氧浓度对hESCs三维培养形成神经视网膜及前体细胞的影响及机制研究

发布时间：2018-08-11 21:24

【摘要】：背景:视网膜色素变性(retinitis pigmentosa,RP)是一类以感光细胞凋亡为特征的遗传性致盲性眼病,目前尚无治愈方法,通过移植干细胞来源的感光细胞或视网膜色素上皮细胞成为RP治疗最有希望的新方法之一。视网膜是免疫豁免器官,加之玻璃体视网膜手术进行细胞移植是成熟技术,细胞移植后的视网膜功能和植入细胞的情况可以通过无创方法进行检测,使得眼睛成为细胞移植研究的理想器官。受制于伦理等因素,目前涉及从直接从人体组织获得干细胞进行临床研究的进展较缓慢。人胚胎干细胞(human embryonic stem cells,hESCs)具有向内中外三胚层分化的潜能,是一类理想的种子细胞。体外三维诱导hESCs形成类器官的方法较好地解决了种子细胞来源问题,同时,由于三维诱导hESCs形成类器官的过程很好地重演了胚胎发育发育过程中器官发生的过程,已经使其成为发育学研究的良好模型。目前,利用三维培养离体诱导hESCs已经可以形成包括视杯在内的多种器官组织,但三维培养形成神经视网膜(neural retina,NR)仍存在有以下几点问题亟待解决:1、hESCs三维诱导形成的NR与在体发育过程中形成的NR仍存在结构和功能的差异;2、影响三维培养hESCs所形成的NR的因素不明,诱导效率偏低。此外,由于诱导形成的NR尚处发育阶段,其内细胞组分混杂,缺乏特异膜标记物直接定位和分选视网膜前体细胞(retinal progenitor cells,RPCs)。基于此,选择合适的手段改善三维培养方法并选择恰当的膜标记物分离细胞是当下研究的关键点。氧对生命的维系至关重要,一方面,生命体通过氧化磷酸化的有氧呼吸方式大量产生ATP保障了生命活动的高效运转;另一方面,机体发育伴随了外环境氧浓度的变化,而干细胞的命运与氧浓度的高低密切相关。通常认为:低氧是决定干细胞调节自我更新以及维持干性的重要因素;高氧则会促进干细胞的分化。三维诱导hESCs形成NR的过程通常都在常氧条件下进行,但胚胎发育过程中,中枢神经系统发育先于心血管系统,因此在血液循环建立前后,视网膜的发育会经历一个氧浓度由低升高的过程。目前,大多数三维培养没有涉及氧浓度的转变过程。此外,理想的可移植细胞应具备以下条件:1、处于前体细胞阶段,便于移植前离体扩增以及移植后分化为目的细胞;2、无胚胎源性,不会在移植入器官后无限增殖形成畸胎瘤。研究发现,器官来源的络氨酸蛋白激酶受体(tyrosine-protein kinase Kit,C-Kit)阳性细胞是一类具有自我更新和分化潜能的干细胞,将其移植入对应的器官可发挥组织修复功能。而阶段特异的胚胎抗原4 (stage-specific embryonic antigen 4, SSEA4)为人器官组织在胚胎阶段表达的表面标记物,SSEA4将有利于鉴别胚胎源性的细胞。目的:hESCs三维培养以探究其形成的NR的细胞和组织构筑特点;研究氧浓度升高对三维诱导hESCs形成NR效率的影响,从而通过调控氧浓度改良三维诱导方法;研究从三维诱导hESCs形成NR中分离的C-Kit+/SSEA4-细胞的生物学特点以及低氧对其干性维持的影响,进而获取状态良好和足够数量的RPCs;利用全基因谱扫描分析从三维诱导hESCs形成NR分离的C-Kit+/SSEA4-细胞的基因表达谱,全面深入了解该细胞的特点。方法与结果:本研究分为三个部分:第一部分:hESCs的三维培养及神经视网膜的自发形成1、利用细胞免疫荧光、流式细胞分析研究hESCs细胞系H1的细胞特性。发现H1-hESCs成克隆聚集样生长,核分裂相明显。高达95%以上的细胞表达胚胎干细胞标志物 OCT4, SOX2 和 SSEA4。2、通过免疫荧光染色鉴定三维诱导hESCs形成的视泡、视杯样结构。可见拟胚体(embryonic bodies, EBs)在8天左右即可形成视泡样结构,继续培养24天后,视泡的形态更加清晰,并可观察到双层凹陷的视杯样结构,三维诱导38D的hESCs来源NR可表达增殖标记物Ki67; RPCs标记物:RAX、PAX6、CHX10;神经前体细胞标记物:SOX2、NESTIN;神经节细胞(retinal ganglion cells, RGCs)标记物 Tuj1及感光前体细胞标记物Crx,其中RAX在全神经视网膜均有表达。第二部分:氧浓度对三维诱导hESCs形成神经视网膜发育的影响1、利用免疫荧光染色分析高氧对三维诱导hESCs自发形成NR的影响,发现40%O2的高氧可以明显促进神经视网膜内细胞的增殖,且40%O2组中NR内增殖细胞在尖-底(apical-basal)端两侧间迁移增加,高氧可促进细胞核动态迁移(Interkinetic nuclear migration)的发生。2、利用免疫荧光染色研究高氧对三维诱导hESCs自发形成NR中神经玫瑰花节(neural rossetes)尖-底端极性的影响,发现在20%O2组中,神经玫瑰花结内侧尖端面会翻转至外侧,而40%O2组中神经玫瑰花节可按照正常生理的尖-底端极性发育。3、利用免疫荧光染色研究高氧对三维诱导hESCs自发形成NR中RPCs的影响,发现氧浓度变化并不会改变神经视网膜标记物的表达模式,但40%O2下PAX6阳性视网膜祖细胞数量较20%O2显著增加,RPCs向神经内层迁移更为明显,且40%O2显著促进RGCs的形成。相比20%O2的常氧处理,高氧下三维诱导hESCs形成的RGCs的突起更长,向基底侧迁移增加。第三部分:氧浓度对hESCs三维诱导NR来源C-Kit+/SSEA4- (hESC-NR-C-Kit+/SSEA4-)细胞的增殖的影响及其生物学特性分析1、利用改进的三维诱导方法诱导hESCs形成NR,研究氧浓度的影响对NR标记物表达的影响,发现改进的三维诱导方法所得EBs形成相对较慢,其形成的NR细胞与组织构筑特点没有显著变化,高氧对EBs生长仍有促进作用。2、利用免疫荧光染色研究改进的三维诱导hESCs形成NR中C-Kit的表达的时空分布特点,发现C-Kit阳性细胞主要分布于神经视网膜的内层,C-Kit阳性细胞同时表达干细胞标记Nestin、PAX6、RAX,但不表达CHX10。随着诱导时间的进展,NR内C-Kit的表达水平不断下降。3、利用细胞生存实验分析hESC-NR-C-Kit+/SSEA4-细胞增殖特性,发现hESC-NR-C-Kit+/SSEA4-细胞在3%O2的低氧下细胞增殖明显增加,在诱导30D、45D及60D分选细胞,以30D分选的细胞增殖活性最好。4、利用细胞免疫荧光染色,研究hESC-NR-C-Kit+/SSEA4的-细胞的特性,发现hESC-NR-C-Kit+/SSEA4-细胞可以表达包括 Nestin、PAX6、RAX 等多种 RPCs 的标记物和增殖标记物Ki67,且可以诱导分化形成RGCs、双极细胞、感光细胞及Muller细胞。5、利用全基因组转录谱表达分析,研究hESC-NR-C-Kit+/SSEA4-细胞的基因表达谱特点,发现30D、45D及60D分选的hESC-NR-C-Kit+/SSEA4-视网膜前体细胞与人胚眼分离的RPCs之间的Pearson相关系数均在0.88以上,且以30D最高(0.908),hESC-NR-C-Kit+/SSEA4-细胞较RPCs增殖和迁移相关基因表达增加,p53信号通路激活增加,但细胞粘附分子通路激活降低。结论:1、三维诱导hESCs形成的NR可以表达各类视网膜干细胞标记物,其发育类似在体视网膜发育过程。选用改进的(BMP4介导)三维诱导方法,其诱导过程操作简单,NR形成更稳定,表明改进的三维诱导方法更利于后期临床运用。2、氧浓度升高对于三维诱导hESCs形成NR的主要影响有包括促进NR的增殖、促进神经玫瑰花结按照正常生理的尖-底端极性发育、促进PAX6阳性RPCs的形成和迁移以及促进RGCs的形成、成熟和迁移。表明高氧处理有利于获得更多、更接近在体发育的NR。3、低氧培养下,诱导30D分选的hESC-NR-C-Kit+/SSEA4-细胞的状态和增殖活性较常氧培养有明显改善,表明低氧培养有利于细胞的稳定扩增。4、三维诱导hESCs形成的NR中C-Kit阳性细胞可以同时表达Nestin、PAX6、RAX等视网膜干细胞标记,但不表达胚胎抗原,可分化为感光细胞,双极细胞,Muller 细胞及 RGCs,表明 hESC-NR-C-Kit+/SSEA4-细胞为一类 RPCs。5、基因组表达谱扫描结果显示hESC-NR-C-Kit+/SSEA4-细胞与从人胚眼中分离的RPCs非常类似(90%),但其增殖和迁移相关基因表达高于RPCs,提示hESC-NR-C-Kit+/SSEA4-细胞是接近于在体发育的一类RPCs。为干细胞移植治疗视网膜变性疾病提供了成瘤风险低、分化潜能好、有标准化产业化条件新的种子细胞。
