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There is great interest in the potential of the human endocrine pancreas for regeneration by β-cell replication or neogenesis. Our aim was to explore this potential in adult human pancreases and in both islet and exocrine tissue transplanted into mice. The design was to examine pancreases obtained from cadaver donors, autopsies, and fresh surgical specimens and compare these findings with those obtained from islet and duct tissue grafted into the kidney. Islets and exocrine tissue were transplanted into normoglycemic ICR-SCID mice and studied 4 and 14 weeks later. β-Cell replication, as assessed by double staining for insulin and Ki67, was 0.22±0.03% at 4 weeks and 0.13±0.03% at 14 weeks. In contrast, no evidence of β-cell replication could be found in 11 cadaver donor and 10 autopsy pancreases. However, Ki67 staining of β-cells in frozen sections obtained at surgery was comparable to that found in transplanted islets. Evidence for neogenesis in transplanted pancreatic exocrine tissue was supported by finding β-cells within the duct epithelium and the presence of cells double stained for insulin and cytokeratin 19 (CK19). However, β-cells within the ducts never constituted more than 1% of the CK19-positive cells. With confocal microscopy, 7 of 12 examined cells expressed both markers, consistent with a neogeneic process. Mice with grafts containing islet or exocrine tissue were treated with various combinations of exendin-4, gastrin, and epidermal growth factor; none increased β-cell replication or stimulated neogenesis. In summary, human β-cells replicate at a low level in islets transplanted into mice and in surgical pancreatic frozen sections, but rarely in cadaver donor or autopsy pancreases. The absence of β-cell replication in many adult cadaver or autopsy pancreases could, in part, be an artifact of the postmortem state. Thus, it appears that adult human β-cells maintain a low level of turnover through replication and neogenesis.

Bone marrow and tissue precursor cells have been postulated to replenish grafts of transplanted islets. Several investigators have reported that bone marrow cells can promote the regeneration of injured islets. In this study, we investigated the potential of recipient-derived precursor cells to form new pancreatic endocrine cells in islet grafts transplanted under the kidney capsule. Mouse insulin promoter (MIP)-green fluorescence protein (GFP) mice, which express GFP only in β-cells, or β-actin GFP mice, which express GFP ubiquitously, were used to determine if the recipient-derived cells differentiate into β-cells or other types of endocrine cells. We transplanted MIP-GFP islets into wild-type mice, wild-type islets into MIP-GFP mice, β-actin GFP islets into wild-type mice, and wild-type islets into β-actin GFP mice. β-Actin GFP bone marrow cells were then injected into wild-type mice to evaluate the potential role of bone marrow stem cells to provide new islet cells to the graft. No β-cells with green fluorescence were seen in the graft when wild-type islets were transplanted into MIP-GFP mice. When wild-type islets were transplanted into β-actin GFP mice, no β-cells with GFP staining could be identified in the grafts. Similarly, no endocrine cells with GFP staining could be identified in the grafts after injection of β-actin GFP bone marrow cells into wild-type islet-transplanted wild-type mice. This study provides further support for the concept that recipient precursor cells do not produce new β-cells in grafts of transplanted islets.

Neonatal porcine pancreas has considerable capacity for growth and differentiation, making it an attractive potential source of islet tissue for xenotransplantation. Pancreases from 1–3-day-old newborn pigs were digested with collagenase and cultured for 8 days. The resulting cellular aggregates are called porcine neonatal pancreatic cell clusters (NPCCs). The mean yield of NPCCs from a newborn pig was 28,200 ± 1700 islet equivalents. Cytokeratin 7 (CK7) was used as a marker for the immunostaining of pancreatic duct cells. In neonatal pancreas, 18% of the insulin-positive cells co-stained for CK7, thus being protodifferentiated. NPCCs also contained protodifferentiated cells; insulin/PP and insulin/somatostatin co-stained cells were more common than insulin/glucagon cells. Between 1 and 8 days of culture, the DNA content of the NPCCs fell to 16% and the insulin content to 33% of the starting value, mainly due to the preferential loss of exocrine cells. Transplantation of 2000 or 4000 NPCCs into diabetic nude mice typically normalized glucose values in 10–20 weeks. Mice with successful grafts had lower fasting blood glucose levels than normal mice and accelerated glucose clearance after an IP glucose load. The starting NPCCs consisted of 17% insulin-staining cells, but the grafts of mice with reversed diabetes consisted of 94% beta cells, with some co-stained for CK7, indicating that the grafts still contained immature cells. The mass of insulin-producing cells rose from 0.22 ± 0.08 mg 1 week after transplantation to 4.34 ± 0.27 mg in mice sacrificed at 27–35 weeks. In summary, NPCCs contain mostly islet precursor cells, which when transplanted into nude mice undergo striking differentiation and beta cell expansion.