Proteomics Analysis Of Marrow Mesenchymal Stem Cells Induction Into Photoreceptors Like Cells In Vitro

Mesenchymal Stem Cells (MSCs) could be induced to retinal photoreceptor cell.However , the features of MSCs as well as the exact mechnism that proteins interact with each other during its differention into photoreceptor-like cells have not yet been well discribed. We established an in vitro model for the survival of retinal explants, induced Bone Mesenchymal Stem Cells BMSCs into photoreceptor-like cells and identified proteins that are significantly different during BMSCs differentiation, to investigate the differentiation markers and the mechanism of related proteins in regulating the induction of BMSCs to differentiate into photoreceptor-like cells.Part 1 Induction of Human Bone Marrow Mesenchymal Stem Cells to Differentiate into Photoreceptor-like Cells in vitroObjective: To study the expression of cell-specific markers as rhodopsin and recoverin during the induction of BMSCs to differentiate into photoreceptor-like cells in vitro.Methods: The second to the fourth passage human BMSCs were divided into two groups Induced group and control group. Induction group BMSCs were cultured for 7d in conditioned medium mixed with 20ng/mlbFGF, 20ng/mlEGF, 20ng/mlBDNF and cultured RPE medium experimentally. For the control group, cells were cultured in the prepared MSCM only. Immunocytochemistry was carried out during different induction time (3d, 5d, 7d) for the detection of rhodopsin expression rate. Detected by Realtime-PCR of the cell expression of mRNA of rhodopsin and recoverin after 7-days induction.Results: Rhodopsin can be detected since the third day of inducing differentiation by immunocytochemistry, and rhodopsin-positive rate arrived to (28.87±0.43)% on day 7 . While rhodopsin can't be detected in all control groups.The difference between induced group and control group has statistical significance, p<0 01. The mRNA of rhodopsin and recoverin can be detected after after 7-days induction by Realtime-PCR,while it can't detected in control group . Conclusion:This study confirms BMSCs which co-culture with RPE in the culture medium containing cytokines in vitro can be induced to differentiate into photoreceptor cells, and express their specific markers rhodopsin and recoverin.Part 2 A Proteomic Study on The Differentiation of BMSCs into Photoreceptor-like Cells in vitroObjective: To investigate the marker proteins which are responsible for the differention of BMSCs into photoreceptor-like cells and the molecular mechanism that regulate the differentiation of BMSCs in vitro.Methods: HBMSCs from passage 4 to passage 6 were divided into two groups for experiment and control respectively. The induction of hBMSCs to differetiation were conducted as previous described. Two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry were used to analyse the protein diversity between the groups. Western-blot was conducted for a further confirmation of the significantly different proteins.Results: Thirty two proteins were detected significantly different in expression, among which 11 were up-regulated, while the others down-regulated. The proteomic database was established in photoreceptor-like differentiated BMSCs. These proteins were generally classified as follows, cytoskeletons, chaperones, metabolism kinases, signal proteins, proliferation, differentiation and apoptosis associated proteins, calcium binding proteins, ion chanels and others. The down-regulation of -tropomyosin, p38, chain C Human PCNA and the up-regulation of myosin, mitochondrial heat shock 60kD protein 1 variant 1, thioredoxin domain-containing protein 5 isoform 3 suggest that these proteins might play significant roles in the differentiation of hBMSCs into photoreceptor-like cells. The expression of Zyxin and tropomyosin was confirmed down-regulated by Western-blot.Conclusions: Together with these approaches, we established the diversity of the proteomic database when MSCs differentiated into photoreceptor-like cells and pointed out the marker proteins in responsible for the differentiation as well as its possible mechanism.