Direct Conversion of Adult Skin Fibroblasts to Endothelial Cells by Defined Factors
Direct Conversion of Adult Skin Fibroblasts to Endothelial Cells by Defined Factors
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DOI：
10.1161/circulationaha.113.007727
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发表时间：
2014-09-30
期刊：
影响因子：
37.8
通讯作者：
Kim, Hyo-Soo
中科院分区：
文献类型：
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作者：
Han, Jung-Kyu;Chang, Sung-Hwan;Kim, Hyo-Soo
以细胞为基础的增加内皮细胞（ECs）的疗法具有很大的治疗前景。在这里，我们报告了一种直接从成纤维细胞产生功能性ECs的新方法。方法与结果：筛选了11个内皮细胞发育的关键调控基因。从Tie2-GFP（+）小鼠制备绿色荧光蛋白（GFP）阴性的皮肤成纤维细胞，并用慢病毒感染，使所有11种因子同时过表达。流式细胞术检测Tie2- gfp +细胞（0.9%），代表Tie2基因激活。连续逐步筛选发现5个关键因子（Foxo1, Er71, Klf2， Tal1和Lmo2）是将皮肤成纤维细胞有效重编程为Tie2-GFP+细胞所必需的（4%）。这种重编程策略不涉及多能性诱导，因为Oct4和Nanog在5个关键因子转导后都没有表达。采用荧光活化细胞分选分离Tie2-GFP（+）细胞，并将其命名为诱导ECs （iECs）。内皮细胞呈内皮样鹅卵石状，并表达内皮细胞分子标记。iECs具有内皮功能，如单纯带状带-1凝集素结合、乙酰化低密度脂蛋白摄取、在基质上形成毛细血管和产生一氧化氮。由于VE-cadherin和Tie2基因的启动子被去甲基化，iec的表观遗传谱与真正的ec相似。mRNA谱分析显示，iECs具有真实的内皮细胞和高度富集的内皮基因。在小鼠后肢缺血模型中，iEC植入增加了毛细血管密度，增强了肢体灌注，证明了iEC的体内活力和功能。结论：我们首次证实了成纤维细胞向功能性内皮细胞的直接转化。这些结果为缺血性血管疾病的细胞治疗提供了一种新的治疗方式。
Background-Cell-based therapies to augment endothelial cells (ECs) hold great therapeutic promise. Here, we report a novel approach to generate functional ECs directly from adult fibroblasts.Methods and Results-Eleven candidate genes that are key regulators of endothelial development were selected. Green fluorescent protein (GFP)-negative skin fibroblasts were prepared from Tie2-GFP(+) mice and infected with lentiviruses allowing simultaneous overexpression of all 11 factors. Tie2-GFP+ cells (0.9%), representing Tie2 gene activation, were detected by flow cytometry. Serial stepwise screening revealed 5 key factors (Foxo1, Er71, Klf2, Tal1, and Lmo2) that were required for efficient reprogramming of skin fibroblasts into Tie2-GFP+ cells (4%). This reprogramming strategy did not involve pluripotency induction because neither Oct4 nor Nanog was expressed after 5 key factor transduction. Tie2-GFP(+) cells were isolated using fluorescence-activated cell sorting and designated as induced ECs (iECs). iECs exhibited endothelium-like cobblestone morphology and expressed EC molecular markers. iECs possessed endothelial functions such as Bandeiraea simplicifolia-1 lectin binding, acetylated low-density lipoprotein uptake, capillary formation on Matrigel, and nitric oxide production. The epigenetic profile of iECs was similar to that of authentic ECs because the promoters of VE-cadherin and Tie2 genes were demethylated. mRNA profiling showed clustering of iECs with authentic ECs and highly enriched endothelial genes in iECs. In a murine model of hind-limb ischemia, iEC implantation increased capillary density and enhanced limb perfusion, demonstrating the in vivo viability and functionality of iECs.Conclusions-We demonstrated the first direct conversion of adult fibroblasts to functional ECs. These results suggest a novel therapeutic modality for cell therapy in ischemic vascular disease.