract good quality RNA from pancreas for qRT-PCR studies. Thanks to the central facilities at NIRRH of Confocal Microscopy and Flow Cytometry for their expert help. Authors also thank Shri Vaibhav Shinde for help with the art work. DB is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Author Contributions

SAM has done all the work, data interpretations and helped with manuscript preparation. SMM performed all the surgeries involved in the study and participated in the discussions. DB conceptualized the project, arranged the funds, helped with study design data, interpretation and manuscript preparation. All the authors critically reviewed the manuscript and provided intellectual inputs.

Funding Information

Thanks to ICMR Centenary PDF Scheme (3/1/3/PDF [16]/−2017-HRD-5) to support this work at NIRRH, Mumbai. NIRRH Accession Number RA/755/03–2019.

Compliance with Ethical Standards

Conflict of Interest

No potential conflicts of interest relevant to this article were reported.

Supplementary material

12015_2019_9919_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1310 kb)


  1. 1.
    Rezanejad, H., Lock, J. H., Sullivan, B. A., & Bonner-Weir, S. (2018). Generation of pancreatic ductal organoids and whole-mount immunostaining of intact organoids. Current Protocols in Cell Biology, 83, e82. Scholar
  2. 2.
    Zhou, Q., & Melton, D. A. (2018). Pancreas regeneration. Nature., 557, 351–358.PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Dor, Y., Brown, J., Martinez, O. I., & Melton, D. A. (2004). Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature., 429, 41–46.PubMedCrossRefGoogle Scholar
  4. 4.
    Xiao, X., Chen, Z., Shiota, C., Prasadan, K., Guo, P., El-Gohary, Y., et al. (2013). No evidence for β cell neogenesis in murine adult pancreas. The Journal of Clinical Investigation, 123, 2207–2217.PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Qadir, M. M. F., Álvarez-Cubela, S., Klein, D., Lanzoni, G., García-Santana, C., Montalvo, A., Pláceres-Uray, F., Mazza, E. M. C., Ricordi, C., Inverardi, L. A., Pastori, R. L., & Domínguez-Bendala, J. (2018). P2RY1/ALK3-expressing cells within the adult human exocrine pancreas are BMP-7 expandable and exhibit progenitor-like characteristics. Cell Reports, 22, 2408–2420.PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Bhartiya, D., Mundekar, A., Mahale, V., & Patel, H. (2014). Very small embryonic-like stem cells are involved in regeneration of mouse pancreas post-pancreatectomy. Stem Cell Research & Therapy, 5, 106.CrossRefGoogle Scholar
  7. 7.
    Zuba-Surma, E. K., Kucia, M., Wu, W., Klich, I., Lillard, J. W., Jr., Ratajczak, J., & Ratajczak, M. Z. (2008). Very small embryonic like stem cells are present in adult murine organs: image stream based morphological analysis and distribution studies. Cytometry., 73, 1116–1127.CrossRefGoogle Scholar
  8. 8.
    Starzyńska, T., Dąbkowski, K., Błogowski, W., Zuba-Surma, E., Budkowska, M., Sałata, D., Dołęgowska, B., Marlicz, W., Lubikowski, J., & Ratajczak, M. Z. (2013). An intensified systemic trafficking of bone marrow-derived stem/progenitor cells in patients with pancreatic cancer. Journal of Cellular and Molecular Medicine, 17, 792–799.PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Ratajczak, M. Z., Ratajczak, J., & Kucia, M. (2019). Very small embryonic-like stem cells (VSELs). Circulation Research, 124, 208–210.PubMedCrossRefGoogle Scholar
  10. 10.
    Bhartiya, D., Patel, H., Ganguly, R., Shaikh, A., Shukla, Y., Sharma, D., & Singh, P. (2018). Novel insights into adult and cancer stem cell biology. Stem Cells and Development, 27, 1527–1539.PubMedCrossRefGoogle Scholar
