Pancreatic progenitors (PP) derived in vitro from human pluripotent embryonic stem cells (hESC) are being investigated as a cell therapy for insulin-dependent diabetes. Placing PP in microcapsules, modified from those previously used in clinical trials, is a strategy we are pursuing to prevent rejection of the cells by diabetic recipients. In this study, we examined the ability of PP to differentiate into insulin-producing cells by transplanting them into immunodeficient NOD/SCID mice.
hESC were differentiated into PP in vitro using a modification of a published technique (Nat Biotech 2008; 26: 443). On the final day of differentiation, PP cells were characterised by 1) routine immunohistochemistry which showed >70% of cells stained positively for PDX-1 and >50% for SOX9; and 2) qPCR analysis which showed an up-regulation of the genes for endocrine and precursor pancreas PDX-1, SOX9, HNF6, NKX6.1, NGN3 and NKX2.2 during the culture period peaking at the end of the differentiation process with low expression of insulin gene.
PP were transplanted into non-diabetic mice either using encapsulation in barium alginate with infusion into the peritoneal cavity (n=16) or mixing PP in a blood clot and grafting beneath the renal capsule (n=16). Mice were euthanized at 8, 12 and 16 weeks post transplantation and cardiac blood collected for measurement of human C-peptide by ELISA. Encapsulated cells retrieved from the peritoneal cavity and grafts separated from the mouse kidney were placed in tissue culture to determine insulin secretion by static stimulation and ELISA.
Human C-peptide was detected in mice engrafted with encapsulated PP from 8 weeks post transplantation and from 12 weeks in mice with kidney grafts. Insulin was detected in grafts immunohistochemically. Static stimulation with 20mM glucose did not enhance insulin production in either group.
In summary, PP that are encapsulated and transplanted into mice can develop into insulin-producing cells.