Allogeneic bone marrow supports human islet beta cell survival and function over six months

Abstract

In this study, we have established a new strategy increasing human islet longevity utilizing allogeneic whole bone marrow (BM) co-cultured with human islets. The cultured islets' function and survival have been evaluated by analysis of insulin secretion in response to high-glucose-challenge, morphological evaluation of cell growth. Human islet only culture failed to reveal evidence of long term survival, growth or function in terms of insulin release or insulin response to glucose challenge. These results indicate that BM increases islet survival and function with the eventual formation of pancreatic endocrine tissue capable of sustaining beta cell fuction.

Figure 1. Islet morphology in short-term culture

(a) BM co-cultured with islets, Day 6 BM cells surround islet forming capsule like structure. (b) BM co-cultured with islets, Day 156. Intact islet morphology is seen in culture. (c) Islet only culture, Day 6. Cells spilling from islet, eventually to form monolayer (see following). (d) Islet only culture, Day 156. Complete loss of islet morphology with only monolayer of cells noted.

Figure 2. Bone marrow retains islet function in culture

(a.) Evaluation of baseline insulin release until 204 days culture and (b.) insulin secretion in response to a high glucose challenge (20 mM for 30 minutes) as measured for 204 days.

Figure 3. Pancreatic endocrine tissue generated from long-term BM co-cultures (a)

shows tissue generated from islet in long-term bone marrow coculture (190 days), and the bar represents a length of 1 cm. The sample is about 1.8 cm long with clear three-dimensional structure. The histological structure of a 5 μm section from the frozen tissue in (b), stained with hematoxylin and eosin in (c), shows islet-like structure (arrow indicating) surrounded by porous scaffold-like tissue. Insulin content is 56307 uU/per mg protein (ELISA assay). The islet-like tissue contains primarily β cells and a few α cells. In (d) fluorescence immunohistochemistry reveals β cells with anti-human insulin antibody (red, b arrow), α cells with anti-human glucagon antibody (green, a arrow), and nucleus staining (DAPI, blue, magnification × 20). As indicated in Figures (e) and (f), evaluation of the tissue's insulin release and insulin in response to high glucose challenge before histological evaluation shows that the regenerated tissue has function. These data suggest that human islet tissue in coculture is generated not only because bone marrow facilitates islet aggregation but also because bone marrow stimulates islet cells to form islet tissues. B = bone marrow; I = islet * = p<0.05 vs. islet only culture.