Bone marrow mesenchymal stem cells (BMSCs) have a therapeutic role in retinal degeneration (RD). However, heterogeneity of BMSCs may be associated with differential therapeutic effects in RD. In order to confirm this hypothesis, two subsets of rat BMSCs, termed rBMSC1 and rBMSC2, were obtained, characterized and functionally evaluated in the treatment of RD of Royal College of Surgeons (RCS) rats. Both subpopulations expressed mesenchymal stem cells (MSC) markers CD29 and CD90, but were negative for hemacyte antigen CD11b and CD45 expression. In comparison with rBMSC2, rBMSC1 showed higher rate of proliferation, stronger colony formation, and increased adipogenic potential, whereas rBMSC2 exhibited higher osteogenic potential. Microarray analysis showed differential gene expression patterns between rBMSC1 and rBMSC2, including functions related to proliferation, differentiation, immunoregulation, stem cell maintenance and division, survival and antiapoptosis. After subretinal transplantation in RCS rats, rBMSC1 showed stronger rescue effect than rBMSC2, including increased b-wave amplitude, restored retinal nuclear layer thickness, and decreased number of apoptotic photoreceptors, whereas the rescue function of rBMSC2 was essentially not better than the control. Histological analysis also demonstrated that rBMSC1 possessed a higher survival rate than rBMSC2 in subretinal space. In addition, treatment of basic fibroblast growth factor, an accompanying event in subretinal injection, triggered more robust increase in secretion of growth factors by rBMSC1 as compared to rBMSC2. Taken together, these results have suggested that the different therapeutic functions of BMSC subpopulations are attributed to their distinct survival capabilities and paracrine functions. The underlying mechanisms responsible for the different functions of BMSC subpopulation may lead to a new strategy for the treatment of RD.
Genetic manipulation of the porcine genome to produce genetically modified pigs for various biomedical and agriculture applications has been hampered by the lack of an ideal cell type that can grow in culture for a long period of time and is amenable to various genetic manipulations with high efficiency. The cell type currently used for various genetic manipulations is fetal fibroblast. These cells have very limited life span in culture and the efficiency of gene targeting is very low. There is no report of isolation of functional embryonic stem cells (ESC) from pig that would have been used to produce transgenic pigs with high efficiency as has been possible in mice. Recently, porcine induced pluripotent stem cells (iPSC) have been reported by 3 groups. However, they have yet to be tested for genetic manipulations and production of transgenic pigs. In this study, we developed a simple but novel strategy to recover stem cells from adult porcine liver and adipose tissues. Small colonies with few cells became visible as early as 2 to 3 days under reduced oxygen conditions on collagen-coated plates, and a full-grown colony with a fibroblast-like morphology took 10 to 14 days to form. Ten single colonies per tissue were isolated, subcultured, and monitored for growth and gene expression. Both of these cell types maintained steady growth through 70 population doublings (at the time of writing) and are still growing without any change in their morphology. Reverse transcription PCR was used to monitor gene expression. Both cell types show strong expression of c-Myc and KLF4. Moreover, low expression of Oct-4 and Lin28, 2 important genes related to pluripotency, was also detected in cells derived from adipose tissues, indicating that these cells could be easily reprogrammed to pluripotent state. These cells maintained a normal karyotype after long-term culture. Cell lines with stable genetic modifications and extended expression of transgene were obtained when these cells were transfected with a plasmid containing the neomycin resistance gene and selected under G418. Further, these 2 cell types, liver stem cells (LSC) and fat stem cells (FSC), and fetal fibroblasts (FF) as a control were used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos. The average fusion rates were 87, 81, and 89% for LSC, FSC, and FF, respectively. Of 2 recipients receiving nuclear transfer embryos produced with each cell type, one established pregnancy at Day 30 (50%). Efficiencies were 5% (11 fetuses/223 embryos transferred), 1.8% (4 fetuses/228 embryos transferred),and 5% (11 fetuses/219 embryos transferred) for LSC, FSC, and FF, respectively. Thus, these adult liver and fat stem cells are attractive cell types for cloning valuable adult animals with high efficiency and for SCNT transgenesis.
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