Recent progress in islet transplantation coupled with the extremely limited supply of primary human islets has spurred the search for alternative sources of beta-cells for transplantation therapy in treating diabetes. Many potential sources of cells are being explored, including embryonic and adult stem cells, identification of intrapancreatic precursor cells, and human beta-cell lines. Here, we review the promise and problems with those cell sources, focusing on our studies in developing functional human beta-cell lines. Those efforts involve a two-step process in which the first is to introduce growth stimulatory genes that induce human beta-cells to enter the cell cycle. Immortalization can then be achieved by expressing the hTERT telomerase subunit. The second step is to induce differentiation. This involves a complex set of manipulations, including the expression of the important beta-cell transcription factor PDX-1. Although PDX-1 is critical for promoting beta-cell differentiation, we do not find increased expression of the glucagon-like peptide-1 receptor, a gene that has been reported to be induced by PDX-1. Further understanding of the factors governing beta-cell development are likely to be required before a robust cell-based therapy is available for the treatment of diabetes.
[Show abstract][Hide abstract] ABSTRACT: Our objective was to address current cell source limitations in engineering pancreatic â-cells for the treatment of type 1 diabetes by investigating retroviral genetic modification of murine embryonic stem cells (mESC) with a murine stem cell virus (MSCV) encoding proendocrine transcription factor Neurogenin 3 (Ngn3). We found that expression of Ngn3 and the enhanced green fluorescent protein (eGFP) reporter gene were both significantly silenced in genetically modified mESCs. To overcome this obstacle and enhance the efficiency of retroviral gene transfer to mESCs in general, we employed a virus-polymer complexation method to deliver more transgenes to mESCs. Despite increased transgene delivery and integration in mESCs, transgene expression did not increase. Results suggest mESCs may be restricted in several steps of retrovirus transduction. We then investigated which steps of the virus lifecycle restrict efficient transduction of mESCs by using a recombinant MMuLV-derived retrovirus and a recombinant HIV-1-derived lentivirus to compare three major steps in the transduction of mESCs and NIH 3T3 cells - virus binding, virus integration, and transgene expression. We found that retroviruses and lentiviruses similarly bind 3 or 4-fold less efficiently to R1 mES cells than to NIH 3T3 fibroblasts. We also detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels in NIH 3T3 cells, suggesting the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels, suggesting lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. We then investigated whether depletion of linker histone 1 in mESCs would alleviate silencing of retrovirus transgenes and improve gene transfer by transducing histone H1c, H1d, H1e triple null mESCs with different recombinant vectors. We found this did not improve viral gene transfer. This research is significant for improving protocols for gene transfer to ES cells and facilitating the use of modified ES cells in regenerative medicine. Ph.D. Committee Chair: Joseph Le Doux; Committee Member: Anthanassios Sambanis; Committee Member: David Archer; Committee Member: Michelle LaPlaca; Committee Member: Steve Stice; Committee Member: Todd McDevitt
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem (ES) cells are pluripotent, possessing the unique property to differentiate into any somatic cell type while retaining the ability to proliferate indefinitely. Due to their ability to recapitulate embryonic differentiation, ES cells are an ideal tool to study the process of early embryogenesis in vitro. Signalling cascades and genes involved in differentiation can be easily studied, and functional genomics approaches aim to identify the regulatory networks underlying lineage commitment. Their unique ability to differentiate into any cell type make ES cells a prime candidate for cell replacement therapy (CRT) of various degenerative disorders. Results from various disease models are promising and have demonstrated their principal suitability as a therapeutic agent in diseases such as myocardial infarctions, diabetes mellitus and Parkinson's disease. Prior to clinical trials in humans, two issues remain to be solved: due to their high proliferative potential, ES cells can form teratocarcinomas in the recipient, and depending on the source of the cells, ES cell grafts may be rejected by the host organism. This review discusses the current state of basic ES cell research with a focus on cardiac differentiation and gives an overview of their use in CRT approaches.
[Show abstract][Hide abstract] ABSTRACT: Recent advances in clinical islet transplantation have allowed patients with type 1 diabetes to become insulin independent, but the procedure is limited since islets from two donors per recipient are typically required. This limitation arises because within a few days of the islets being embolized into the portal circulation, at least half of the transplanted beta-cells have undergone apoptotic cell death triggered by hypoxic and chemokine/cytokine-mediated stress. We hypothesized that the survival of beta-cells in the early post-transplant period would be enhanced if naturally occurring inhibitor of apoptosis proteins (IAPs) were transiently overexpressed in the grafts. In the present study, we used a growth-regulatable beta-cell line (betaTC-Tet) as a model for beta-cells within islets, and examined whether adenovirally delivered XIAP (X-linked IAP-a highly potent IAP) could enhance beta-cell survival. In vitro, XIAP-expressing betaTC-Tet cells were markedly resistant to apoptosis in an ischemia-reperfusion injury model system and following exposure to cytokines. When Ad-XIAP transduced betaTC-Tet cells were transplanted subcutaneously into immunodeficient mice, the grafts were able to reverse diabetes in 3 days, vs. 21 days for Ad-betaGal transduced cells. This approach may allow more efficient use of the limited existing supply of human islets.
American Journal of Transplantation 07/2005; 5(6):1297-305. DOI:10.1111/j.1600-6143.2005.00891.x · 5.68 Impact Factor
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