, Volume 56, Issue 9, pp 1987–1998 | Cite as

Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice

  • Jennifer E. Bruin
  • Alireza Rezania
  • Jean Xu
  • Kavitha Narayan
  • Jessica K. Fox
  • John J. O’Neil
  • Timothy J. KiefferEmail author



Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo.


The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice.


Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells.


Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.


Cell therapy Diabetes Encapsulation Human embryonic stem cells Insulin Islets 



Chemokine [C-X-C motif] receptor 4


Glucagon-like peptide 1 receptor


Haematoxylin and eosin


Human embryonic stem cells


v-Maf musculoaponeurotic fibrosarcoma oncogene homolog A (avian)


NK2 homeobox 2


NK6 homeobox 1


Stage 4




University of British Columbia



This work was funded by the Canadian Institutes of Health Research (CIHR) Regenerative Medicine and Nanomedicine Initiative, Stem Cell Network (SCN), JDRF, and Stem Cell Technologies. We would like to thank Ali Asadi for his technical assistance with immunofluorescent staining.


TJK was supported by a senior scholarship from the Michael Smith Foundation for Health Research and received financial support from Janssen R&D LLC. JEB was funded by a JDRF postdoctoral fellowship, a CIHR postdoctoral fellowship and the CIHR Transplantation Training Program. JEB also received a L’Oréal Canada for Women in Science Research Excellence Fellowship.

Duality of interest

AR, JX, KN and JJO’N are employees of Janssen R&D LLC, and TJK received financial support from Janssen R&D LLC for the research described in this article. The other authors confirm that there is no duality of interest associated with their contribution to this manuscript.

Contribution statement

JEB wrote the manuscript. AR, JEB, JX and TJK contributed to conception and design of experiments. AR, JEB, JX, KN, JJO’N and JKF were responsible for the acquisition, analysis and interpretation of data. All authors contributed to manuscript revisions and approved the final version of the manuscript.

Supplementary material

125_2013_2955_MOESM1_ESM.pdf (115 kb)
ESM Methods (PDF 115 kb)
125_2013_2955_MOESM2_ESM.pdf (61 kb)
ESM Table 1 (PDF 60 kb)
125_2013_2955_MOESM3_ESM.pdf (83 kb)
ESM Table 2 (PDF 82 kb)
125_2013_2955_MOESM4_ESM.pdf (74 kb)
ESM Table 3 (PDF 74 kb)
125_2013_2955_MOESM5_ESM.pdf (227 kb)
ESM Fig. 1 (PDF 226 kb)
125_2013_2955_MOESM6_ESM.pdf (370 kb)
ESM Fig. 2 (PDF 370 kb)
125_2013_2955_MOESM7_ESM.pdf (4.4 mb)
ESM Fig. 3 (PDF 4512 kb)
125_2013_2955_MOESM8_ESM.pdf (4.3 mb)
ESM Fig. 4 (PDF 4371 kb)
125_2013_2955_MOESM9_ESM.pdf (1.6 mb)
ESM Fig. 5 (PDF 1647 kb)
125_2013_2955_MOESM10_ESM.pdf (3 mb)
ESM Fig. 6 (PDF 3044 kb)
125_2013_2955_MOESM11_ESM.pdf (4.5 mb)
ESM Fig. 7 (PDF 4593 kb)
125_2013_2955_MOESM12_ESM.pdf (2.3 mb)
ESM Fig. 8 (PDF 2346 kb)