  11. 11.
    Lahlil, R., Scrofani, M., Barbet, R., Tancredi, C., & Aries, A. (2018). Hénon. VSELs maintain their pluripotency and competence to differentiate after enhanced ex vivo expansion. Stem Cell Reviews, 14, 510–524.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Havens, A. M., Sun, H., Shiozawa, Y., Jung, Y., Wang, J., Mishra, A., Jiang, Y., O'Neill, D. W., Krebsbach, P. H., Rodgerson, D. O., & Taichman, R. S. (2014). Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo. Stem Cells and Development, 23, 689–701.PubMedCrossRefGoogle Scholar
  13. 13.
    Monti, M., Imberti, B., Bianchi, N., Pezzotta, A., Morigi, M., Del Fante, C., et al. (2017). A novel method for isolation of pluripotent stem cells from human umbilical cord blood. Stem Cells Development., 26, 1258–1269.PubMedCrossRefGoogle Scholar
  14. 14.
    Shaikh, A., Anand, S., Kapoor, S., Ganguly, R., & Bhartiya, D. (2017). Mouse bone marrow VSELs exhibit differentiation into three embryonic germ lineages and germ & hematopoietic cells in culture. Stem Cell Reviews and Reports, 13, 202–216.CrossRefGoogle Scholar
  15. 15.
    Kucia, M., Campbell Reca, R., Zuba-Surma, F. R., Majka, E., Ratajczak, M. J., et al. (2006). A population of very small embryonic-like (VSEL) CXCR4 (+) SSEA-1(+) Oct-4+ stem cells identified in adult bone marrow. Leukemia, 20, 857–869.PubMedCrossRefGoogle Scholar
  16. 16.
    White, M. G., Al-Turaifi, H. R., Holliman, G. N., Aldibbiat, A., Mahmoud, A., & Shaw, J. A. (2011). Pluripotency-associated stem cell marker expression in proliferative cell cultures derived from adult human pancreas. The Journal of Endocrinology, 211, 169–176.PubMedCrossRefGoogle Scholar
  17. 17.
    Zhao, M., Amiel, S. A., Christie, M. R., Muiesan, P., Srinivasan, P., Littlejohn, W., Rela, M., Arno, M., Heaton, N., & Huang, G. C. (2007). Evidence for the presence of stem cell-like progenitor cells in human adult pancreas. The Journal of Endocrinology, 195, 407–414. Scholar
  18. 18.
    Ashizawa, S., Brunicardi, F. C., & Wang, X. P. (2004). PDX-1 and the pancreas. Pancreas., 28, 109–120.PubMedCrossRefGoogle Scholar
  19. 19.
    Pedica, F., Beccari, S., Pedron, S., Montagna, L., Piccoli, P., Doglioni, C., & Chilosi, M. (2014). PDX-1 (pancreatic/duodenal homeobox-1 protein 1). Pathologica., 106, 315–321.PubMedGoogle Scholar
  20. 20.
    Li, D.-S., Yuan, Y.-H., Tu, H.-J., Liang, Q.-L., & Dai, L.-J. (2009). A protocol for islet isolation from mouse pancreas. Nature Protocols, 4, 1649–1652.PubMedCrossRefGoogle Scholar
  21. 21.
    Bhartiya, D., Ali Mohammad, S., Guha, A., Pushpa, S., Diksha, S., & Ankita, K. (2019). Evolving definition of adult stem/progenitor cells. Stem Cell Reviews and Reports, 15, 1–3. Scholar
  22. 22.
    Zuba-Surma, E. K., Kucia, M., Ratajczak, J., & Ratajczak, M. Z. (2009). “Small stem cells” in adult tissues: very small embryonic-like stem cells stand up! Cytometry. Part A, 75, 4–13.CrossRefGoogle Scholar
  23. 23.
    Chomczynski, P., & Sacchi, N. (2006). The single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroformextraction: twenty-something years on. Nature Protocols, 1(2), 581–585.PubMedCrossRefGoogle Scholar
  24. 24.
    Bhartiya, D., Shaikh, A., Anand, S., Patel, H., Kapoor, S., Sriraman, K., Parte, S., & Unni, S. (2016). Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead. Human Reproduction Update, 23(1), 41–76.PubMedCrossRefGoogle Scholar
  25. 25.
