Neural Stem Cell Properties Of Muller Glia Cells In Rat Retina: Regulation By Wnt And Notch Signaling Pathway

Objectives1. To simplify the procedure of primary cultivation and purification of retinal Muller cells in vitro.2. To study the mechanism and regulation underlying the activation of the latent neural stem cell properties of Muller cells.3. To study the roles of Wnt and Notch signaling in the activation of neural stem cell properties in Muller glia.Methods1. Eyeballs were enucleated from newborn rats and retinas were mechanically dissociated into small aggregates and cultured in DMEM/F12 (Dulbecco's Modified Eagle's Medium/Nutrient Mixture F12 Ham's) medium containing 10%FBS (fetal bovine serum) for 8-10 days. After that, the floating reinal aggregates and debris were removed and the medium was changed every 2-3 days to get more putified cell population. The cells were passaged when they were confluent. Immunofluorescence assay was performed to identify the cultured Muller cells, and FACS(Fluorescence activated cell sorting) was carried out to determine the purity.2. Muller cells were dissociated using 0.25%Trypsin-0.05%EDTA and cultured in Serum Free Medium (DMEM/F12 containing lx N2 supplement,2x B27 supplement,2 mM L-glutamine,100 U/ml penicillin and 100μg/ml streptomycin) supplemented with 10 ng/ml bFGF(basic fibroblast growth factor) and 20 ng/ml EGF (epidermal growth factor) at a density of about 1 x 105 cells/cm2 for 3-5 days to generate neurospheres, refreshed the medium and grow factors every 2 days. Immunofluorescence assay, Real time RT-PCR and Western blotting were performed to test the expression of stem cell markers such as Nestin, Musashi-1 and Pax6 in the neurospheres, CCK-8 assay was to test the proliferation of the neurospheres. To examine the differentiation potential ability, neurospheres were shifted into the differentiation medium (DMEM/F12 containing 10%FBS, lx N2 supplement,2x B27 supplement,2mM L-glutamine,100 U/ml penicillin and 100μg/ml streptomycin) with the absence of EGF and bFGF, culture was continued for 5-7 days, immunofluorescence assay was to test the expression of GFAP in the redifferentiated cells.3. The mRNA levels of the components in Wnt and Notch pathway such as Fzdl, Fzd2, Lefl, Notch 1, Delta 1 and Hesl in the neurospheres were quantified by Real time RT-PCR analysis compared to which in the primary cultured Muller cells. During the dedifferentiation assay in Muller cells, Western blotting was performed to test the protein levels of Wnt2,β-catenin, Notch I, Pax6 and Nestin in d0, d1, d2, d3, d4 and d5.4. Pathway agonist (Wnt-3a, Notch1) and antagonist (Dkk-1, DAPT) were to be added to perturbed the Wnt and Notch signaling for studing their regulation effects in activating the neural stem cell properties in Muller glia. FACS was performed to test the Pax6 positive cells in the neurospheres. CCK-8 assay was to reveal the proliferating ability of the neurospheres by interfering the Wnt and Notch signaling.Results1. Cultured Muller cell had large cell bodies and relatively richer cytoplasma. About 97.2±1.43%of the cells were glutamine synthetase (GS) positive, and about 90.3±2.17%of the cells were Vimentin positive. FACS analysis showed that 99.7% of the cultured cells were GS positive after 3 passages.2. After 3-5 days cultured in the serum free medium, most of the Muller cells proliferated and formed neurospheres with an average size of 1OOμm in diameter. Immunofluorescence analysis show that, most of the neurospheres express the stem cell markers such as Nestin, Musashi-1 and Pax6. The rusults of Real time RT-PCR showed that, Pax6 and Nestin mRNA levels in neurospheres was about 5.57-and 3.98-fold compared to the Muller glia (p<0.05). Western blotting analysis showed that the protein levels of Pax6 and Nestin were also increased in the neurospheres. In the FACS assay, the positive cells of Pax6 and Nestin in the neurospheres were 80.2%and 60.4%versus 7.8%and 7.3%in the Muller glia. CCK-8 assay revealed that the neurospheres have self-renewing ability and can redifferentiate in vitro cultrue expressing GFAP.3. Real time RT-PCR analysis showed that, the mRNA levels of the components in the Wnt and Notch pathways were increased, Fzdl, Fzd2, Lefl, Notchl, Deltal and Hesl was 2.44-,2.82-,4.62-,3.24-,2.64-and 5.71-fold compared to the Muller glia (p<0.05). Western blotting analysis showed that, the protein levels of Wnt2, β-catenin, Notch 1, Pax6 and Nestin was increased accordingly from d0 to d5.4. In the Wnt-3a+Notch1 group, there were significant more and larger neurospheres, while there were less and smaller in the Dkk-1+DAPT group. FACS analysis showed that, the Pax6 positive cells in the Wnt-3a+Notch 1 group was 94.1%, the Wnt-3a group was 87.1%and the Notch1 group was 76.2%, which were higher than the conrol (63.7%), while the Dkk-1 group(38.4%) and the DAPT group (31.7%) were lower. CCK-8 assay showed that, the neurospheres proliferate more quickly in the Wnt-3a+Notch 1, besides, Wnt-3a and Notch 1 group were still both higher than the cells in control group, while in the Dkk-1 and the DAPT group, they proliferated more slowly compared to the control group.Conclusions1. The modified procedure intoduced in our experiment is a simple and practical method for culturing retinal Muller cells.2. Retinal Muller cells are potential stem cell source in the adlut retina. New born SD rats Muller cells can generate clonal stem cell neruospheres with the sitimulation of growth factors in vitro and display cardinal features of neural stem cells.3. The neurospheres derived from retinal Muller cells exhibite the neural stem cell characteristics and have the ability of self-renewing and redifferentiation.4. The Wnt signaling plays an important roles in the activation of the stem cell properties in Muller cells in concert with Notch.. Up regulation of both the signaling can facilitate the dedifferentiation process of dedifferentiation and also can promote the proliferation of the neurospheres derived from Muller glia.