Experiment Research For Directed Differentiation Of Human Adult Stem Cells Toward Retinal Photoreceptor-like Cells

【Objective】 To compare the differentiation ability of human orbital adipose tissuederived stem cells and human periodontal ligament derived stem cells, toward retinalphotoreceptor cell. To research the protocol for isolating adipose tissue-derived stem cellsfrom human orbital adipose tissue by explant method and enzyme method. To compare thedifferentiation ability, toward retinal photoreceptor cell, of human orbital adipose tissuederived stem cells from enzyme method and explant method.【Methods】 Human orbital adipose tissue derived stem cells and human periodontalligament derived stem cells were cultured. The two cell lineages were cultured followingspecial differentiation protocol. The culture medium was added with IGF, b-FGF, DKK-1and Noggin at different time point. At the end of the differentiation period,100mMVitamin A was used to treat the cells. The cells were fixed and harvested at presurposedtime points. Stem cell specific and retinal photoreceptor specific gene expression andsurface marker were investigated by reverse transcription-polymerase chain reaction andimmunofluorescent staining. Expression time points of the genes or protein were compared.Adipose tissue-derived stem cells were isolated by enzymatic method and explant method.The cells were cultured following special differentiation protocol. Stem cell specific andretinal photoreceptor specific gene expression and protein products were investigated. Tocompare the differentiation ability of these human adult stem cells.【Results】After directed differentiation protocol, human orbital adipose tissue derivedstem cells showed neuron specific synapsis connection and the structure similar to outersegment of rod cell. However, human periodontal ligament derived stem cells did not showany morphology change. Expression of Pax6was much earlier in human periodontalligament derived stem cells. However, expression of Rhodopsin was much later in humanperiodontal ligament derived stem cells than in human orbital adipose tissue derived stem explant method and enzyme digestion method successfully. After primary culture, wefinished the directed differentiation process and both of the two adipose tissue derived stemcell lineages expressed stem cell marker. After the directed differentiation process, both ofthe two cells showed the structure similar to outer segment of rod cell. Adipose tissuederived stem cells which were isolated by explant method expressed Pax6and rhodopsinearlier than the cells which were isolated by enzyme digestion method.【Conclusions】 Human orbital adipose tissue derived stem cells which were isolated byexplant method has the potential to differentiate into retinal photoreceptor-like cell in vivo.It has better differentiation ability than human orbital adipose tissue derived stem cellswhich were isolated by enzyme digestion method or human periodontal ligament derived...