[Abstract]:BACKGROUND: Retinal pigmentosa (RP) is a kind of hereditary blinding ophthalmopathy characterized by photoreceptor cell apoptosis. At present, there is no cure method. Transplantation of stem cell-derived photoreceptor cells or retinal pigment epithelial cells (RPE) is one of the most promising new methods for the treatment of RP. Cell transplantation in vitreoretinal surgery is a mature technique. The function of the retina and the condition of the implanted cells can be detected by non-invasive methods, making the eye an ideal organ for cell transplantation. Human embryonic stem cells (hESCs) have the potential to differentiate inward, outward and outward into three embryonic layers, and are a kind of ideal seed cells. The process of organogenesis in embryonic development has been reproduced, which has made it a good model for developmental research. At present, the induction of hESCs by three-dimensional culture in vitro has been able to form a variety of organs and tissues including optic cups. However, the formation of neural retina (NR) by three-dimensional culture still has the following problems to be solved urgently. Solutions: 1. There are still structural and functional differences between NR induced by hESCs and NR formed during in vivo development; 2. The factors affecting NR induced by hESCs are unknown and the induction efficiency is low. In addition, the NR induced by hESCs is still in the developmental stage, and there is no direct localization and sorting of specific membrane markers. Retinal progenitor cells (RPCs). Based on this, choosing appropriate methods to improve the three-dimensional culture method and selecting appropriate membrane markers to isolate cells is the key point of current research. On the other hand, the development of the body is accompanied by changes in oxygen concentration in the external environment, and the fate of stem cells is closely related to oxygen concentration. The process usually takes place under normal oxygen conditions, but during embryonic development, the central nervous system develops prior to the cardiovascular system, so the development of the retina undergoes a process from low to high oxygen concentration before and after the establishment of blood circulation. Cells should be in the precursor cell stage for in vitro expansion before transplantation and differentiation into target cells after transplantation. 