  1. 1.
    Ryan EA, Lakey JR, Rajotte RV et al (2001) Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50:710–719PubMedCrossRefGoogle Scholar
  2. 2.
    Shapiro AM, Lakey JR, Ryan EA et al (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238PubMedCrossRefGoogle Scholar
  3. 3.
    Shapiro AM (2011) State of the art of clinical islet transplantation and novel protocols of immunosuppression. Curr Diab Rep 11:345–354PubMedCrossRefGoogle Scholar
  4. 4.
    Tuduri E, Bruin JE, Kieffer TJ (2012) Restoring insulin production for type 1 diabetes. J Diabetes 4:319–331PubMedCrossRefGoogle Scholar
  5. 5.
    Kelly OG, Chan MY, Martinson LA et al (2011) Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat Biotechnol 29:750–756PubMedCrossRefGoogle Scholar
  6. 6.
    Jiang J, Au M, Lu K et al (2007) Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells 25:1940–1953PubMedCrossRefGoogle Scholar
  7. 7.
    Jiang W, Shi Y, Zhao D et al (2007) In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res 17:333–344PubMedCrossRefGoogle Scholar
  8. 8.
    Zhang D, Jiang W, Liu M et al (2009) Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res 19:429–438PubMedCrossRefGoogle Scholar
  9. 9.
    Nostro MC, Sarangi F, Ogawa S et al (2011) Stage-specific signaling through TGFbeta family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 138:861–871PubMedCrossRefGoogle Scholar
  10. 10.
    D’Amour KA, Bang AG, Eliazer S et al (2006) Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24:1392–1401PubMedCrossRefGoogle Scholar
  11. 11.
    Kroon E, Martinson LA, Kadoya K et al (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol 26:443–452PubMedCrossRefGoogle Scholar
  12. 12.
    Mfopou JK, Chen B, Mateizel I, Sermon K, Bouwens L (2010) Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells. Gastroenterology 138:2233–2245PubMedCrossRefGoogle Scholar
  13. 13.
    Shim JH, Kim SE, Woo DH et al (2007) Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. Diabetologia 50:1228–1238PubMedCrossRefGoogle Scholar
  14. 14.
    Cai J, Yu C, Liu Y et al (2010) Generation of homogeneous PDX1(+) pancreatic progenitors from human ES cell-derived endoderm cells. J Mol Cell Biol 2:50–60PubMedCrossRefGoogle Scholar
  15. 15.
    Rezania A, Riedel MJ, Wideman RD et al (2011) Production of functional glucagon-secreting alpha-cells from human embryonic stem cells. Diabetes 60:239–247PubMedCrossRefGoogle Scholar
  16. 16.
    Xu X, Browning VL, Odorico JS (2011) Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells. Mech Dev 128:412–427PubMedCrossRefGoogle Scholar
  17. 17.
    Rezania A, Bruin JE, Riedel MJ et al (2012) Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes 61:2016–2029PubMedCrossRefGoogle Scholar
  18. 18.
    Johnson JD, Ao Z, Ao P et al (2009) Different effects of FK506, rapamycin, and mycophenolate mofetil on glucose-stimulated insulin release and apoptosis in human islets. Cell Transplant 18:833–845PubMedCrossRefGoogle Scholar
  19. 19.
    Nir T, Melton DA, Dor Y (2007) Recovery from diabetes in mice by beta cell regeneration. J Clin Investig 117:2553–2561PubMedCrossRefGoogle Scholar
  20. 20.
    Zhang N, Su D, Qu S et al (2006) Sirolimus is associated with reduced islet engraftment and impaired beta-cell function. Diabetes 55:2429–2436PubMedCrossRefGoogle Scholar
  21. 21.
    Zahr E, Molano RD, Pileggi A et al (2007) Rapamycin impairs in vivo proliferation of islet beta-cells. Transplantation 84:1576–1583PubMedCrossRefGoogle Scholar
  22. 22.
    Brauker JH, Carr-Brendel VE, Martinson LA, Crudele J, Johnston WD, Johnson RC (1995) Neovascularization of synthetic membranes directed by membrane microarchitecture. J Biomed Mater Res 29:1517–1524PubMedCrossRefGoogle Scholar
