猪的诱导性多潜能干细胞向视网膜感光细胞分化的体内及体外研究
摘要
目的：探讨猪的诱导性多潜能干细胞(piPSC)在体外分化成视网膜神经细胞的方法。研究piPSC移植治疗猪的视网膜损伤的效果。
材料与方法：piPSC在放射处理后的SNL细胞上培养并传代。取第28代、第40代和第43代piPSC分别悬浮培养3天后形成胚胎体,再将胚胎体转至未稀释基质胶,1：10和1：20稀释的基质胶以及层粘连蛋白-纤维连接蛋白基质上粘附培养以使其分化。用免疫荧光染色和实时PCR技术检测piPSC以及分化细胞中干细胞标志物OCT4、ABCG2、Nanog、Sox2,神经细胞标志物TUBB3,感光细胞标志物RCVRN、NRL、RHO、IRBP、ROM1和ARR3以及视杆细胞型双极细胞标志物PRKCA的表达,计数表达上述标志物的细胞的比例以及在形态上具有外节结构细胞的比例。用带IRBP-GFP基因的慢病毒感染分化后的细胞,使视锥型和视杆型感光细胞表达绿色荧光蛋白(GFP)。碘乙酸(IAA)静脉注射建立猪视杆细胞损伤的动物模型,将感染GFP的piPSC分化细胞移植入的一侧病眼视网膜下,对侧病眼视网膜下注射DMEM/F12培养液作为对照。分别在用药前及细胞移植术后三周检测视网膜电生理反应(ERG)。术后3周摘取视网膜,切片,通过免疫荧光染色观察移植后的细胞在病变视网膜中的整合情况。
结果：实时PCR和免疫荧光染色显示分化后的细胞失去干细胞特异性基因OCT4、Nanog和Sox2的表达,转而表达视网膜感光细胞基因RCVRN、NRL、RHO、IRBP、ROM1和cone-arrestin。其中有30.91±7.2%的分化细胞表达转录因子NRL,25.61±8.5%表达RCVRN,6.765±2.083%表达RHO,此外还有2.6±0.4%的细胞表达PRKCA。以层粘连蛋白—纤维连接蛋白为细胞外基质可以分化出更多的RHO阳性细胞,但其外形更类似普通神经细胞。以基质胶为细胞外基质可以分化出更多的具有视杆细胞形态的细胞,即有外节结构的细胞。用携带IRBP-GFP基因的病毒感染分化细胞后,约44%的细胞表达GFP。这些细胞已经定向分化成视网膜感光细胞前体细胞或成熟细胞。移植到视网膜下后可检测到RHO阳性的细胞在视网膜各层均有表达,其主要表达部位在视网膜外核层,即正常的感光细胞所在的位置。在这些整合入视网膜的移植细胞中,有一部分细胞具有外节结构。
结论：piPSC能在体外高效地分化成视网膜神经细胞并且部分细胞具有与原代培养的视杆细胞类似的外节样结构。将这些分化的细胞移植入视杆细胞损伤的猪视网膜下方,移植细胞可以整合入受损视网膜。部分移植细胞在眼内分化出外节结构,这可能有助于视觉功能的恢复。
Purpose:A two-step protocol was developed for efficient differentiation of porcine induced pluripotent stem cells (piPSC) into rod photoreceptors for transplantation into a swine model of rod photoreceptor loss.
Materials and Methods:piPSC were cultured on the irradiate inactivated SNL feeder cell layer. P28, P40 and P43 piPSC were used for the differentiation. The cells were put in floating culture to form embryoid bodies followed by three weeks of differentiation in adherent culture. We examined the effect of substratum for adhesion culture and media composition on differentiation to rod photoreceptor lineage. Real time PCR and immunostaining were used to follow iPSC differentiation and the morphology of the cells was examined as well. We analyzed expression of the stem cell markers Oct4, ABCG2, Nanog, Sox2; rod lineage markers including RCVRN, NRL, RHO, IRBP, ROM1, cone-arrestin and rod bipolar cell marker PRKCA. Differentiated cells were then infected with the IRBP-GFP lentivirus. Differentiated cells were then transplanted into the subretinal space of swine treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, retinal sections were immunostained to follow engrafted cells.
Results:Real time PCR and immunostaining demonstrated loss of expression of the stem cell specification gene OCT4, Nanog, Sox2 and induction of rod photoreceptor gene markers including RCVRN, NRL, RHO, IRBP, ROM1, cone-arrestin and rod bipolar cell marker PRKCA. Immunostaining results and statistic analysis showed among the differentiated cells,30.91±7.2% were positive for NRL,25.61±8.5% were positive for RCVRN,6.765±2.083% expressed RHO, and 2.6±0.4% expressed PRKCA. Adherent culture on laminin-fibronectin led to a higher number of RHO+ cells while Matrigel led to a morphology resembling primary cultures of rod photoreceptors and to concentration of RHO and ROM1 in outer segment-like projections. After infection by the IRBP-GFP lentivirus, about 44% of differentiated cells were GFP+, which reflected rod and cone photoreceptors. After transplantation, RHO+ cells were evident in all retinal layers, but they were concentrated in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had projections resembling outer segments.
Conclusions:Skin-derived swine iPSC can efficiently differentiate to express markers of rod lineage and they morphologically resemble rods in culture concentrating RHO and ROM1 into projections resembling outer segments. These cells can integrate into the outer nuclear layer following rod photoreceptor loss, and some of engrafted cells display outer segment-like projections suggesting transition to functional morphology.
