Human Umbilical Cord-derived Mesenchymal Stem Cells Differentiate In Vitro And Rescure Photoreceptor Degeneration In Vivo

Background Retinitis pigmentosa (RP) is a well-know clinical entity that was named by German physician Franz Donders in 1857. It is characterized by early nyctalopia and non-inflammatory, bilateral, progressive, degenerative pigmentary retinopathy. The loss of photoreceptors, first rods and then cones, is followed by perivascular pigment deposition within the retina. Thus, the word "retinitis" is a misnomer, because retinal inflammation does not play a major role. RP is a heterogeneous group of inherited retinal disorders. More than 160 different mutations ingenes coding for proteins with remarkably diversefunctions result in rod photoreceptor degeneration. The fundus typically show intraretinal pigmentation, referred to as bonespicule deposits, created by the migration of retinal pigment epithelial (RPE) cells and their deposition around retinal vessels. There is currently no cure or effective therapy for the treatment of RP.Mesenchymal stem cells (MSCs) are undifferentiated cells that are able to self-renew and that have a high proliferative capacity. These cells comprise a rare population of multipotent precursors that are capable of supporting hematopoiesis. More recently, some groups have reported success in isolating and establishing MSCs cultures from umbilical cord (UC) vein and UC stroma, also called Wharton's jelly. Moreover, several reports suggest that MSCs were able to differentiate into various cell types, including chondrocytes, osteocytes, adipocytes, myocytes, and neurons. Raymond and colleagues have compared efficacy of four human-derived cell types in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the Royal College of Surgerons (RCS) rat early in the progress of degeneration. Umbilical tissue-derived cells, placenta-derived cells, and mesenchymal stem cells were studied; dermal fibroblasts served as cell controls. Both umbilical-derived and mesenchymal cells significantly reduced the degree of functional deterioration in each test. Umbilical tissue-derived cells gave large areas of photoreceptor rescue; mesenchymal stem cells gave only localized rescure. Donor cells were confined to the subretinal space. There was no evidence of cell differentiation into neurons, of tumor formation or other umtoward pathology. Since the umbilical tissue-derived cells demonstrated the best photoreceptor rescue and, unlike mesenchymal stem cells, were capable of sustained population doubling without karyotypic changes, it is proposed that they may provide utility as a cell source for the treatment of retinal degenerative diseases such as retinitis pigmentosa.Umbilical cord were recently found as a good source of mesenchymal stem cells, so here we want to study whether umbilical cord mesenchymal stem cell could be induced into the neuron-like cells, which could promote the neural function recovery after photoreceptor degeneration.Purposes We plan to explore the feasibility of generation of neuron-like cells induced from umbilical cord, and then transplant these cells into the vitreous cavity of photoreceptor degeneration induced by 60 mg/kg MNU intraperitoneal injection and investigate the survival, distribution and differentiation of the grafted cells in order to provide the theory basis and the experimental data in retinal degeneration clinical application of umbilical cord-derived mesenchymal stem cells.Methods We chose the umbilical cord from healthy neonatal with full-term pregnancy cesarean section using the method of enzymatic digestion and cell adherent to gain the primitive cells. Passage 3 were used in this study.study. The third passage of MSC was placed in 24-well cell culture plates at a density of 1×104/well. After the adherent of cells. the medium was changed to DMEM/F-12 containing different concentration of ATRA (0.25μmol/L,0.5μmol/L,1.0μmol/L, 2.0μmol/L,4.0μmol/L) for 24 h respectively. The cells cultured without ATRA were taken as the coutrol group. After another 24 h, the morphologie changes of induced cells were observed by inverted microscope and cell proliferation, apoptosis of ATRA was analyzed using the MTT colorimetrie assay. We take another control group and ATRA groups to detect the apoptotic and positive stained percentage of induced cells by Annexin V-FITC/PI combining flow cytometry. The optimal concentration of ATRA was determined by all the above mentioned index. In