  23. 23.
    Nyman LR, Wells KS, Head WS et al (2008) Real-time, multidimensional in vivo imaging used to investigate blood flow in mouse pancreatic islets. J Clin Investig 118:3790–3797PubMedCrossRefGoogle Scholar
  24. 24.
    O'Sullivan ES, Vegas A, Anderson DG, Weir GC (2011) Islets transplanted in immunoisolation devices: a review of the progress and the challenges that remain. Endocr Rev 32:827–844PubMedCrossRefGoogle Scholar
  25. 25.
    Lee SH, Hao E, Savinov AY, Geron I, Strongin AY, Itkin-Ansari P (2009) Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies. Transplantation 87:983–991PubMedCrossRefGoogle Scholar
  26. 26.
    Matveyenko AV, Georgia S, Bhushan A, Butler PC (2010) Inconsistent formation and non function of insulin positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats. Am J Physiol Endocrinol Metab 299:E713–E720PubMedCrossRefGoogle Scholar
  27. 27.
    Movassat J, Beattie GM, Lopez AD, Hayek A (2002) Exendin 4 up-regulates expression of PDX 1 and hastens differentiation and maturation of human fetal pancreatic cells. J Clin Endocrinol Metab 87:4775–4781PubMedCrossRefGoogle Scholar
  28. 28.
    Beattie GM, Rubin JS, Mally MI, Otonkoski T, Hayek A (1996) Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact. Diabetes 45:1223–1228PubMedCrossRefGoogle Scholar
  29. 29.
    Biarnes M, Montolio M, Nacher V, Raurell M, Soler J, Montanya E (2002) Beta-cell death and mass in syngeneically transplanted islets exposed to short- and long-term hyperglycemia. Diabetes 51:66–72PubMedCrossRefGoogle Scholar
  30. 30.
    Laybutt DR, Hawkins YC, Lock J et al (2007) Influence of diabetes on the loss of beta cell differentiation after islet transplantation in rats. Diabetologia 50:2117–2125PubMedCrossRefGoogle Scholar
  31. 31.
    De Vos A, Heimberg H, Quartier E et al (1995) Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression. J Clin Investig 96:2489–2495PubMedCrossRefGoogle Scholar
  32. 32.
    Aguayo-Mazzucato C, Koh A, El Khattabi I et al (2011) Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells. Diabetologia 54:583–593PubMedCrossRefGoogle Scholar
  33. 33.
    Kin T, Korbutt GS (2007) Delayed functional maturation of neonatal porcine islets in recipients under strict glycemic control. Xenotransplantation 14:333–338PubMedCrossRefGoogle Scholar
  34. 34.
    Butler AE, Robertson RP, Hernandez R, Matveyenko AV, Gurlo T, Butler PC (2012) Beta cell nuclear musculoaponeurotic fibrosarcoma oncogene family A (MafA) is deficient in type 2 diabetes. Diabetologia 55:2985–2988PubMedCrossRefGoogle Scholar
  35. 35.
    Holz GGT, Kuhtreiber WM, Habener JF (1993) Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7–37). Nature 361:362–365PubMedCrossRefGoogle Scholar
  36. 36.
    Abraham EJ, Leech CA, Lin JC, Zulewski H, Habener JF (2002) Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology 143:3152–3161PubMedCrossRefGoogle Scholar
  37. 37.
    Bai L, Meredith G, Tuch BE (2005) Glucagon-like peptide-1 enhances production of insulin in insulin-producing cells derived from mouse embryonic stem cells. J Endocrinol 186:343–352PubMedCrossRefGoogle Scholar
  38. 38.
    Ku HT, Zhang N, Kubo A et al (2004) Committing embryonic stem cells to early endocrine pancreas in vitro. Stem Cells 22:1205–1217PubMedCrossRefGoogle Scholar
  39. 39.
    Li H, Lam A, Xu AM, Lam KS, Chung SK (2010) High dosage of exendin-4 increased early insulin secretion in differentiated beta cells from mouse embryonic stem cells. Acta Pharmacol Sin 31:570–577PubMedCrossRefGoogle Scholar
  40. 40.
