Neonatal Pig as an Alternative Source of Islets for Transplantation

• Author / Creator

  Huang, Wenlong

• Background
  Islet transplantation is an attractive alternative treatment for type 1 diabetes mellitus (T1DM). However, further application of islet transplantation in the clinic is limited by the shortage of human organ donors and the immune-mediated rejection. Neonatal pig islets (NPI) are being considered as an alternative source of islets for transplantation. The potential human immune-mediated response to pig islet cells necessitates the development of new immunosuppressants. Genetically modified pigs (GMP) have been produced to alleviate the human organ shortage and rejection. However, the effects of genetic modifications on the function of NPI have not been carefully studied. Hence, a better understanding of NPI development may enable the generation of new GMP.
  Objectives of this thesis project were: to investigate the immune response of peripheral blood mononuclear cells (PBMC) from individuals with T1DM to neonatal pig islet cells in vitro, and to explore the potential inhibitory effect of Suramin and Direct Red 80 (DR80) on the proliferation of PBMC after stimulation with neonatal pig islet cells; to investigate the immune response of individuals with or without T1DM to NPI in a humanized mouse model; to characterize and compare the islets from GalTKO, GalTKO/hCD46, GalTKO/hCD46/hCD39 and wild type neonatal pigs; to examine patterns of epithelial-cadherin (E-cadherin), neural-cadherin (N-cadherin) and vascular endothelial-cadherin (VE-cadherin) protein expression as islets develop in culture.
  Methods
  Proliferation curves of human PBMC from individuals with T1DM in response to pig cells were determined through one-way mixed lymphocyte reaction. The effect of Suramin and/or DR80 on this proliferation was traced with CFSE.
  The rejection of NPI by human PBMC was tested in NOD.SCID gamma (NSG) mouse model. After achieving normoglycemic, mice were reconstituted with 15 million human PBMC from individuals with or without T1DM. Islet xenografts were harvested at 1-, 2- or 3-week post-reconstitution and analyzed for the presence of endocrine cells.
  The difference in islet gene expression among GalTKO, GalTKO/hCD46, GalTKO/hCD46/hCD39 islets was evaluated by microarray analysis and function of islets were evaluated in an NSG mouse model.
  Islets were isolated from 1-, 3-, 7- and 10-day-old neonatal pigs. Morphology of islets in culture was evaluated. In addition, islet samples were collected on day 0, 1, 3, 5, and 7 of culture to evaluate the expression of E-, N- and VE-cadherins by RT-PCR and western blot.

  Results
  Suramin inhibited NPI-induced proliferation of human PBMC with IC50~62.5 to 125 μg/ml, and IC50~125 to 250 μg/ml when induced by pig PBMC. Also, DR80 inhibited the proliferation of human PBMC stimulated with ConA, NPI cells or pig PBMC with IC50~31.2 to 62.5 μg/ml.
  Blood glucose levels were not increased in mice reconstituted with human PBMC, while hyperglycemia was observed in mice at 2 to 3 weeks post-reconstitution with NOD splenocytes. Immunohistochemistry revealed massive insulin-positive cells in islet grafts at 1-week post-reconstitution, which became sporadic and less at 2- and 3-weeks post-reconstitution. Macrophage, CD4+ T cell, CD8+ T cells were identified in grafts of reconstituted mice.
  Microarray results showed that fewer genes were altered as more genetic modifications were introduced into the pigs. A delay in reversing hyperglycemia was observed when more genetic modifications were applied to islets. Islet xenografts of normoglycemic mice contained various insulin and glucagon-positive cells, while less insulin- and glucagon-positive cells were identified in grafts of mice that remained hyperglycemic.
  Islets from 1-, 3-, 7- and 10-day-old pigs matured and formed oval to round structure on day 3 of culture, while dramatical increase in E-cadherin, N-cadherin and VE-cadherin was observed on day 3 of culture, suggesting these cadherin molecules may play an important role in post-natal islet development.

  Conclusions
  These results demonstrate the inhibitory effect of DR80 and Suramin on the in vitro human immune response to pig cells. NPI rejection by human immune cells started at 2-3 weeks post-reconstitution and involved macrophage, CD4+ and CD8+ T cells. Islets from GMP have therapeutic effect on hyperglycemia. However, variations in the ability of these islet xenografts to reverse the diabetic state of recipient mice was observed and this may be due in part to the difference in patterns of their gene expression. E-cadherin, N-cadherin and VE-cadherin play important roles in the formation and maintenance of pig islet structure and may be important molecules to preserve when designing genetic engineered pigs.

• Subjects / Keywords

  • Neonatal Pig
  • Islet Xenotransplantation

• Graduation date

  Fall 2019

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-rmex-z433

• License

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