Effects of bone marrow on the microenvironment of the human pancreatic islet: A Protein Profile Approach.

Stem cells are a new therapeutic modality that may support the viability and function of human organs and tissue. Our previous studies have revealed that human allogeneic bone marrow (BM) sustains pancreatic ß cell function and survival. This paper examines whether BM creates a microenvironment that supports human pancreatic islets in vitro by evaluating 107 proteins in culture media from BM, islet, and islet/bone marrow (IB) with mass spectrometry. Proteins were considered up- or down-regulated if p-values < 0.05 and fold change was greater than 2 fold I VS. IB. In addition, proteins identified that were uniquely found in islets co-cultured with bone marrow, but not in islets or bone marrow. A 95% protein probability was used as a threshold. Twenty three proteins were upregulated, and sixteen proteins were downregulated. The function of each protein is listed based on the protein database, which include structural proteins (9 upregulated, 4 downregulated); anti-protease and anti-endopeptidase enzymes (8 upregulated); cation binding proteins (6 up-regulated). Six proteins were uniquely identified in islet co-cultured with bone marrow. Three are anti-proteases or anti-endopeptidases, and 1 is a structural protein. These findings suggest that BM, by changing culture media proteins, may be one of mechanisms to maintain human islet function and survival.

Anti-apoptotic Effects of Bone Marrow on Human Islets: A Preliminary Report.

Apoptosis is one of the major factors contributing to the failure of human islet transplantation. Contributors to islet apoptosis exist in both the pre-transplantation and post transplantation stages. Factors include the islet isolation process, deterioration in vitro prior to transplantation, and immune rejection post transplantation. Previous studies have demonstrated that co-cultured bone marrow cells with human islets not only significantly enhanced the longevity of human islets but also maintained function. We hypothesized that the protective effects of bone marrow cells on human islets are through mechanisms related to preventing apoptosis. This study observed the levels of inflammatory factors such as interleukin-1ß (IL-1ß), the release of extracellular ATP in vitro, and expression levels of P2X7 ATP receptor (P2X7R), all of which lead to the occurrence of apoptosis in human islets. When human islets were co-cultured with human bone marrow, there was a reduction in the rate of apoptosis correlated with the reduction in inflammatory factors, extra cellular ATP accumulation, and ATP receptor P2X7R expression versus human islets cultured alone. These results suggest that co-culturing bone marrow cells with human islets inhibits inflammation and reduces apoptosis, thus protecting islets from self-deterioration.

Human Bone Marrow Subpopulations Sustain Human Islet Function and Viability In vitro.

AIMS: Allogeneic bone marrow (BM) has been shown to support human islet survival and function in long-term culture by initiating human islet vascularization and ß-cell regeneration. Various BM subpopulations may play different roles in human islet functions and survival. In this paper we investigated the effects of BM and its subpopulations, endothelial progenitor cells (E) and mesenchymal (M) cells on human islet's ß-cell function and regeneration. STUDY DESIGN: Isolation and identification of subpopulations from human bone marrow and culture with allogeneic human islet to investigate effects of different cell population on human islet function and regeneration. PLACE AND DURATION OF STUDY: Department of Medicine, Center for Stem Cell & Diabetes Research, RWMC, Providence, RI, USA, between 2010 - 2014. METHODOLOGY: Human islets were distributed from Integrated Islet Distribution Program (IIDP) and human bone marrow (BM) was harvested by Bone marrow transplantation center at Roger Williams Hospital. BM subpopulation was identified cell surface markers through Fluorescence-activated cell sorting, applied in flow cytometry (FACS), islet function was evaluated by human ELISA kit and ß cell regeneration was evaluated by three methods of Cre-Loxp cell tracing, ß cell sorting and RT-PCR for gene expression. RESULTS: Four different BM and seven different islet donates contributed human tissues. We observed islet ß-cell having self regeneration capability in short term culture (3∼5 days) using a Cre-Loxp cell tracing. BM and its subtype E, M have similar benefits on ß cell function during co-culture with human islet comparison to islet only. However, only whole BM enables to sustain the capability of islet ß-cell self regeneration resulting in increasing ß cell population while single E and M individual do not significantly affect on that. Mechanism approach to explore ß-cell self regeneration by evaluating transcription factor expressions, we found that BM significantly increases the activations of ß-cell regeneration relative transcription factors, the LIM homeodomain protein (Isl1), homologue to zebrafish somite MAF1 (MAFa), the NK-homeodomain factor 6.1 (NKX6.1), the paired box family factors 6 (PAX6), insulin promoter factor 1 (IPF1) and kinesin family member 4A (KIF4a). CONCLUSION: These results suggest that BM and its derived M and E cells enable to support human islet ß-cell function. However, only BM can sustain the capability of ß-cell self regeneration through initiating ß-cell transcriptional factors but not individual E and M cells suggesting pure E and M cells less supportive for islet long-term survival in vitro.

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

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.