The combination of bFGF and CHIR99021 maintains stable self-renewal of mouse adult retinal progenitor cells
The combination of bFGF and CHIR99021 maintains stable self-renewal of mouse adult retinal progenitor cells
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bFGF 和 CHIR99021 的组合可维持小鼠成年视网膜祖细胞的稳定自我更新。
DOI：
10.1186/s13287-018-1091-y
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发表时间：
2018-12-13
影响因子：
7.5
通讯作者：
Xu, Guo-Tong
中科院分区：
文献类型：
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作者：
Jin, Caixia;Ou, Qingjian;Xu, Guo-Tong
背景数以百万计的人患有最终导致失明的视网膜疾病，视网膜祖细胞（RPC）移植是一种有前途的治疗途径。然而，RPC的扩展和潜在的监管mechanisms仍然hailing.MethodsAdult小鼠神经RPC（mNRPCs）的分离和扩增与碱性成纤维细胞生长因子（bFGF）和糖原合成酶激酶3（GSK 3）抑制剂CHIR 99021的组合。移植前采用RT-PCR、免疫细胞化学（ICC）、Western blot、流式细胞术和转录组分析评估祖细胞特征。结果在bFGF和CHIR 99021存在下，mNRPCs能自我更新，并具有明显的RPC特征。bFGF通过促进G1/S和G2/M期转换而促进细胞周期。bFGF联合CHIR 99021激活非经典Wnt 5A/Ca 2+通路，形成钙稳态。此外，自我更新mNRPCs可以在体外诱导分化为视杆细胞样细胞和视网膜色素上皮细胞样细胞。当将绿色荧光蛋白（GFP）标记的细胞移植到Pde 6 b（rd 1）小鼠（也称为RD 1小鼠或无杆小鼠）的视网膜下腔（SRS）中时，细胞存活超过12周并迁移到视网膜中。部分受体视网膜可见光感受器标记物视紫红质的阳性表达。移植的细胞可以迁移到视网膜，主要迁移到内细胞层（INL）和神经节细胞层（GCL）。一些细胞可以分化为星形胶质细胞和无长突细胞。结论体外培养的mNRPCs在NOD/SCID小鼠体内移植6个月后未形成肿瘤。本研究建立了一种维持成人视网膜组织中RPC长期自我更新的方法，并揭示了非经典Wnt 5A/Ca 2+通路的激活可能参与调控RPC的自我更新。这项研究提出了一个非常有前途的平台，以扩大RPC的未来治疗应用。
BackgroundMillions of people are affected with retinal diseases that eventually cause blindness, and retinal progenitor cell (RPC) transplantation is a promising therapeutic avenue. However, RPC expansion and the underlying regulation mechanisms remain elusive.MethodsAdult mouse neural RPCs (mNRPCs) were isolated and amplified with the combination of basic fibroblast growth factor (bFGF) and glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021. The progenitor characteristics were evaluated with RT-PCR, immunocytochemistry (ICC), western blot, flow cytometry, and transcriptome analysis prior to transplantation. By treating cells with or without bFGF and CHIR99021 at different time points, the mechanism for mNRPCs' self-renewal was investigated by transcriptome analysis and western blot assay.ResultsmNRPCs were self-renewing in the presence of bFGF and CHIR99021 and showed prominent RPC characteristics. bFGF was essential in promoting cell cycle by facilitating G1/S and G2/M transitions. bFGF combined with CHIR99021 activated the non-canonical Wnt5A/Ca2+ pathway and form a calcium homeostasis. In addition, the self-renewing mNRPCs could differentiate into rod photoreceptor-like cells and retinal pigment epithelium (RPE)-like cells by in vitro induction. When green fluorescent protein (GFP)-labeled cells were transplanted into the subretinal space (SRS) of Pde6b (rd1) mice (also known as RD1 mice, or rodless mice), the cells survived for more than 12weeks and migrated into the retina. Parts of the recipient retina showed positive expression of photoreceptor marker rhodopsin. Transplanted cells can migrate into the retina, mainly into the inner cell layer (INL) and ganglion cell layer (GCL). Some cells can differentiate into astrocytes and amacrine cells. Cultured mNRPCs did not form tumors after transplanted into NOD/SCID mice for 6months.ConclusionsPresent study developed an approach to maintain long-term self-renewal of RPCs from adult retinal tissues and revealed that activation of the non-canonical Wnt5A/Ca2+ pathway may participate in regulating RPC self-renewal in vitro. This study presents a very promising platform to expand RPCs for future therapeutic application.