材料与方法：piPSC在放射处理后的SNL细胞上培养并传代。取第28代、第40代和第43代piPSC分别悬浮培养3天后形成胚胎体,再将胚胎体转至未稀释基质胶,1：10和1：20稀释的基质胶以及层粘连蛋白-纤维连接蛋白基质上粘附培养以使其分化。用免疫荧光染色和实时PCR技术检测piPSC以及分化细胞中干细胞标志物OCT4、ABCG2、Nanog、Sox2,神经细胞标志物TUBB3,感光细胞标志物RCVRN、NRL、RHO、IRBP、ROM1和ARR3以及视杆细胞型双极细胞标志物PRKCA的表达,计数表达上述标志物的细胞的比例以及在形态上具有外节结构细胞的比例。用带IRBP-GFP基因的慢病毒感染分化后的细胞,使视锥型和视杆型感光细胞表达绿色荧光蛋白(GFP)。碘乙酸(IAA)静脉注射建立猪视杆细胞损伤的动物模型,将感染GFP的piPSC分化细胞移植入的一侧病眼视网膜下,对侧病眼视网膜下注射DMEM/F12培养液作为对照。分别在用药前及细胞移植术后三周检测视网膜电生理反应(ERG)。术后3周摘取视网膜,切片,通过免疫荧光染色观察移植后的细胞在病变视网膜中的整合情况。
结果：实时PCR和免疫荧光染色显示分化后的细胞失去干细胞特异性基因OCT4、Nanog和Sox2的表达,转而表达视网膜感光细胞基因RCVRN、NRL、RHO、IRBP、ROM1和cone-arrestin。其中有30.91±7.2%的分化细胞表达转录因子NRL,25.61±8.5%表达RCVRN,6.765±2.083%表达RHO,此外还有2.6±0.4%的细胞表达PRKCA。以层粘连蛋白—纤维连接蛋白为细胞外基质可以分化出更多的RHO阳性细胞,但其外形更类似普通神经细胞。以基质胶为细胞外基质可以分化出更多的具有视杆细胞形态的细胞,即有外节结构的细胞。用携带IRBP-GFP基因的病毒感染分化细胞后,约44%的细胞表达GFP。这些细胞已经定向分化成视网膜感光细胞前体细胞或成熟细胞。移植到视网膜下后可检测到RHO阳性的细胞在视网膜各层均有表达,其主要表达部位在视网膜外核层,即正常的感光细胞所在的位置。在这些整合入视网膜的移植细胞中,有一部分细胞具有外节结构。
结论：piPSC能在体外高效地分化成视网膜神经细胞并且部分细胞具有与原代培养的视杆细胞类似的外节样结构。将这些分化的细胞移植入视杆细胞损伤的猪视网膜下方,移植细胞可以整合入受损视网膜。部分移植细胞在眼内分化出外节结构,这可能有助于视觉功能的恢复。
Purpose:A two-step protocol was developed for efficient differentiation of porcine induced pluripotent stem cells (piPSC) into rod photoreceptors for transplantation into a swine model of rod photoreceptor loss.
Materials and Methods:piPSC were cultured on the irradiate inactivated SNL feeder cell layer. P28, P40 and P43 piPSC were used for the differentiation. The cells were put in floating culture to form embryoid bodies followed by three weeks of differentiation in adherent culture. We examined the effect of substratum for adhesion culture and media composition on differentiation to rod photoreceptor lineage. Real time PCR and immunostaining were used to follow iPSC differentiation and the morphology of the cells was examined as well. We analyzed expression of the stem cell markers Oct4, ABCG2, Nanog, Sox2; rod lineage markers including RCVRN, NRL, RHO, IRBP, ROM1, cone-arrestin and rod bipolar cell marker PRKCA. Differentiated cells were then infected with the IRBP-GFP lentivirus. Differentiated cells were then transplanted into the subretinal space of swine treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, retinal sections were immunostained to follow engrafted cells.
Results:Real time PCR and immunostaining demonstrated loss of expression of the stem cell specification gene OCT4, Nanog, Sox2 and induction of rod photoreceptor gene markers including RCVRN, NRL, RHO, IRBP, ROM1, cone-arrestin and rod bipolar cell marker PRKCA. Immunostaining results and statistic analysis showed among the differentiated cells,30.91±7.2% were positive for NRL,25.61±8.5% were positive for RCVRN,6.765±2.083% expressed RHO, and 2.6±0.4% expressed PRKCA. Adherent culture on laminin-fibronectin led to a higher number of RHO+ cells while Matrigel led to a morphology resembling primary cultures of rod photoreceptors and to concentration of RHO and ROM1 in outer segment-like projections. After infection by the IRBP-GFP lentivirus, about 44% of differentiated cells were GFP+, which reflected rod and cone photoreceptors. After transplantation, RHO+ cells were evident in all retinal layers, but they were concentrated in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had projections resembling outer segments.
Conclusions:Skin-derived swine iPSC can efficiently differentiate to express markers of rod lineage and they morphologically resemble rods in culture concentrating RHO and ROM1 into projections resembling outer segments. These cells can integrate into the outer nuclear layer following rod photoreceptor loss, and some of engrafted cells display outer segment-like projections suggesting transition to functional morphology.
引文
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