Laboratory of Stem Cells and Restorative Neurology, University Hospital, Lund, Sweden.
Lund Stem Cell Center, University Hospital BMC B10, Lund University, SE-221 84, Lund, Sweden.
Stem Cell Res Ther. 2017 Sep 29;8(1):207. doi: 10.1186/s13287-017-0658-3.
Human fibroblasts can be directly converted to several subtypes of neurons, but cortical projection neurons have not been generated.
Here we screened for transcription factor combinations that could potentially convert human fibroblasts to functional excitatory cortical neurons. The induced cortical (iCtx) cells were analyzed for cortical neuronal identity using immunocytochemistry, single-cell quantitative polymerase chain reaction (qPCR), electrophysiology, and their ability to integrate into human neural networks in vitro and ex vivo using electrophysiology and rabies virus tracing.
We show that a combination of three transcription factors, BRN2, MYT1L, and FEZF2, have the ability to directly convert human fibroblasts to functional excitatory cortical neurons. The conversion efficiency was increased to about 16% by treatment with small molecules and microRNAs. The iCtx cells exhibited electrophysiological properties of functional neurons, had pyramidal-like cell morphology, and expressed key cortical projection neuronal markers. Single-cell analysis of iCtx cells revealed a complex gene expression profile, a subpopulation of them displaying a molecular signature closely resembling that of human fetal primary cortical neurons. The iCtx cells received synaptic inputs from co-cultured human fetal primary cortical neurons, contained spines, and expressed the postsynaptic excitatory scaffold protein PSD95. When transplanted ex vivo to organotypic cultures of adult human cerebral cortex, the iCtx cells exhibited morphological and electrophysiological properties of mature neurons, integrated structurally into the cortical tissue, and received synaptic inputs from adult human neurons.
Our findings indicate that functional excitatory cortical neurons, generated here for the first time by direct conversion of human somatic cells, have the capacity for synaptic integration into adult human cortex.
人类成纤维细胞可以直接转化为几种神经元亚型，但尚未产生皮质投射神经元。
在这里，我们筛选了潜在的转录因子组合，可以将人类成纤维细胞转化为功能性兴奋性皮质神经元。通过免疫细胞化学、单细胞定量聚合酶链反应（qPCR）、电生理学以及它们在体外和体内通过电生理学和狂犬病毒追踪整合到人类神经网络中的能力，来分析诱导的皮质（iCtx）细胞的皮质神经元特性。
我们表明，三种转录因子 BRN2、MYT1L 和 FEZF2 的组合能够直接将人类成纤维细胞转化为功能性兴奋性皮质神经元。通过使用小分子和 microRNAs 处理，将转化率提高到约 16%。iCtx 细胞表现出功能性神经元的电生理特性，具有类似锥体的细胞形态，并表达关键的皮质投射神经元标志物。对 iCtx 细胞的单细胞分析揭示了一个复杂的基因表达谱，其中一个亚群具有与人类胎脑皮质神经元非常相似的分子特征。iCtx 细胞从共培养的人类胎脑皮质神经元接收突触输入，包含棘突，并表达突触后兴奋性支架蛋白 PSD95。当体外移植到成人人类大脑皮质的器官型培养物中时，iCtx 细胞表现出成熟神经元的形态和电生理特性，结构上整合到皮质组织中，并从成人人类神经元接收突触输入。
我们的发现表明，通过人类体细胞的直接转化首次产生的功能性兴奋性皮质神经元具有整合到成人人类皮质中的突触能力。
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