The potential of human adipose tissue derived mesenchymal stem cells in cell therapy for retinal degenerative diseases

Resum

In the dry form of age related macular degeneration (AMD) disease, the subretinal environment consists of damaged and dysfunctional retinal pigment epithelium (RPE) and degenerating neuroretina. The subretinal injection of human adipose tissue derived mesenchymal stem cells (hAD¿MSCs) to delay the process of disease progression in early onset of AMD could be one of the promising strategies under advance cell therapy options. The fate of injected hAD¿MSCs and their paracrine immunomodulatory effects might be beneficial for AMD and other retinal degenerative diseases. It would be better if these cells could be directed to RPE phenotypes before moving to their subretinal injection. There is one published report, in which vasoactive intestinal peptide (VIP) has been used to differentiate hAD¿MSCs towards RPE phenotypes but only partial success was achieved. It shows that there is a possibility to differentiate hAD¿MSCs to RPE cells but it requires to explore other differentiation inducing factors and their combinations with the previously used factor (VIP), which could stimulate hADMSCs to RPE like cells especially in the artificial culture environment partially similar to the subretinal environment of AMD. Along the process of differentiation, it would be logical and novel to investigate that hAD¿MSCs themselves in these environments may produce their positive or negative effects over the degenerating retina (neuroretina and RPE) under in vitro condition. The purpose of this work is to explore the potential of hAD¿MSCs as a future candidate for retinal tissue repair and replacement together with neuroprotection in cellular therapy for retinal degenerative diseases like dry AMD and others. In this work, hAD¿MSCs and RPE cells had been isolated from donated human lipoaspirates and post¿mortem eyes respectively and they had been characterized and maintained in culture condition and finally they had been subjected to many different environments slightly similar to the in vivo subretinal environment present in the AMD patients where both RPE and neural retina are under degeneration process; further these in vitro environments were supplemented with different differentiation factors (VIP, NIC and ATRA) and their combinations to analyze their influences on cell behavior of hAD¿MSCs and retinal tissue. For this purpose, the hAD¿MSCs had been co¿cultivated directly and indirectly with the inactivated and degenerating RPE and with degenerating human neuroretina (hNR) in the presence and absence of chosen factors (VIP, NIC and ATRA) and their combinations. In all these different conditions of differentiation, the fate of hADMSCs along with their effects on degenerating RPE cells and neural retina had been investigated by using different methods. The fate of hAD¿MSCs subjected to different conditions was investigated by phase contrast observations and by analyzing the expression of RPE protein markers in them by immunofluorescence and western blot (WB) studies. The effects of these differentiation cells along with mentioned factor(s) were studied over the in vitro degenerating hNR explants through histochemical staining and immunohistostaining with toluidine blue and with different neuroretinal markers respectively. The effects of some of these differentiating conditions over the MMC inactivated (stressed and dying cells) RPE cells were evaluated by cell viability/cytotoxicity assay and cell proliferation assay (alamarBlue® assay). It has been demonstrated in these experiments that hAD¿MSCs enhance their expression of RPE markers such as RPE65, Ezrin, Bestrophin, CK8/18, PEDF and others while they reduces their expression of MSCs¿specific marker CD90 significantly when they are co¿cultured directly or indirectly with MMC treated RPE cells (hRPE and ARPE19) and these effects of differentiation was further enhanced when supplemented with combinations of differentiating factors in the culture medium. These experiments demonstrated that hAD¿MSCs stimulate the proliferation and survival of MMC treated RPE cells in co¿culture conditions. The hAD¿MSCs have also demonstrated the neuroprotective effects over the in vitro degenerating neuroretinal explants and these neuroprotective effects were significantly enhanced in the presence of combinations of factors (VIP+NIC and VIP+NIC+ATRA). This study concludes that the hAD¿MSCs in combination with the factors of differentiation (VIP, NIC and ATRA) along with molecules secreted by the RPE cells not only can partially delay or rescue the degeneration of RPE and neural retina but also can induce the differentiation in hAD¿MSCs to RPE phenotypes and hence could replace the diseased and degenerated RPE cells to some extent.