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Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters
Published on: May 19, 2014
Characterization of endocrine-rich monolayers of human fetal pancreas that display reduced immunogenicity
A M Simpson1, B E Tuch, P C Vincent
1Department of Medicine, University of Sydney, New South Wales, Australia.
Insights
Researchers developed fibroblast-free human fetal pancreas cell monolayers with reduced immunogenicity for diabetes transplantation. Further immunosuppression and cell preservation are needed for clinical success.
Area of Science:
- Endocrinology
- Cell Biology
- Immunology
Background:
- Human fetal pancreas cells are a potential source for beta-cell replacement therapy in diabetes.
- Establishing functional, immunologically compatible pancreatic cell cultures is challenging.
- Fibroblast overgrowth and immune rejection are significant hurdles in pancreatic transplantation.
Purpose of the Study:
- To create fibroblast-free, insulin-secreting monolayers from human fetal pancreas islet-like cell clusters (ICCs).
- To assess the immunogenicity and functional characteristics of these novel pancreatic cell monolayers.
- To evaluate the potential of these monolayers for future diabetes transplantation strategies.
Main Methods:
- Human fetal pancreases (14-20 weeks gestation) were processed to obtain islet-like cell clusters (ICCs).
- ICCs were cultured on bovine corneal matrix-coated plates to form endocrine-rich monolayers.
- Mixed-lymphocyte reactions with human peripheral blood lymphocytes (HPBLs) were used to assess immunogenicity.
- Insulin release in response to secretagogues was measured.
Main Results:
- Fibroblast-free monolayers derived from ICCs showed significantly reduced immunogenicity compared to single-cell suspensions.
- These ICC-derived monolayers maintained endocrine cell populations (65% +/- 13%) with substantial beta-cell presence (51%).
- Cells exhibited stimulated insulin release with theophylline, calcium, and phorbol ester, but not glucose, and synthesized DNA, proinsulin, and protein.
Conclusions:
- Establishment of an endocrine-rich human fetal pancreas monolayer with reduced immunogenicity is feasible.
- Residual immune activity necessitates further immunosuppression strategies for successful transplantation in diabetic patients.
- Cryopreservation and subculturing techniques may be required to generate sufficient beta-cell numbers for clinical application.
Abstract:
Fibroblast-free insulin-secreting monolayers of human fetal pancreas (14-20 wk of gestation) were formed by plating isletlike cell clusters (ICCs) obtained from partially digested pancreases on plates coated with bovine corneal matrix. Human fetal pancreatic cells, freshly digested with collagen, displayed a 17-fold response to human peripheral blood lymphocytes (HPBLs) in mixed-lymphocyte culture. After 14 days in culture, monolayers derived from ICCs exhibited a smaller, twofold response to HPBLs. By comparison, in monolayers produced from single-cell suspensions, fibroblast overgrowth remained a problem. The endocrine component of the monolayers was 65 +/- 13 and 43 +/- 8%, respectively, with the number of beta-cells being 51 and 9%. Cells from both monolayers displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+, and 0.6-1.3 microM 12-O-tetradecanoylphorbol-13-acetate but not to 20 mM glucose. Monolayers derived from ICCs synthesized DNA, proinsulin, and protein. This study showed that it is possible to establish an endocrine-rich monolayer of human fetal pancreas that has greatly reduced immunogenicity. The existence of residual activity to HPBLs suggests some additional form of immunosuppression is required to prevent rejection of this tissue when grafted into diabetic patients. Subculturing and cryopreservation may also be needed to achieve adequate numbers of beta-cells for clinical transplantation.
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