addition, to further determine the function of umbilical cord-derived mesenchymal stem cells, we transplanted these cells into the vitreous cavity of photoreceptor degeneration induced by MNU intraperitoneal injection. Sprague-Dawley rats were intraperitoneally injected with 60 mg/kg mediating N-methyl-N-nitrosourea (MNU) at 7 weeks of age, and then 12h after umbilical cord-derived mesenchymal stem cells were given. Rats were killed 6h,12h,24h,3d and 7 d after MNU, and 3d and 7d after MNU-treated and hUC-MSCs vitreous cavity injected. Apoptosis in photoreceptor cells was detected by performing formamide-induced DNA denatration and staining with anti-single-stranded DNA antibody. Retinal morphologies were compared and evaluted morphometrically using the photoreceptor cell ratio and retinal damage ratio as indices to evaluate the efficacy of hUC-MSCs. We examined expression of the rhodopsin and recoverin in retinas of MNU-treated rat concurrently treated with or without hUC-MSCs, compared with nomal retinas as control.Results1. Compared to tile contml group. ATRA at tile concentration of 0.25μmol/L did not inhibit the proliferation of umbilical cord MSC obviously (t=0.72,1.32, P>0.05). Part of MSC were floating instantly at the moment of adding ATRA of 4.0μmol/L and no adherent cells were observed after 24 h culture. Exposed to ATRA at the concentration of≥1.0μmol/L for 24 h, the proliferation of MSC were significantly inhibited, showing a dose-dependent manner (t=8.8,18.9,22.1; P<0.01).0.5μmol/L of ATRA did not affect the proliferation of cells and its morphology remained normal; 1.0μmol/L of ATRA affected very few cells:but 2.0μmol/L of ATRA cultured for 24 h inhibited the proliferation of cells obviously than 1 h and tile cells increased in size and became flattened. Flow cytometry showed tllat the rate of apoptosis between the control group and≥1.0μmol/L groups were significantly different (t=9.88,19.95,31.61; P<0.01).2. H&E staining of retinal section s showed no substantial differences in the retinal structures 6 h after MNU injection. The first changes obseved 12 h after injection of MNU were expansion of extracellular space with vacuoles in the outer nuclear layer (ONL) and the outer segment (OS). By day 1, the thicknesses of the ONL and OS were slightly thinner than those in retinas from rats before MNU treatment. These changes became more severe over 7 days. Numerous positive TUNEL staining cells were also observed in the ONL one day after MNU treatment, but not in any other layers. No TUNEL-stained cells were detected in retinas before MNU treatment, and no other obvious structural changes were observed in the MNU rats. Rats treated with hUC-MSCs showed ameliorative effect and significant effect at 3 d. Also, as evaluated by retinal damage ratio, hUC-MSCs completely rescued photoreceptor cell damage.3. hUC-MSCs survived following transplantation into the vitreaous cavity in SD rats induced by MNU. The hUC-MSCs had migrated as far as the ONL. The number of surviving cells in the degenerating SD rat retina was significantly higher compared to that in the normal rat retina. Moreover, hUC-MSCs rescured MNU-induced photoreceptor cell apoptosis via up-regulating recoverin and rhodopsin mRNA expression.Conclusion1. Umbilical cord that is the easist obtainable source of MSCs, easily cultured to a high purity and have favourable biological characteristics and multipotency. These stem cells are suitable candidates for further experimental manipulation. In the process of inducing umbilical cord MSC into neuron-like cells 0.5μmol/L ATRA was the optical concentration.≥1.0μmol/L ATRA can inhibit the cell proliferation, increase the apoptosis of cells significantly and caused obvious damages. 2. A single administration of MNU induces photoreceptor cell loss in SD rats. Disease progression is rapid in that the active signs of photoreceptor cell apoptosis have ended by day 7. MNU is a direct acting alkylating agent and induces retinal damage through 7-methyldeoxyguanosine DNA adduct formation in photoreceptor nuclei. Thus, our MNU-treated rats comprise a valid model of photoreceptor cell degeneration.3. hUC-MSCs treatment rescured MNU-induced photoreceptor cell apoptosis via up-regulating recoverin and rhodopsin mRNA expression. The beneficial effect of hUC-MSCs on MNU-induced retinal injury suggests that hUC-MSCs plays an important role in photoreceptor cell apoptosis, and provides a new direction for clinical transplantation in the future.