CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China.
Stem Cell Res Ther. 2021 Mar 18;12(1):188. doi: 10.1186/s13287-021-02238-4.
Neural stem cell (NSC) therapy remains one of the most potential approaches for the treatment of neurological disorders. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized the technique of cell therapy. Meanwhile, it is often required that NSCs are stored and transported to a long distance for research or treatment purposes. Although high survival rates could be maintained, conventional methods for cell transportation (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore, the establishment of a safe, affordable, and low-cost strategy to store and transport easily accessible hiPSCs and hiNSCs, with characteristics that match fetal hNSCs, is incredibly urgent.
We reprogrammed human urinary cells to iPSCs using a non-integrating, virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis, and in vitro as well as in vivo differentiation capabilities, etc.).
Here, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique. Next, we demonstrated that the iNSCs differentiated into mature cerebral cortical neurons and neural networks. Interestingly, hiNSCs survived longer as neurospheres at ambient temperature (AT) than those cultured in a monolayer. Within 7 days approximately, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to AT that were placed under standard culture conditions (37 °C, 5% CO) recovered their typical morphology, and retained their proliferation and differentiation abilities.
In this study, we provided a simple method for the storage of NSCs as neurospheres at AT as an alternative method to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at AT and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.
神经干细胞（NSC）疗法仍然是治疗神经紊乱的最有潜力的方法之一。人类诱导多能干细胞（hiPSC）的发现和 hiPSC 衍生的人神经干细胞（hiNSC）的建立彻底改变了细胞治疗技术。同时，通常需要将 NSCs 储存和运输到远距离，用于研究或治疗目的。尽管可以保持高存活率，但传统的细胞运输方法（干冰或液氮）既不方便又昂贵。因此，建立一种安全、经济实惠且低成本的策略来储存和运输易于获取的 hiPSC 和 hiNSC，并具有与胎儿 hNSC 相匹配的特性，是非常紧迫的。
我们使用非整合、无病毒技术将人尿液细胞重编程为 iPSC，并在符合良好生产规范（GMP）的条件下将 iPSC 分化为 iNSC/神经球和神经元。iPSC 和 iNSC 的多能性通过一系列经典方法（表面标志物、核型分析以及体外和体内分化能力等）进行了表征。
在这里，我们的结果表明，我们成功地通过无病毒技术从更易得、非侵入性和更易接受的尿液细胞中生成了 hiNSC/神经球。接下来，我们证明了 iNSC 分化为成熟的大脑皮质神经元和神经网络。有趣的是，hiNSC 作为神经球在室温（AT）下比在单层培养中存活时间更长。在大约 7 天内，神经活力保持在>80%，而在单层培养中的 hiNSC 几乎立即死亡。暴露于 AT 并置于标准培养条件（37°C，5% CO）下的神经球恢复了其典型形态，并保持了其增殖和分化能力。
在这项研究中，我们提供了一种简单的方法，可以将 NSCs 作为神经球在 AT 下储存，作为更昂贵和不方便的传统冷冻保存方法的替代方法。这将使 hiNSC 能够在 AT 下远距离运输，并促进 NSCs 作为神经球的治疗应用，而无需进一步治疗。
