| Objective:Retinitis pigmentosa(RP) is one of the leading causes of blindness in the aging population all over the world,and now effective treatments are still lacking.The relatively few documented cases of visual improvement following RPE transplantation demonstrated that cellular therapy is feasible and may be a useful approach for treating selected RP patients.Retinal progenitor cells(RPCs) derivation from embryonic stem cells(ESCs) can differentiate into 5 neural retinal cells、RPE and 1 glia cells.As a stem cells receptor,c-kit+ cells derivation from progenitor cell populations exhibit the capacity for self-renew and multipotent differentiation.As it is a surface antigen,we can isolate c-kit+ cells by using flow cytometry.This study isolates c-kit+/ssea1- cells during differentiation from m ESCs into RPE cells,because we identify c-kit+/SSEA1- cells as late stage retinal progenitor cells.The retina pigment epithelium is a highly polarized and specialized monolayered epithelium.RPE are critical to photoreceptor viability.The RPE create the blood-retina barrier and have multiple roles in maintaining photoreceptor health and visual function. RPE phagocytosis rod outer segments, absorb of stray light, secrete tro-phic factors, and assist in visual cycle retinol con-version and nutrient diffusion from the choroid. Loss or dysfunction of macular retinal pigmented epithelum,which provides crucial supportive functions for the photoreceptors,is thought to play a crucial role in disease progression.Therefore, dysfunctional RPE leads to the subsequent damage and death of photoreceptors. Pho-toreceptor loss within the macula causes central vision impairment with disease progression ex-panding the extent of vision loss.We are looking for the proficiency of inducing c-kit+/SSEA1- retinal progenitor cells derivation from mouse embryonic stem cells differentiate into retina pigment epitheliumcells in vitro and to provide a new way of cell transplantation replacement therapy to the RP disease.Methods:Part I: Discussion on the method of inducing mouse embryonic stem cells differentiate into c-kit+/SSEA1- RPCs and the identification of c-kit+/SSEA1- RPCs1. Culture mouse embryonic stem cells AB1.Identify stem cell marker SSEA1 after 21、26、31 generation.2. By culturing m ESCs under serum-free suspension conditions in the presence of Dkk1 and Lefty A,we differentiate it into RPCs.Identify marker c-kit on Day 9.3. Use flow cytometry to sort c-kit+/SSEA1- cells out of RPCs on Day 9.Identify stem cell markers Nestin、Pax6、c-kit and Ki67.Part II: Differentiating c-kit+/SSEA1- RPCs into retinal pigment epithelium cells and the identification of retinal pigment epithelium cells.1. Differentiating c-kit+/SSEA1- RPCs into retinal pigment epithelium cellsunder adherent condition.2. Use flow cytometry to identify the positive rate of maker of retinal pigment epithelium progenitor cells Mitf、Pax6 on Day 0、Day 10 and Day 40.3. Use flow cytometry to identify the positive rate of maker of retinal pigment epithelium cells Bestrophin、CRALBP and RPE65 on Day 0、Day 10 and Day 40.Results:Part I: Discussion on the method of inducing mouse embryonic stem cells differentiate into c-kit+/SSEA1- RPCs and the identification of c-kit+/SSEA1- RPCs1. Through P21、P26 and P31 stem cell marker SSEA1 expresses positive and the positive rate of SSEA1 respectively are(80.3±4.9)%、(74.9±1.1)% and(72.4±2.3)%.2. Floating m ESCs form aggregates within 1 day,and aggregates gather volume in following days.Maker c-kit expresses positive on Day 9.3. Sorted c-kit+/SSEA1- cells adhere to the bottom of the dish,and stem cell markers Nestin、Pax6、c-kit and Ki67 express positive.Part II: Differentiating c-kit+/SSEA1- RPCs into retinal pigment epithelium cells and the identification of retinal pigment epithelium cells.1. On Day 25 we can observe typical cobble stone phenotype and pigmentogenesis in the cells.2. The positive rate of Mitf respectively are(1.7±1.5)%、(28.5±7.2)% and(5.7±2.1)% on Day 0、Day10 and Day 40..The positive rate of Pax6 respectively are(6.5±0.2)%、(60.2±15.6)% and(4.6±0.6)% on Day 0、Day10 and Day 40.3. The positive rate of Bestrophin respectively are(1.2±0.2)%、(45.5±1.2)% and(61.6±2.4)% on Day 0、Day10 and Day 40..The positive rate of CRALBP respectively are(2.3±0.3)%、(6.0±0.1)% and(31.0±3.9)% on Day 0、Day10 and Day 40.The positive rate of RPE65 respectively are(1.3±0.1)%、(4.3±2.2)% and(57.8±2.0)% on Day 0、Day10 and Day 40.Conclusion:1. By culturing m ESCs under serum-free suspension conditions in the presence of Dkk1 and Lefty A,we could differentiate mouse embryonic stem cells AB1 into retinal progenitor cells in vitro.2. Sorted c-kit+/SSEA1- cells possessed retinal progenitor cells features,and the positive rate of c-kit+ cells in the retinal progenitor cells was around 20%.3.By adherent culturing c-kit+/SSEA1-retinal progenitor cells can be differentiated into retinal pigment epithelium cells.Triton-X-100美国Serva公司0.1mmol l-1β-巯基乙醇、90%Knockout DMEM。... |
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