    Shaikh, A., Bhartiya, D., Kapoor, S., & Nimkar, H. (2016). Delineating the effects of 5-fluorouracil and follicle-stimulating hormone on mouse bone marrow stem/progenitor cells. Stem Cell Research & Therapy, 7, 59.CrossRefGoogle Scholar
  26. 26.
    Hardikar, A. A., Karandikar, M. S., & Bhonde, R. R. (1999). Effect of partial pancreatectomy on diabetic status in BALB/c mice. The Journal of Endocrinology, 162, 189–195.PubMedCrossRefGoogle Scholar
  27. 27.
    Anand, S., Bhartiya, D., Sriraman, K., & Mallick, A. (2016). Underlying mechanisms that restore spermatogenesis on transplanting healthy niche cells in busulphan treated mouse testis. Stem Cell Reviews, 12, 682–697.CrossRefGoogle Scholar
  28. 28.
    Yu, K., Fischbach, S., & Xiao, X. (2016). Beta cell regeneration in adult mice: controversy over the involvement of stem cells. 2016. Current Stem Cell Research & Therapy, 11, 542–546.CrossRefGoogle Scholar
  29. 29.
    Domínguez-Bendala, J., Qadir, M. M. F., & Pastori, R. L. (2019). Pancreatic progenitors: there and back again. Trends in Endocrinology and Metabolism, 30(1), 4–11.PubMedCrossRefGoogle Scholar
  30. 30.
    Bhartiya, D., & Patel, H. (2015). Very small embryonic-like stem cells are involved in pancreatic regeneration and their dysfunction with age may lead to diabetes and cancer. Stem Cell Research & Therapy, 6, 96–102.CrossRefGoogle Scholar
  31. 31.
    Karaoz, E., Ayhan, S., Gacar, G., Aksoy, A., Duruksu, G., Okçu, A., Demircan, P. Ç., Sariboyaci, A. E., Kaymaz, F., & Kasap, M. (2010). Isolation and characterization of stem cells from pancreatic islet: pluripotency, differentiation potential and ultrastructural characteristics. Cytotherapy., 12, 288–302.PubMedCrossRefGoogle Scholar
  32. 32.
    Zanini, C., Bruno, S., Mandili, G., Baci, D., Cerutti, F., Cenacchi, G., Izzi, L., Camussi, G., & Forni, M. (2011). Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype. PLoS One, 6, e28175.PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Bhartiya, D. (2013). Are mesenchymal cells indeed pluripotent stem cells or just stromal cells? OCT-4 and VSELs biology has led to better understanding. Stem Cells International, 2013, 547501.PubMedPubMedCentralGoogle Scholar
  34. 34.
    Clevers, H., & Watt, F. M. (2018). Defining adult stem cells by function, not by phenotype. Annual Review of Biochemistry, 87, 1015–1027.PubMedCrossRefGoogle Scholar
  35. 35.
    Furuyama, K., Chera, S., van Gurp, L., Oropeza, D., Ghila, L., Damond, N., Vethe, H., Paulo, J. A., Joosten, A. M., Berney, T., Bosco, D., Dorrell, C., Grompe, M., Ræder, H., Roep, B. O., Thorel, F., & Herrera, P. L. (2019). Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells. Nature., 567(7746), 43–48.PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Cho, J., D'Antuono, M., Glicksman, M., Wang, J., & Jonklaas, J. (2018). A review of clinical trials: mesenchymal stem cell transplant therapy in type 1 and type 2 diabetes mellitus. American Journal of Stem Cells, 7(4), 82–93.PubMedPubMedCentralGoogle Scholar
  37. 37.
    Sipp, D., Robey, P. G., & Turner, L. (2018). Clear up this stem-cell mess. Nature, 561, 455–457.PubMedCrossRefGoogle Scholar
  38. 38.
    Caplan, A. I. (2019). There is no “stem cell mess”. Tissue Engineering. Part B, Reviews, 25, 291–293. Scholar
  39. 39.
    Banakh, I., Gonez, L. J., Sutherland, R. M., Naselli, G., & Harrison, L. C. (2012). Adult pancreas side population cells expand after β cell injury and are a source of insulin-secreting cells. PLoS One, 7, e48977.PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Augstein, P., Loudovaris, T., Bandala-Sanchez, E., Heinke, P., Naselli, G., Lee, L., Hawthorne, W. J., Góñez, L. J., Neale, A. M., Vaillant, F., Thomas, H. E., Kay, T. W., Banakh, I., & Harrison, L. C. (2018). Characterization of the human pancreas side population as a potential reservoir of adult stem cells. Pancreas., 47, 25–34.PubMedCrossRefGoogle Scholar
