Transplantation of insulin-producing fetal pancreatic tissue into diabetic recipients has been shown to normalize blood glucose levels after several months. This time period is required for the growth and maturation of the fetal tissue so insulin levels cannot be used as a marker of graft function while the beta-cell is immature. Therefore, we have examined the use of another pancreatic endocrine hormone, pancreatic polypeptide (PP), to monitor graft function. The cell that produces this hormone has been shown to be the first mature endocrine cell in the fetal pancreas. Fetal pig pancreatic tissue, both in the form of 1 mm3 explants and islet-like cell clusters (ICCs), was transplanted into immunodeficient SCID mice and the levels of PP and insulin were measured in plasma and in the graft for up to 12 weeks. PP was detected in the untransplanted explants (0.58 pmol/mg) and ICCs (0.06 pmol/ICC) and the PP to insulin ratio was 2.7% and 5.8%, respectively. PP (but not porcine C-peptide, a marker of insulin secretion) was detectable in the plasma of SCID mice from 4 days to 3 weeks after transplantation, but not thereafter. The highest values were obtained at 4 days to 1 week. In the grafted tissue PP and insulin were present at all time points and the ratio of PP to insulin was 59%, 87%, 75%, 56%, 7%, 8%, and 7% at 4 days, 1, 2, 3, 6, 9, and 12 weeks, respectively. The decline in PP levels 3 weeks after transplantation was associated with beta-cell development in the graft. PP was also secreted by fetal pig pancreatic explants transplanted into diabetic NOD/SCID mice, with plasma levels measurable in the first week after the tissue was grafted. In immunocompetent BALB/c mice transplanted with the tissue, PP was detectable in plasma for 2 days after transplantation but not at 4 days, when cellular rejection commenced, or thereafter. We conclude that plasma PP levels can be used as a marker of the viability of fetal porcine pancreatic tissue in the first 3 weeks after it is transplanted into mice. These findings may have relevance to fetal pancreatic tissue transplanted into humans if suitable techniques can be developed to separate pig from human PP.

Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 ± 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 ± 2 vs. 31 ± 2 min). The size of ICCs was similar for both collagenase (83 ± 0.5 µm) and Liberase (79 ± 0.4 µm) as was the number of ICCs produced per pancreas (7653 ± 1297 vs. 8101 ± 1177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 ± 1% vs. 76 ± 1%, p < 0.05). Responsiveness to β-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at 1 month after transplantation of 1500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.

The pancreatic polypeptide cell, the only mature endocrine cell in the fetal pig pancreas, produces equimolar amounts of two peptides, pancreatic polypeptide and pancreatic icosapeptide, from the same precursor. The amino acid sequence of pancreatic polypeptide is more homogeneous among species, whereas pancreatic icosapeptide is heterogeneous. We determined the 19-amino acid sequence of porcine pancreatic icosapeptide, which is markedly different from that of known sequences (e.g. 47% homology with human). We developed an ELISA that can measure porcine pancreatic icosapeptide levels in the range of 7.2-480 pmol/liter. Actual levels of pancreatic icosapeptide in pig sera were 9.6-25 pmol/liter. The assay requires relatively small amounts of nonextracted samples, and human and mouse sera do not cross-react. Levels of pancreatic icosapeptide rose in response to hypoglycemia in pigs and to carbachol in fetal porcine pancreatic cells in vitro. When fetal porcine pancreatic tissue was transplanted into nonobese diabetic-severe combined immune deficiency mice, porcine pancreatic icosapeptide (but not C peptide) was detectable in mouse sera for up to 3 wk after transplantation, with levels highest on d 4. Porcine pancreatic icosapeptide and insulin were detectable in grafts removed from the mice. Therefore, porcine pancreatic icosapeptide may be used as a marker of the viability of xenotransplanted fetal pig pancreatic tissue in the immediate posttransplant period.