A Study On The Effect Of Human Eyelid Adipose-derived Mesenchymal Stem Cells On Proliferation And Differentiation Of Human Retinal Progenitor Cells

Objective: Retinal degeneration,including retinitis pigmentosa(RP)and age related macular degeneration(AMD)results in vision malfunctioned.The death of retinal photoreceptors and retinal pigment epithelium is the main cause of retinal degeneration,while no effective treatment is currently available.Cell replacement has been demonstrated as a potentially useful way of treating retinal degeneration diseases.Most efforts have been reported that cell replacement therapies have the capacities to restore and replace lost or damaged tissues.Retinal progenitor cells(RPCs),which are a type of progenitor cell,are isolated from the retina.The advantages of RPCs are not only the ability of self-renew but also can maintain the multi-differentiation potential.The application of RPCs is simplicity,accessibly and safety than other stem cells.However,the issues of cell expansion and cell predisposed to photoreceptor cell fate remain a major challenge for the use of RPCs.Previous studies have proved that the secretome of Mesenchymal stem cells,including more active cytokines and neurotrophic factors,have the paracrine potential of enhancing proliferation and differentiation in several cell types.human eyelid adiposederived mesenchymal stem cells(HEASCs)are a new source of autologous mesenchymal stem cells,which are derived from neuroectoderm and potential applied in the tissue regeneration.The aim of this study firstly was to harvest the HEASCs and to investigate whether h RPCs could effectively proliferate,adherent and differentiate toward retinal specific cell types by treated with the condition medium of h UCMSCs or h EASCs.Finally,we identify the neurotrophic factors of h UCMSCCM and h EASCs and explore neurotrophic factors which can promote the RPCs differentiate to photoreceptor cells.Methods: The eyelid adipose tissue samples were extracted and cultured by explant culture method and the effect of age on the proliferative capacity of HEASCs was evaluated in vitro.Flow cytometry was used to detect the specific stem cell-related cell surface markers of HEASCs among three groups.The differentiation abilities of HEASCs were evaluated and quantified.The condition medium of HEASCs and UMSCs were collected under N2 neural supplement conditions.The proliferation,adhesion and neuronal differentiation capacities of h RPCs treated with h UCMSCCM and h EASCCM were evaluated by cell cycle analysis,quantitative polymerase chain reaction and immunofluorescence staining.To measure the concentration of specific cytokines,chemokines and other proteins,condition mediums were analyzed using antibody-based assays.Futhermore,we evaluated the effect of Activin A on growth and differentiation of h RPCs.To investigate the underlying mechanisms of Activin A promote the photoreceptor differentiation of h RPCs.Results: The HEASCs successfully outgrew from all donor age eyelid adipose tissues after explant culture.In culture,the cell populations were capable of forming adherent cells,a characteristic of other stromal stem cell populations and retain their bipolar shape.The proliferative rate,osteogenic and chondrogenic differentiation capacity were influenced by age increase.A shift in favor of expression of CD90 surfaces marker and adipogenic differentiation with increased age were observed.These results have shown culture under h UCMSCCM or h EASCCM increases the proliferation rate and the S and G2 phase cells and promotes the adhesion of h RPCs.This increased proliferation potential is coincident with an upregulation of Ki67 expression.Moreover,the upregulation expression of NF,Recoverin and Rhodopsin demonstrate that h UCMSCCM or h EASCCM favor h RPCs differentiation towards specialized retinal cells,including ganglion cells and photoreceptors.Under differentiation conditions,h RPCs treated with h UCMSCCM or h EASCCM increase the expression of retinal neuron and photoreceptor specific markers.The results of antibody-based assay showed that the total of 230 positive cytokines,including growth factors,cytokines,chemokines,cell receptors and inflammation factors.Only 62 positive cytokines were specific expressed in the condition medium of HEASCs and 59 positive cytokines were secret by UMSCs.Activin A was only highly expressed by HEASCs.CCK-8 cell proliferation expressions and Western blot demonstrated that there was no significantly change in proliferative rate of h PRCs stimulated by Activin A.Conversely,we found that Activin A treatment of h RPCs significantly differentiated into rod photoreceptors.The promotion effects were blocked by PI3K/AKT inhibitor(LY294002)and the level of phosphor-ERK and phosphor-AKT were decreased by inhibitor treatment.Conclusion: HEASCs were successfully isolated and cultured by an explant culture method.The proliferative rates,osteogenic and chondrogenic differentiation potentials significantly decreased as age increased.However,the expression of CD90 antigen and the adipogenic differentiation showed an age-related increases in HEASCs.The h UMSCCM and h EASCCM can stimulate RPC proliferation,promote it adherence and support RPC neuronal and photoreceptor differentiation.The results suggest that the effects of h UMSCCM and h EASCCM on h RPCs properties may provide a new strategy to improve the viability of h RPCs and photoreceptor differentiation capacities.Activin A plays an important role in the regulation of differentiation of h RPCs.Activin A promotes h RPCs differentiation via PI3K/AKT/ERK pathways.