2. There is no embryonic origin, and no infinite proliferation of teratoma after transplantation. Studies have found that organ-derived tyrosine-protein kinase Kit (C-Kit) positive cells are Stage-specific embryonic antigen 4 (SSEA4) is a surface marker expressed in human organs during the embryonic stage. SSEA4 will be helpful to identify embryonic cells. S three-dimensional culture was used to explore the cell and tissue architecture of NR; the effect of elevated oxygen concentration on NR formation efficiency of three-dimensional induction of hESCs was studied to improve the three-dimensional induction method by adjusting oxygen concentration; the biological characteristics of C-Kit+/SSEA4-cells isolated from three-dimensional induction of NR formation of hESCs and the maintenance of their dryness by hypoxia were studied. Methods and Results: This study was divided into three parts: the first part: three-dimensional culture of hESCs and neural retina. Spontaneous formation 1. Immunofluorescence and flow cytometry were used to study the cell characteristics of hESCs cell line H1. It was found that H1-hESCs grew in clonal aggregates with obvious mitotic phase. Visible embryonic bodies (EBs) in about 8 days to form the structure of the optic vesicles, continue to culture 24 days, the morphology of the optic vesicles more clear, and can be observed double-decked cup-like structure, three-dimensional induction of 38D hESCs source NR can express proliferation markers Ki67; RPCs markers: RAX, PAX6, CHX10; neural precursor cell markers; Notes: SOX2, NESTIN; retinal ganglion cells (RGCs) marker Tuj1 and photoreceptor precursor cell marker Crx, in which RAX was expressed in the whole nervous retina. Part II: Effect of oxygen concentration on the development of three-dimensional inducible hESCs to form neural retina 1. Immunofluorescence staining was used to analyze the effect of hyperoxia on three-dimensional inducible hESCs spontaneously. The effect of hyperoxia on the formation of NR was found to promote the proliferation of neuroretinal cells. In 40% O2 group, the proliferation of NR cells increased between apical-basal ends. Hyperoxia could promote the occurrence of nuclear migration. 2. Immunofluorescence staining was used to study the effect of hyperoxia on three-dimensional induction. The effect of nerve rossetes tip-to-bottom polarity on spontaneous formation of NR in hESCs was observed. In 20% O2 group, the medial tip of the nerve rossetes turned to the lateral side, while in 40% O2 group, the nerve rossetes developed according to the normal physiological tip-to-bottom polarity. 3. Immunofluorescence staining was used to study the effect of hyperoxia on three-dimensional induction of hESCs self-polarity. The changes of oxygen concentration did not change the expression pattern of neuroretinal markers, but the number of PAX6-positive retinal progenitor cells increased significantly in 40% O2 compared with 20% O2, and the migration of RPCs to the inner layer of the nerve was more obvious. 40% O2 significantly promoted the formation of RGCs. Compared with 20% O2, the three-dimensional induction of hESCs in hyperoxia induced the formation of hESCs. The process of RGCs was longer and the migration to basal side increased. Part 3: The effect of oxygen concentration on the proliferation of NR-derived C-Kit+/SSEA4-cells induced by hESCs and its biological characteristics were analyzed. Part 1: The NR formation of hESCs was induced by an improved three-dimensional induction method. It was found that the formation of EBs by the improved three-dimensional induction method was relatively slow, and the characteristics of NR cells and tissue architecture were not significantly changed. Hyperoxia still promoted the growth of EBs. 2. Immunofluorescence staining was used to study the temporal and spatial distribution of C-Kit expression in NR induced by improved three-dimensional induction of hESCs. C-Kit positive cells expressed stem cell markers Nestin, PAX6 and RAX but not CHX10 in the inner layer of the retina. With the development of induction time, the expression of C-Kit in NR decreased. 