| BACKGROUNDRetinal Pigment Epithelium is very complex in the construction and its function is special. RPE can phagocytosis the shedding rodoutersegmeni,which is very important in the regeneration of photoreceptor cells and the maintenance of normal vision function. The abnormal Retinal Pigment Epithelium can lead to severe disease of retina and some severe genetic eye diseases, such as Best disease, Stargardt disease and Leber congential blindness, age-related macular degeneration. The RPE cells transplantation in the therapy of eye diseases is a hot area in the future. The problem is that how we can get RPE cells that can be used for transplantation.The rise of cell sheet engineering especially the stem cell engineering provides a new method for the therapy of retinal damage. The use of stem cell was discussed several times,it is also a hot area in nowadays.Recently, Kucia group of Louisville university separated a kind of embryonic-like multi-lineage potential cell cells from mouse bone marrow and human umbilical cord blood,named very small embryonic like stem cells. These cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). The morphology of VSEL-SCs is similar to embryonic stem cells and they have the potential of multi-lineage differentiation as the embryonic. VSELSCs can be induced into endoderm, mesoderm and ectoderm cells, they are powerful in the potential of differentiation, and then become a nice candidate for the clinical stem cell. They can also avoid the ethical issues and become useful in the clinical field.Recently, our group purified a rare population of primitive Scal+/Lin-/CD45-cells from murine bone marrow by employing Magnetic. Based on flow cytometric and gene expression analysis, we want to find a resource of tissue-special stem cells.Absolutely, the most widely used seed cells are embryonic stem cells.Embryonic stem cells are powerful in the potential of differentiation. Recently, the induction of ESC into RPE in vitro is success in some experiments, The induction of ESC into RPE can be a resource of retina transplantation, which may be widely used in future. But the method of induction is limited.We use PA6 cells as the feeder cells in the condition similar to the induction of skin melanin cells. The ESC can be differentiate to several kinds of RPE like cells full of pigment when cultured with PA6 cells. There is no better method to obtain RPE except co-culture and tissue transplantation. So we want to build a system that free of feeder cells that can induce ESC to RPE and photoreceptor with high efficiency.OBJECTIVE1 To Separate scal+lin-CD45-cells from the tissue of neonatal murine retina through the method of magnetic bead sorting.The cells showed the same characterization as the embryonic stem cells.2 To Analyse the morphology and differences of gene expression of murine retinal multipotential stem-like cells and embryonic stem cells.3 To explore the induction of RPE from murine retinal multipotential stem-like cells and estabish a stepwise feeder-free culture method of generating RPE from mouse ES cells with high efficiency.METHODS1 The purification of mouse retinal retinal multipotential stem-like cells:We separated the tissue of murine nerve-retina from new bron C57 mice and made it to monoplast, then sortted the Lin-cells and then Scal+cells through magnetic beads sorting and got scal+lin-CD45-cells.The FACS and immunofluorescence showed they had the same characterization as the embryonic stem cells.2 The cultivation of mouse retinal multipotential stem-like cells,mouse ES and RPE:We cultured mouse embryonic stem cells, retinal multipotential stem-like cells with mouse ES medium and NeuroCult Basal Medium, and got the RPE by induction,then observed the characterization. 3 The identification of mouse retinal multipotential stem-like cells,mouse ES and RPE from induction:We identified the ES and mouse retinal multipotential stem-like cells by AKP,immunofluorescence, and we cultured mouse ES with feeder-free method and defined factors for 26 days, then detected embryonic stem cell-derived retinal cells through cell immunohistochemistry, Real Time-PCRand Transcription-PCR.4 The detection of the differences of gene expression of murine ES,mouse retinal multipotential stem-like cells and RPE and the murine ES and ES-induced RPE:We detected the expression of the three totipotent genes Oct4,Nagnog,Sox2 on the embryonic stem cells, mouse retinal multipotential stem-like cells and retinal pigment epithelium cells through Real Time-PCR. The level of MITF, TYR, RPE65, TYRP1 genes were also analyzed on ES, retinal multipotential stem-like cells and retinal pigment epithelium cells through Reverse Transcription-PCR.RESULTS1 The sorting rate of mouse retinal multipotential stem-like cells:We separated scal+lin-CD45-cells from the tissue of neonatal murine retina through the method of magnetic beads sorting and got about 1.2% cells.2 The morphology and immuhistochemisty of mouse retinal multipotential stem-like cells,mouse ES and RPE:Those mouse retinal multipotential stem-like cells expressed Sca1 antigen,their diameter were 3-6um,circle,high nuclear-to-cytoplasm ratio.The mouse ES showed typical morphology and expressed AKP positive. The RPE expressed specific markers.3 The differences of gene expression of murine ES,mouse retinal multipotential stem-like cells and RPE and the murine ES and ES-induced RPE:Real Time-PCR showed that the three totipotent genes Oct4,Nagnog,Sox2 expressed on embryonic stem cells, retinal multipotential stem-like cells but not on retinal pigment epithelium cells.Reverse-Transcription PCR showed that MITF, TYR, TYRP1 RPE65genes had a different expression level on them.4 The induction of RPE from mouse ES:We got RPE cells from mouse ES but not from mouse retinal multipotential stem-like cells through our protocal. The embryonic stem cell-derived retinal cells expressed specific markers.CONCLUSION1 We separated retinal multipotential stem-like cells from the tissue of neonatal murine retina successfully.The cells showed the same characterization as the embryonic stem cells.2 The expression level of potential genes on retinal multipotential stem-like cells was lower than embryonic cells but much higher than RPE,showed the characterization of adult stem cell. 3 We failed to induce mouse retinal multipotential stem-like cells into RPE,but have established a stepwise feeder-free culture method of generating RPE from mouse ES cells in vitro with high efficiency. |
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