    Xu G, Stoffers DA, Habener JF, Bonner-Weir S (1999) Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48:2270–2276PubMedCrossRefGoogle Scholar
  41. 41.
    Baggio LL, Drucker DJ (2006) Therapeutic approaches to preserve islet mass in type 2 diabetes. Annu Rev Med 57:265–281PubMedCrossRefGoogle Scholar
  42. 42.
    Ghofaili KA, Fung M, Ao Z et al (2007) Effect of exenatide on beta cell function after islet transplantation in type 1 diabetes. Transplantation 83:24–28PubMedCrossRefGoogle Scholar
  43. 43.
    Tornehave D, Kristensen P, Romer J, Knudsen LB, Heller RS (2008) Expression of the GLP-1 receptor in mouse, rat, and human pancreas. J Histochem Cytochem 56:841–851PubMedCrossRefGoogle Scholar
  44. 44.
    Merani S, Shapiro AM (2006) Current status of pancreatic islet transplantation. Clin Sci (Lond) 110:611–625CrossRefGoogle Scholar
  45. 45.
    Lammert E, Cleaver O, Melton D (2001) Induction of pancreatic differentiation by signals from blood vessels. Science 294:564–567PubMedCrossRefGoogle Scholar
  46. 46.
    Villasenor A, Cleaver O (2012) Crosstalk between the developing pancreas and its blood vessels: an evolving dialog. Semin Cell Dev Biol 23:685–692PubMedCrossRefGoogle Scholar
  47. 47.
    Sweet IR, Yanay O, Waldron L et al (2008) Treatment of diabetic rats with encapsulated islets. J Cell Mol Med 12:2644–2650PubMedCrossRefGoogle Scholar
  48. 48.
    Tarantal AF, Lee CC, Itkin-Ansari P (2009) Real-time bioluminescence imaging of macroencapsulated fibroblasts reveals allograft protection in rhesus monkeys (Macaca mulatta). Transplantation 88:38–41PubMedCrossRefGoogle Scholar
  49. 49.
    Kumagai-Braescha M, Jacobsonb S, Moria H et al (2012) The TheraCyteTM device protects against islet allograft rejection in immunized hosts. Cell Transplant. doi: 10.3727/096368912X657486 Google Scholar
  50. 50.
    Pepper AR, Gall C, Mazzuca DM, Melling CW, White DJ (2009) Diabetic rats and mice are resistant to porcine and human insulin: flawed experimental models for testing islet xenografts. Xenotransplantation 16:502–510PubMedCrossRefGoogle Scholar
  51. 51.
    Bavamian S, Klee P, Britan A et al (2007) Islet-cell-to-cell communication as basis for normal insulin secretion. Diabetes Obes Metab 9(Suppl 2):118–132PubMedCrossRefGoogle Scholar
  52. 52.
    Harb G, Korbutt GS (2006) Effect of prolonged in vitro exposure to high glucose on neonatal porcine pancreatic islets. J Endocrinol 191:37–44PubMedCrossRefGoogle Scholar
  53. 53.
    Zhu X, Orci L, Carroll R, Norrbom C, Ravazzola M, Steiner DF (2002) Severe block in processing of proinsulin to insulin accompanied by elevation of des-64,65 proinsulin intermediates in islets of mice lacking prohormone convertase 1/3. Proc Natl Acad Sci U S A 99:10299–10304PubMedCrossRefGoogle Scholar
  54. 54.
    Stoffers DA, Desai BM, DeLeon DD, Simmons RA (2003) Neonatal exendin-4 prevents the development of diabetes in the intrauterine growth retarded rat. Diabetes 52:734–740PubMedCrossRefGoogle Scholar
  55. 55.
    Ham JN, Crutchlow MF, Desai BM, Simmons RA, Stoffers DA (2009) Exendin-4 normalizes islet vascularity in intrauterine growth restricted rats: potential role of VEGF. Pediatr Res 66:42–46PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Jennifer E. Bruin
    • 1
  • Alireza Rezania
    • 2
  • Jean Xu
    • 2
  • Kavitha Narayan
    • 2
  • Jessica K. Fox
    • 1
  • John J. O’Neil
    • 2
  • Timothy J. Kieffer
    • 1
    • 3
    Email author
  1. 1.Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences InstituteUniversity of British ColumbiaVancouverCanada
  2. 2.BetaLogics VentureJanssen R&D LLCRaritanUSA
  3. 3.Department of SurgeryUniversity of British ColumbiaVancouverCanada

Personalised recommendations