| PartⅠ: Isolation and Pre-induction with Neonatal Retina of Human Peripheral Blood Mononuclear CellsPurpose: Isolate mononuclear cells from adult human peripheral blood and pre-induce them with retina of 1-day-old SD rats. Then study the changes from their morphology and gene expression.Method: The human peripheral blood mononuclear cells were obtained from peripheral blood of healthy humans through density gradient centrifugation. Then, human peripheral blood mononuclear cells were seeded at the bottom of transwell plates and co-cultured with retinal tissue of 1-day-old SD rats for 4 days. Quantitative real-time polymerase chain reaction was performed to reveal alterations in the expression levels of specific RNAs following pre-induction. After pre-induction, human peripheral blood mononuclear cells were transplantated into the subretinal space of the mice.Results: Freshly isolated human peripheral blood mononuclear cells were spheroid in suspension. After four days’ incubation, several morphologically distinct cell subtypes were observed: spheroid suspended mononuclear cells, cells with one or two shortprotrusions and cell masses. The results of quantitative real-time polymerase chain reaction revealed that RNA expression levels of Nestin、βⅢ-tubulin、Rhodopsin、MAP2、GFAP、Synapsin、CD14、CD44、CD45、CD11b and CD19 increased after pre-induction, while expression of CD16、CD3、CD34 and Vimentin decreased. Among them, the changes of MAP2 、 Rhodopsin 、 CD19 and Vimentin were significant.Conclusions: After 4 days of pre-induction, huamn peripheral blood mononuclear cell can differentiate into early-stage retinal precursor cells and primarily rhodopsin-positive photoreceptor-like cells.Part Ⅱ: Survival and Function of Pre-induced Peripheral Blood Mononuclear Cells After Subretinal Transplantation to Retinal Degenerative MicePurpose: To investigate the survival and function of pre-induced Peripheral blood mononuclear cells after subretinal transplantation.Method: Pre-induced peripheral blood mononuclear cells were transplanted into the subretinal space of rds and rd1 mice. Then, six months after transplantation, treated eyes of rds mice were collected and cell phenotype studied by immunofluorescent staining. To assess retinal function, Electroretinography responses of rd1 mice were recorded one, three, and five months after subretinal transplantation. After electroretinography measurements, a subgroup of treated mice was sacrificed, and the treated eyes were harvested to measure the expression of human mitochondrial cytochrome b gene.Results: At 6 months after human peripheral blood mononuclear cells injection, many transplanted cells still expressed nestin, vimentin, βIII-tubulin, MAP2, or rhodopsin. Most of them were dispersed as single cells and had spread into the inner nuclear layer and the ganglion cells layer. However, a subgroup transplantated cells-derived cells were found that did not express the ptotein neural markers. Human mitochondrial cytochrome b gene was still expressed in mouse retina one, three, and five months after transplantation as shown by electrophoretograms of PCR products. That indicated the peripheral blood mononuclear cells survived at these time points. The electroretinography measurement showed that both eyes of the rd1 mice showed flat electroretinography responses after one month of injection. Three months after transplantation, all treated mice examined had clear responses in both eyes. Five months after transplantation, however, none of the untreated contralateral eyes exhibited a light response, while only part of treated eyes did.Conclusions: Pre-induced human peripheral blood mononuclear cells can survive in the subretinal space of degenerated mouse retina for at least six months and exhibit the protein expression phenotypes of retinal neurons and photoreceptors. The electroretinography results also demonstrates that retinal function can be restored five months after pre-induced human peripheral blood mononuclear cells transplantation. |
PDF Full Text Request