Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
Mol Ther Nucleic Acids. 2012 Jul 10;1(7):e32. doi: 10.1038/mtna.2012.25.
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.
转分化是指分化细胞在不经过中间增殖性干细胞样阶段的情况下被重新编程为另一种谱系，这是再生医学的下一个前沿领域。Wernig 等人首次描述了将成纤维细胞直接转化为功能性诱导神经元细胞（iNs）。随后有报道称将其转化为临床上相关的神经元亚型，进一步证实了自体细胞疗法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，这可能使其无法在临床上转化。相反，我们使用生物可还原的线性聚（酰胺胺）将编码神经元转录因子（Brn2、Ascl1、Myt1l）的质粒递送到原代小鼠胚胎成纤维细胞中。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水平的转基因表达。以 48 小时的间隔连续给予 0.5μg cm(-2) 的重编程因子，可产生高达 7.6%的 Tuj1（+）（神经元特异性 III 类β-微管蛋白）细胞，其中一部分表达 MAP2（微管相关蛋白 2）、tau 和突触小体蛋白。一个突触小体红色荧光蛋白（RFP）报告基因有助于鉴定更成熟、电生理活性的细胞，其中 24/26 个被膜片钳钳制的 RFP（+）细胞可产生动作电位。观察到一些非病毒诱导的神经元细胞（NiNs）可产生多个和自发性动作电位。这项研究证明了非病毒神经元转分化的可行性，并且可能适用于其他转分化过程。
