A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors
A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors
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DOI：
10.1186/s13287-019-1163-7
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发表时间：
2019-03-12
影响因子：
7.5
通讯作者：
Marsala, Martin
中科院分区：
文献类型：
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作者：
Bohaciakova, Dasa;Hruska-Plochan, Marian;Marsala, Martin
从胚胎干细胞(HESCs)中分离大量临床级别的多潜能人神经干细胞(HNSCs)，目前还没有一种成熟的方法，可以重复、高效、安全地分离。因此，在有损伤或疾病的人类中，将神经源性/胶质源性前体移植到中枢神经系统以达到细胞替代或神经保护的目的尚未得到广泛的测试和实施。方法在这里，我们建立了一种基于神经前体克隆形态的人工选择方法(COMO-NSC)来体外分离高度可扩增的hNSC群体。在免疫缺陷大鼠、免疫抑制ALS大鼠(SOD1(G93A))和脊髓损伤免疫抑制的小型猪体内移植后，通过NSC标记的表达(流式细胞仪、mRNA测序)、缺乏多能标记及其致瘤/分化情况，验证了已建立和广泛扩增的COMO-NSC的纯度和NSC特性。结果体外分析表明，已建立的COMO-NSC一致表达NSC标记(Sox1、Sox2、Nestin、CD24)，但缺乏多能标记(Nanog)，核型稳定，传代超过15代。基因图谱和组织学显示，脊髓移植的COMO-NSCs在体内2-6个月内分化为神经元、星形胶质细胞和少突胶质细胞，没有形成肿瘤衍生物或异常结构。此外，在免疫缺陷大鼠、免疫抑制的ALS大鼠(SOD1G93A)或脊髓损伤的小型猪等多种宿主环境中，移植的COMO-NSCs形成了带有突触接触和神经胶质细胞的神经元，表明这些细胞具有良好的安全性和分化特性。结论手动选择的COMO-NSCs代表了一个安全且可扩展的NSC群体，可有效地用于预期的人类临床细胞替代试验，用于治疗各种神经退行性疾病，包括ALS、中风、创伤性脊髓或脊髓缺血性损伤。
BackgroundA well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation.MethodsHere, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1(G93A)), or (iii) spinally injured immunosuppressed minipigs.ResultsIn vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics.ConclusionsThese data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.