Nonviral direct conversion of primary mouse embryonic fibroblasts to neuronal cells.
Nonviral direct conversion of primary mouse embryonic fibroblasts to neuronal cells.
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DOI：
10.1038/mtna.2012.25
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发表时间：
2012-07-10
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转分化是再生医学的下一个前沿领域，即分化的细胞在不经过中间增殖干细胞样阶段的情况下被重新编程为另一个谱系。Wernig等人首次描述了成纤维细胞直接转化为功能诱导神经元细胞（iNs）。随后关于转分化为临床相关神经元亚型的报道进一步支持了自体细胞治疗神经退行性疾病的前景。到目前为止，所有已发表的神经元转分化方案都依赖于慢病毒，这可能妨碍了它们的临床转化。相反，我们将编码神经元转录因子（Brn2, Ascl1, Myt1l）的质粒用生物可还原的线性聚胺胺传递给原代小鼠胚胎成纤维细胞。这种基因载体的低毒性和高转染效率允许重复给药以维持高的转基因表达水平。连续0.5µg cm−2剂量的重编程因子以48小时的间隔递送，产生高达7.6%的Tuj1+（神经元特异性III类β-微管蛋白）细胞，其中一个子集表达MAP2（微管相关蛋白2），tau和突触素。突触红荧光蛋白（RFP）报告细胞通过24/26膜片夹紧的RFP+细胞发射动作电位，帮助鉴定更成熟、电生理更活跃的细胞。观察到一些非病毒诱导的神经细胞（NiNs）产生多重自发动作电位。该研究证明了非病毒神经元转分化的可行性，并可能适用于其他转分化过程。
Transdifferentiation, where differentiated cells are reprogrammed into another lineage without going through an intermediate proliferative stem cell-like stage, is the next frontier of regenerative medicine. Wernig et al. first described the direct conversion of fibroblasts into functional induced neuronal cells (iNs). Subsequent reports of transdifferentiation into clinically relevant neuronal subtypes have further endorsed the prospect of autologous cell therapy for neurodegenerative disorders. So far, all published neuronal transdifferentiation protocols rely on lentiviruses, which likely precludes their clinical translation. Instead, we delivered plasmids encoding neuronal transcription factors (Brn2, Ascl1, Myt1l) to primary mouse embryonic fibroblasts with a bioreducible linear poly(amido amine). The low toxicity and high transfection efficiency of this gene carrier allowed repeated dosing to sustain high transgene expression levels. Serial 0.5 µg cm−2 doses of reprogramming factors delivered at 48-hour intervals produced up to 7.6% Tuj1+ (neuron-specific class III β-tubulin) cells, a subset of which expressed MAP2 (microtubule-associated protein 2), tau, and synaptophysin. A synapsin-red fluorescent protein (RFP) reporter helped to identify more mature, electrophysiologically active cells, with 24/26 patch-clamped RFP+ cells firing action potentials. Some non-virally induced neuronal cells (NiNs) were observed firing multiple and spontaneous action potentials. This study demonstrates the feasibility of nonviral neuronal transdifferentiation, and may be amenable to other transdifferentiation processes.
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