In Vivo Transplantation of Enteric Neural Crest Cells into Mouse Gut; Engraftment, Functional Integration and Long-Term Safety.
In Vivo Transplantation of Enteric Neural Crest Cells into Mouse Gut; Engraftment, Functional Integration and Long-Term Safety.
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DOI：
10.1371/journal.pone.0147989
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Thapar N
中科院分区：
文献类型：
--
作者：
Cooper JE;McCann CJ;Natarajan D;Choudhury S;Boesmans W;Delalande JM;Vanden Berghe P;Burns AJ;Thapar N
肠神经病变是严重的胃肠道疾病，结局不令人满意。我们的目的是通过将小鼠肠神经嵴细胞（ENCC）移植到体内神经节细胞和无神经节细胞的小鼠肠道中，并分析功能整合和长期安全性，来研究肠神经干细胞治疗此类疾病的潜力。将从出生后Wnt 1-cre; R26 R-YFP/YFP鼠肠道中选择的表达黄色荧光蛋白（YFP）的ENCC产生的神经球移植到野生型同窝小鼠的神经节后肠或Ednrbtm 1 Ywa小鼠（缺乏功能性内皮素受体B型）的无神经节后肠中。然后使用免疫组织化学评估肠道的ENCC整合和分化，使用钙成像评估细胞功能，并使用PCR检测脱靶YFP表达的长期安全性。YFP+ ENCC在受体神经节肠内移植、增殖并分化为肠神经元和神经胶质。移植的细胞和它们的突起沿着内源性肌间神经丛扩散，形成分支网络。内源性神经纤维的电穴位刺激导致YFP+移植的ENCC（用TTX消除）中的钙瞬变（F/F0 = 1.16±0.01;43个细胞，n = 6）。长期随访（24个月）显示，移植的ENCC没有引起肿瘤或扩散到其他器官（肠外部位PCR阴性）。在无神经节肠中，ENCC同样扩散和分化形成神经元和神经胶质网络，其投射与过渡区的内源性神经网络密切相关。移植的ENCC成功植入受体神经节和无神经节肠道，显示出适当的扩散、定位以及重要的功能整合，而没有任何长期安全性问题。这项研究为肠神经干细胞疗法的开发和使用提供了关键支持。
Enteric neuropathies are severe gastrointestinal disorders with unsatisfactory outcomes. We aimed to investigate the potential of enteric neural stem cell therapy approaches for such disorders by transplanting mouse enteric neural crest cells (ENCCs) into ganglionic and aganglionic mouse gut in vivo and analysing functional integration and long-term safety. Neurospheres generated from yellow fluorescent protein (YFP) expressing ENCCs selected from postnatal Wnt1-cre;R26R-YFP/YFP murine gut were transplanted into ganglionic hindgut of wild-type littermates or aganglionic hindgut of Ednrbtm1Ywa mice (lacking functional endothelin receptor type-B). Intestines were then assessed for ENCC integration and differentiation using immunohistochemistry, cell function using calcium imaging, and long-term safety using PCR to detect off-target YFP expression. YFP+ ENCCs engrafted, proliferated and differentiated into enteric neurons and glia within recipient ganglionic gut. Transplanted cells and their projections spread along the endogenous myenteric plexus to form branching networks. Electrical point stimulation of endogenous nerve fibres resulted in calcium transients (F/F0 = 1.16±0.01;43 cells, n = 6) in YFP+ transplanted ENCCs (abolished with TTX). Long-term follow-up (24 months) showed transplanted ENCCs did not give rise to tumours or spread to other organs (PCR negative in extraintestinal sites). In aganglionic gut ENCCs similarly spread and differentiated to form neuronal and glial networks with projections closely associated with endogenous neural networks of the transition zone. Transplanted ENCCs successfully engrafted into recipient ganglionic and aganglionic gut showing appropriate spread, localisation and, importantly, functional integration without any long-term safety issues. This study provides key support for the development and use of enteric neural stem cell therapies.