逆转录病毒感染法对RP患者人体细胞向iPS细胞和iPS—RPE细胞诱导分化的研究

A pilot study on indunction and differentiation of human fibroblasts to iPS cells and iPS-RPE cells mediated by retrovirus infection

背景视网膜色素上皮（RPE）细胞移植是目前人类尝试治疗视网膜色素变性（RP）的主要手段。诱导多能干细胞（iPSCs）将成为移植细胞的一个重要来源，人胚胎干细胞（hESCs）可以无限期地自我更新和分化，是RPE细胞移植的重要供体来源。此外，iPS-RPE细胞为患者自身细胞成为治疗源组织提供了个性化治疗平台。目的评估利用逆转录病毒将Oct4、Sox2、c—Myc和K归4种基因导入RP患者皮肤成纤维细胞后诱导入iPSCs的可行性，并建立将正常人iPSCs诱导分化为RPE细胞的方法。方法分别采集基因突变点为SNRNP200p．S1087L的RP患者及无SNRNP200p．S1087L突变受试者的大腿皮肤组织块约2cm-3cm。采用胰蛋白酶消化和组织块培养法分离纯化得到成纤维细胞并进行传代，取第3代细胞用于研究，分别采用形态学和免疫荧光技术对培养细胞进行鉴定。采用含OCT4、SOX2、C—MYC和KLF44种cDNA的质粒病毒转染的成纤维细胞，并用hESCs条件培养液诱导iPSCs，采用细胞形态学、碱性磷酸酶（AP）染色、干细胞表面多潜能标志物表达对所得到的iPSCs和iPS—RPE细胞进行鉴定，并通过细胞在SCID小鼠的体内种植考察iPSCs的成瘤性。采用诱导分化拟胚体（EB）法将不含SNRNP200p．S1087L突变的iPSCs诱导分化为RPE细胞，分别采用免疫荧光技术和实时荧光定量PCR法检测细胞中特异性蛋白的表达。结果用皮肤组织块培养的成纤维细胞呈梭形和多角形，光学显微镜下可见细胞间排列紧密，细胞形态和大小趋于一致，细胞中特异性蛋白Vimentin表达阳性。经逆病毒转染的成纤维细胞培养后第5～7天可见小的细胞聚集，细胞形态由梭形变为圆形；培养后20d出现类似hESCs集落形态的iPSCs克隆；培养后25～30d细胞中的标志物SSEA3、SSEA4、TRA—1-60、TRA-1-81及Nanog均呈阳性表达，培养后12周形成的iPSCs克隆中AP染色阳性，接种到SCID小鼠体内后形成畸胎瘤。免疫荧光染色显示，诱导分化后30d时iPS—RPE细胞中RPE细胞特异性标志物RPE65、闭锁小带蛋白1（ZO-1）和卵磷脂视黄醇酰基转移酶（LRAT）蛋白均呈阳性表达。结论利用逆转录病毒将Oct4、Sox2、c-Myc和Kif44种基因转入SNRNP200p.S1087L突变的RP患者皮肤成纤维细胞后可以获得iPSCs，建立的iPSCs有ESCs的形态和多能分化的特征。采用同样的转染技术可在无SNRNP200p．S1087L突变的人皮肤成纤维细胞中建立体外高分化、高效能的iPS-RPE细胞。

Background Retinal pigment epithelium （RPE） cell transplantation is the primary means of human trial for the treatment of retinal degeneration. Induced pluripotent stem cells （iPSCs） will become an important source for cell transplantation. In addition,iPS-RPE cells may provide a personalized treatment platform for the patient＇s own cells treatment. Objective This study was to evaluate the feasibility of human fibroblasts differentiate toward iPSCs from retinitis pigmentosa （RP） patients and toward iPSC-RPE cells from non-RP individual by retroviral transfection of Oct4,Sox2,c-Myc and KLF4 genes. Methods Human thigh skin tissues were obtained from a RP patient with hotspot mutation of SNRNP200 p. S1087L and individual without SNRNP200 p. S1087L mutation, respectively,with the size 2 cm×3 cm. Human dermal fibroblasts were isolated and cultured by trypsin digestion and explant method. The fibroblasts were transfeeted by a series of retrovirus and cultured by human embyonie cell- conditioned medium to generate and induce iPSCs, and then the iPSCs were identified by morphology, alkaline phosphatase （AP） staining and immunofluorescenee assay of specific markers of pluripotent stem cells, iPSCs suspension were injected into SCID mouse to observe the tumorigenesis. The iPSCs from non-RP subject were induced to differentiate toward iPS-RPE cells by embryonic body （EB） inducing method,and iPS-RPE cells were identified by detecting the expression of RPE65 ,zonula occludens protein 1 （ZO-1） and lecithin retinol acyltransferase （LRAT）. Results Cultured human dermal fibroblasts showed fusiform or polygon shape and intercellular close arrangement, and Vimentin was positively expressed in the cells. Small cell colonies were harvested 5-7 days after infected by retroviruses,and the morphology changed from spindle into round mass. The hESC-like iPSCs clonies appeared 20 days after cuhivation, and the positive expressions of hESC-specific surface antigens including SSEA3 ,SSEA4,TRA-1-60, TRA-1-81 and Nanog were found in the cells 25-30 days after cultivation, and the positive staining of AP was obtained 12 weeks after cultivation. A teratoma was formed at the injection site of iPSCs suspension in SCID mouse. Immunofluorescence technique showed that RPE cell-specific proteins including RPE65, ZO-1 and LRAT proteins were positively expressed in iPS-RPE cells at 30 days after differentiation. Conclusions Mutation SNRNP200 p. S1087L of RP patient-specific iPSCs can be induced from human dermal fibroblast by retrovirus infection method. The function and morphology of the iPSCs were similar to hESCs. Human iPSCs cell line generated from the dermal fibroblasts of non-RP individuals can differentiate into iPS-RPE cells.