3. Cell survival assay was used to analyze the proliferation characteristics of hESC-NR-C-Kit+/SSEA4-cells. It was found that hESC-NR-C-Kit+/SSEA4-cells were fine under 3% O2 hypoxia. Cell proliferation was significantly increased in the induction of 30D, 45D and 60D sorted cells, and 30D sorted cells had the best proliferative activity. 4. Immunofluorescence staining was used to study the characteristics of hESC-NR-C-Kit+/SSEA4-cells. It was found that hESC-NR-C-Kit+/SSEA4-cells could express markers and proliferative markers Ki67 including Nestin, PAX6, RAX and other RPCs. RGCs, bipolar cells, photoreceptor cells and Muller cells were induced to differentiate into RGCs. Pearson correlation coefficients between hESC-NR-C-Kit+/SSEA4-retinal precursor cells and RPCs isolated from human embryonic eyes were found by genome-wide transcription analysis. HESC-NR-C-Kit+/SSEA4-cells expressed more genes related to proliferation and migration than RPCs, and the activation of p53 signaling pathway increased, but the activation of cell adhesion molecule pathway decreased. Conclusion: 1. NR induced by three-dimensional hESCs can express various kinds of retinal stem cell markers, and its development is similar to that of retinal development in vivo. The improved three-dimensional (BMP4-mediated) induction method is simple and stable, which indicates that the improved three-dimensional induction method is more conducive to the later clinical application. 2. The main effects of elevated oxygen concentration on three-dimensional induction of NR in hESCs include promoting the proliferation of NR and promoting the rosette formation in accordance with the normal physiological tip-to-bottom. The results showed that hyperoxia treatment was conducive to the formation, maturation and migration of PAX6-positive RPCs, which was closer to NR.3 in vivo. Under hypoxia culture, the state and proliferative activity of 30D-sorted hESC-NR-C-Kit+/SSEA4-cells were significantly improved than those in normoxia culture. C-Kit positive cells could express Nestin, PAX6, RAX and other retinal stem cell markers at the same time, but did not express embryonic antigen. They could differentiate into photoreceptor cells, bipolar cells, Muller cells and RGCs, indicating that hESC-NR-C-Kit+/SSEA4-cells were a class of RPCs. The results showed that hESC-NR-C-Kit+/SSEA4-cells were very similar to RPCs isolated from the eyes of human embryos (90%), but the expression of proliferation and migration-related genes was higher than that of RPCs, suggesting that hESC-NR-C-Kit+/SSEA4-cells were a class of RPCs close to in vivo development. There are new seed cells with standardized industrialization conditions.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R774.1

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