Abstract
Transplantation of isolated pancreatic islets in type 1 diabetes is limited by rareness of donor organs. Generation of insulin producing cells from human pancreatic islet derived nestin+ precursors has been demonstrated but is complicated by heterogeneity of stem cell populations. Furthermore, cultures enriched with nestin+ progenitor cells grown out from human pancreatic islets according to the established protocol of Zulewski et al. (Diabetes 50:521, 2001) feature a mean life span of 16 passages with continuous decrease of growth from passage 6 until growth arrest. Therefore we tested the growth enhancing properties of the proliferation associated protein p8 in human nestin+ hMSC-TERT bone marrow derived adult stem cells. hMSC-TERT cells were transfected with CMV promoter driven p8-IRES-GFP or mock-IRES-GFP vectors. Transient transfection results in significantly increased numbers of GFP+ cells after 12 and 18 hours which remain elevated for up to 30h. Loss of growth induction at later timepoints is caused by dilution of cellular plasmid content during mitosis. In contrast, stably transfected and selected subclones with p8 overexpression display durable significant augmentation of proliferation (BrdU+ cells) and growth index (cell numbers) by 3 and 1.75 fold mock, respectively. This approach was transferred to nestin+ precursor enriched cultures using the full length nestin promoter. Preliminary data of transient transfection display results similar to the stem cell model hMSC-TERT. Nestin promoter driven constructs further allow selection of nestin+ cells from mixed primary cultures. Once selected, nestin+ subclones with p8 overexpression may have an extended life span. This approach provides a tool for screening of heterogeneous nestin+ stem cell cultures for subpopulations suitable for differentiation into insulin producing cells in the light of limited availability of human donor material for treatment of diabetes mellitus type 1.
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