| Within the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal stem or mesenchymal progenitor cells. Mesenchymal stem cells (MSC) are a population of pluripotent cells within the human, bird or rodent bone marrow microenvironment defined by their ability, either in vitro or in vivo, to differentiate into the types of the osteogenic, chondrogenic, tendonogenic, adipogenic, neural cells and myogenic lineages. The methodologies to isolate and culture-expand MSC from human bone marrow for establishing the cellular or tissue aspects of differentiation model in vitro have been set up. The multipotential of these cells, the easy isolation and culture property, as well as their high ex vivo expansive potential makes these cells an attractive therapeutic tool. Mere we report that bone marrow mesenchymal stem cells, after subretinally transplanted into normal or Nd: YAG laser-injured rat eyes, can integrate into retina pigmented epithelial (RPE) , photoreceptor cell layer, bipolar cell layer and ganglion layer.We tested the hypothesis that MSC can be differentiated into retinal cells by transplanting MSC in vivo into subretinal space. This hypothesis was tested by three approaches (i) subretinal transplantation of MSC (STM) from male rats into the females and the detection of donor cells in the recipients by means of DNA probes to the Y chromosome sry region and (ii) STM from male MSC transduced by recombinant adeno-associated virus (rAAV) carrying green fluorescence protein (GFP) into the females and the detection of the GFP fluorescence by means of fluorescence microscope, (iii) STM by DAPI labeling and the detection of the GFP fluorescence by means of fluorescence microscope. In situ hybridization and fluorescence microscopeobservation were used to distinguish donor cells from recipient cells. Then we verified that the MSC-derived cells could express CK, NSE and glial fibrillary acidic protein (GFAP) respectively by immunohistochemistry analysis.Female rats were subretinally injected with male MSC engrafts. 4 weeks later the rats were sacrificed and the eyes were enucleated and embedded in paraffin. Using polymerase chain reaction (PCR), a digoxigenin-labeled 459 base pair sized sry gene, which is a gene of the Y chromosome in rat, was synthesized for the sex determination. The probe was then used to detect male mouse cells in tissue sections by in situ hybridization. This constituted a system in which the presence of cells originating from the donor in the recipient retina could be easily detected. Y chromosome sry gene hybridization was finally changed into a color reaction. Some nuclei showed positive signal for the sry gene PCR probe. Under optical microscope observation the ivy-positive cells were found in the outer and inner nuclear layers and photoreceptor cells.Another approach to examine the differentiation of MSC was GFP detection. AAW-gfp was transduced into MSC in vitro. Adenovirus was added into the culture to show that MSC can express GFP. STM was performed from the AAV-gfp-transduced male MSC into female retina. 4 weeks later the female rats were sacrificed and the eyes were enucleated and were made into frozen sections. Fluorescence microscope observation in 490nm wavelength showed that GFP positive cells were dispersed in RPE layer, photoreceptor layer and ganglion cell layer, whereas the untreated female retina has no GFP positive cells. This result showed that the STM has made the donor MSC integrated in the subretinal space without disturbing the retinal organization and lamination and AAV-gfp-MSC can be differentiated into retina-like cells, which have the same results as the ISH showed. We also found that some of the vessel endothelial cells in choroid showed GFP fluorescence, which means that the GFP-labeled cells might take part in the vessel construction (data not shown).DAPI labeling was used to trace how MSC change in the retina. We found that on day 10 DAPI positive cells were found mainly around the injected site in either...
|
PDF Full Text Request