  41. 41.
    Berrocal, T., Luque, A. A., Pinilla, I., & Lassaletta, L. (2005). Pancreatic regeneration after near-total pancreatectomy in children with nesidioblastosis. Pediatric Radiology, 35(11), 1066–1070.PubMedCrossRefGoogle Scholar
  42. 42.
    Menge, B. A., Tannapfel, A., Belyaev, O., Drescher, R., Müller, C., Uhl, W., et al. (2008). Partial pancreatectomy in adult humans does not provoke β-cell regeneration. Diabetes, 57, 142–149.PubMedCrossRefGoogle Scholar
  43. 43.
    Menge, B. A., Breuer, T. G., Ritter, P. R., Uhl, W., Schmidt, W. E., & Meier, J. J. (2012). Long-term recovery of β-cell function after partial pancreatectomy in humans. Metabolism., 61, 620–624.PubMedCrossRefGoogle Scholar
  44. 44.
    Wang, P., Karakose, E., Liu, H., Swartz, E., Ackei, C., Zlatanic, V., et al. (2018). Combined inhibition of DYRK1A, SMAD, and Trithorax pathways synergizes to induce robust replication in adult human beta cells. Cell Metabolism S1550–4131; 18:30742–3.Google Scholar
  45. 45.
    Swartz, F. J., & Carstens, P. H. (1986). An islet of Langerhans located within the epithelium of a human pancreatic duct. Histology and Histopathology, 1, 111–117.PubMedGoogle Scholar
  46. 46.
    Tainaka, K., Kubota, S. I., Suyama, T. Q., Susaki, E. A., Perrin, D., Ukai-Tadenuma, M., Ukai, H., & Ueda, H. R. (2014). Whole-body imaging with single-cell resolution by tissue decolorization. Cell., 159, 911–924.PubMedCrossRefGoogle Scholar
  47. 47.
    Bhartiya, D. (2019). Clinical translation of stem cells for regenerative medicine a comprehensive analysis. Circulation Research, 124, 840–842.PubMedCrossRefGoogle Scholar
  48. 48.
    Shapiro, A. M. J. (2018). Gearing up for stem cell-derived beta cells-are we ready? Transplantation, 102(8), 1207–1208.PubMedCrossRefGoogle Scholar
  49. 49.
    Odorico, J., Markmann, J., Melton, D., Greenstein, J., Hwa, A., Nostro, C., Rezania, A., Oberholzer, J., Pipeleers, D., Yang, L., Cowan, C., Huangfu, D., Egli, D., Ben-David, U., Vallier, L., Grey, S. T., Tang, Q., Roep, B., Ricordi, C., Naji, A., Orlando, G., Anderson, D. G., Poznansky, M., Ludwig, B., Tomei, A., Greiner, D. L., Graham, M., Carpenter, M., Migliaccio, G., D’Amour, K., Hering, B., Piemonti, L., Berney, T., Rickels, M., Kay, T., & Adams, A. (2018). Report of the key opinion leaders meeting on stem cell-derived beta cells. Transplantation., 102(8), 1223–1229.PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Pepper, A. R., Bruni, A., Pawlick, R., O'Gorman, D., Kin, T., Thiesen, A., & Shapiro, A. M. J. (2019). Posttransplant characterization of long-term functional hESC-derived pancreatic endoderm grafts. Diabetes., 68(5), 953–962.PubMedCrossRefGoogle Scholar
  51. 51.
    Pawitan, J. A., Yang, Z., Wu, Y. N., & Lee, E. H. (2018). Towards standardized stem cell therapy in type 2 diabetes mellitus: a systematic review. Current Stem Cell Research & Therapy, 13(6), 476–488.CrossRefGoogle Scholar
  52. 52.
    Bhartiya, D. (2017). Pluripotent stem cells in adult tissues: struggling to be acknowledged over two decades. Stem Cell Reviews, 13(6), 713–724.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Stem Cell Biology DepartmentICMR- National Institute for Research in Reproductive HealthMumbaiIndia
  2. 2.Experimental Animal FacilityICMR-National Institute for Research in Reproductive HealthMumbaiIndia

Personalised recommendations