基于NGRM技术产生的视网膜前体细胞的生物学特性及其对RP的神经免疫调节作用及机制研究

Stem cell based cell therapy is an effective means to repair and restore the neural function in neurodegenerative and damage diseases in the central nervous system(CNS). However, it is currently facing the bottleneck such as low survival rate of the grafted stem cell derived seed cells, low differentiation efficiency, and the therapeutic effect of stem cell was reported to be maintained only for a relatively short period. It might be resulted from the techniques of induction the stem cell in vitro are based on the two-dimensional(2D) induction which only partially mimics the development of CNS. Most recent, a technique termed Next Generation Regenerative Medicine(NGRM) has been developed which was established through three-dimensional(3D) culture combined with low concentrations of growth factors induction of embryonic stem cells (ESCs). The daughter cells from the ESCs obtained with NGRM technique were proved to be higher plasticity and ability to integrate into the circuit of the host when grafted in the CNS. However, it has not been clarified the differences between the traditional 2D induced ESCs-derived cells and the cells obtained from ESCs with NGRM and their therapeutic effect, especially the neuroimmunomodulation funtion in Retinitis Pigmentosa (RP). Therefore, after establishment the NGRM techniques and successful self-organiztion of the optic vesicle and optic cup from human ESCs and sucessfully isolated the retinal pigment epithelial(RPE) with this techniques, we design to sort the retinal precursor cells(RPCs) and analyze the cell biological and exosome secretion characteristics, as well as their microglia regulational function in vitro. And the effect and mechanisms of these hESCs-derived cells in restoration the visual function, and teir immunoregulation on the microglia in the degenerative retinas of RD1 mice and Royal College of Surgeon’s (RCS ) rats. Our results might clarify the function and mechanisms of therapeutic cells from ESCs with NGRM techniques in neural regeneration medicine.

干细胞移植是修复视网膜变性和损伤的有效手段，目前面临移植细胞存活和分化效率低下、治疗效果不能长久维持等瓶颈问题，可能在于目前采用的干细胞来源移植细胞大多为在体外二维诱导得到。最近采用基于三维培养、低贴附结合低浓度因子序贯诱导的下一代再生医学（NGRM）技术得到的胚胎干细胞（ESCs）来源的视网膜前体细胞（RPCs）具有更高的可塑性和神经整合潜能，但尚不清楚和传统二维诱导的ESCs来源细胞相比，这类细胞对视网膜色素变性（RP）的治疗作用及其神经免疫调节机制。为此，本研究在前期成功采用NGRM技术诱导人ESCs形成视泡和视杯并分选出视网膜色素上皮细胞（RPE）的基础上，分选出RPCs并分析其细胞生物学特性、微囊泡分泌特点以及对小胶质细胞的调节作用；移植RCS大鼠和RD1小鼠视网膜后，观察其对变性视网膜的功能修复、免疫调节作用及机制。研究结果为阐明NGRM技术在神经修复中的机制提供新的理论依据。

干细胞移植是修复变性视网膜的有效手段，目前已进入临床应用阶段。基于干细胞移植的视网膜再生修复目前面临的主要问题是植入变性视网膜的干细胞存活和分化效率低下，治疗效果不能持久。这可能与目前用于移植的种子细胞多为体外二维诱导自胚胎干细胞（ESCs）或诱导性多能干细胞（iPSCS）所致，这种诱导方法和体内干细胞发育分化的三维环境完全不同。另外，变性视网膜中小胶质细胞激活所致的炎症环境也不利于植入干细胞的存活和分化。采用下一代再生医学（NGRM）技术得到的ESCs来源的视网膜前体细胞（RPCs）被证明具有更好的可塑性，但尚不清楚和传统二维诱导的ESCs来源RPCs相比，对视网膜色素变性（RP）的治疗作用及其神经免疫调节机制有何差异。本研究采用NGRM技术诱导人ESCs形成神经视网膜并分选出RPCs，分析其细胞生物学特性、微囊泡分泌特点以及对小胶质细胞的调节作用，移植RCS大鼠和RD1小鼠视网膜后，观察其对变性视网膜的功能修复、免疫调节作用及机制。获得以下主要结果：.①采用转录组方法、细胞生物学方法比较了经NGRM 技术获得的 hESCs-RPCs(3D)和 hESCs-RPCs(2D)在细胞增殖、分化以及移植后在视网膜的整合功能和对变性视网膜中小胶质细胞调节作用的差异，结果显示hESCs-RPCs(3D)在转录组上和分离自胚胎的RPCs更为接近，具有更好的可塑性和更强的神经免疫调节能力。.②采用形态学方法比较了人RPCs、小鼠神经干细胞微囊泡和外泌体分泌能力，经外泌体组学分析上述两类干细胞的微囊泡中的micro RNA差异。证实干细胞来源的外泌体可被变性视网膜中的小胶质细胞特异性吞噬并抑制其免疫功能，从而发挥对变性视网膜的保护作用。.③ 经NGRM 技术获得的 hESCs-RPCs(3D)和 hESCs-RPCs(2D)移植RCS 大鼠和RD1小鼠视网膜后能显著延缓视网膜的变性过程，发挥神经保护作用。其中hESCs-RPCs(3D)能产生更为持久的视网膜保护作用，这一作用部分是通过对变性视网膜中小胶质细胞的免疫调节达到的。.④ 本研究共发表标注资助的国外SCI 论文10 篇。培养博士硕士研究生各2 名。.研究结果为阐明NGRM技术在神经修复中的机制提供了新的理论依据。