Differentiation Of The Human Umbilical Cord Mesenchymal Stem Cells Into Retinal Pigment Epithelial Cells By Co-culturing With Sertoli Cells

Retinalpigmentepithelium(RPE) is a neuroectodermal derivative essentialfor the survival of photoreceptors, and supplies nutrition and provides severaltrophic factors that help maintain the normal physiology within theneurosensory retina and photoreceptors. Retinal damage causes permanent lossof vision, and photoreceptor loss in retinal degeneration is the major cause ofblindness.However, recent studies in regenerative medicine have given us thehope of rescuing visual function. One of the strategies is transplantation ofretinal cells, especially photoreceptors or RPE cells. However, the clinicalapplication of cell transplantation is hampered by the fact that cells are oftencultured with materials from other animals, such as feeder cells, serum andrecombinant proteins, and this poses a risk in terms of adverse immuneresponses. In the present study, we have established a method of inducingretinal differentiation of human umbilical cord mesenchymal stem cells（huc-MSCs） using the chemical compounds. These chemical compoundsare non-biological, do not trigger immune responses, have stable activity,show little difference between production lots, and are inexpensive. Therefore,retinal differentiation methods using chemical compounds are ideal for clinicalapplications. Umbilical cord mesenchymal stem cells are similar in biologyand immunological properties with bone marrow mesenchymal stem cells. Butin the proliferation and differentiation umbilical cord mesenchymal stem cellshave more potential abilities. Bone marrow mesenchymal stem cells havegreat limitations, based on in disease,age, injury of donor has affected itsfurther application. Umbilical cord mesenchymal stem cells source isconvenient and has no adverse effects on the donor. At the same time, therehas no ethical restrictions. Therefore, two kinds of cells in contrast, mesenchymal stem cells probably has a wider application prospect.In conclusion, we established a simple and effective protocol forthe differentiation of retinal pigment epithelium from huc-MSCs. Furtherstudy is warranted to establish selection methods, analyze photoreceptorfunctions, and transplant huc-MSCs-derived retinal cells in animal models ofretinal degeneration. However, the huc-MSCs/Sertoli cell coculture systemcan serve as a promising method for therapeutic applications and basicresearch on retinal degeneration disease.Objectives: In the present study, we investigated the induction of retinalpigment epithelial cells(RPE) by co-culturing human umbilical cordmesenchymal stem cells (huc-MSCs) with Sertoli cells(SCs). Which lays thetheoretical and experimental foundation for inducing huc-MSCsdifferentiation into photoreceptors induced by huc-MSCs-derived pigmentedcells.Methods:1Subculture of identified huc-MSCs: Identified huc-MSCs wereconventionally cultured. The growth and morphology of huc-MSCs weremonitored under contrast phase microscope. Huc-MSCs of P3were used toco-culture.2Isolation、purification and identification of SCs from SD rat testis:Testis from19～21-day-old SD rats were removed and decapsulated. Theisolated cells were incubated at37℃in a humidified atmosphere of5%CO2.Cultured cells were treated with Tris-HCl after48hours incubation to increasethe purity. The morphology of cultured cells were monitored daily undercontrast-phase microscope, and were identificated by Gimsa staining, acridineorange(AO) fluorescence staining and Feulgen staining at the seventh day.3Co-culture of huc-MSCs and SCs: SCs were resuspended in culturemedium and seeded at about107cells/cm2in culture dish covered withgelatin. Co-culture supplemented with retinoic acid (RA) was carried out at37℃in a water-saturated atmosphere of95%air and5%CO2. The morphologyof co-culture cells were monitored under contrast phase microscope. After a week incubation, the gene expression of tyrosinase-related protein2(TRP-2),retinal pigment epithelium-specific65kDa protein(RPE65),proto-oncogene tyrosine-protein kinase MER(MERTK)and cellularretinaldehyde-binding protein(CRALBP) was determined by RT-PCR, and theprotein expression of Pax6was detected by indirect immunofluorescence.Results:1Huc-MSCs of P3were all fusiform shape and in a good state.2Primary cultured SCs began to adhere to the wall in the second day.The purity of SCs was above95%7days later. The cell morphologycharacteristics was consistent with those of SCs by eosin staining, AOfluorescent staining and Feulgen staining.3After culturing with Sertoli cells for2weeks, large patches ofpigmented epithelial cells were obtained,and each cell contained a significantamount of melanin pigment granules. The RPE65, Trp-2, Mertk and CRALBPmRNA was present in RPE clusters detected by RT-PCR.The presence of Pax6in RPE clusters was detectable by immunofluorescence.Conclusion: These results indicate that coculturing huc-MSCs with SCsis a useful and efficient method for inducing RPE and providing an insightinto the use of huc-MSCs for retina regeneration.