Division of Cardiology, Department of Internal Medicine, Seoul National University Hospital, Seoul, South Korea.
Strategic Center of Cell and Bio Therapy for Heart, Diabetes and Cancer, Seoul National University Hospital, Seoul, South Korea; Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, South Korea.
Biomaterials. 2021 May;272:120781. doi: 10.1016/j.biomaterials.2021.120781. Epub 2021 Mar 24.
We aimed to directly convert adult human dermal fibroblasts (aHDFs) into functional endothelial cells (ECs). Lentiviral vectors encoding endothelial transcription factors (TFs) were constructed. We examined whether five TFs (FOXO1, ER71, KLF2, TAL1, and LMO2) used for the generation of mouse induced ECs (iECs) could convert the aHDFs into human iECs. Twenty-eight days after transduction with lentiviral constructs, 32.1 ± 5.1% cells expressed vascular endothelial (VE)-cadherin. Factor screening revealed that only three factors (3F: ER71, KLF2, and TAL1) were necessary to induce VE-cadherin (+) cells (49.4 ± 3.5%). However, whole transcriptome sequencing showed that VE-cadherin (+) cells were not completely reprogrammed. Mature iECs double-positive for VE-cadherin/Pecam1 (DP cells) with a cobblestone appearance were obtained at a frequency of only 5.1 ± 0.6%. Using whole transcriptome analysis, the potential factors that could block the conversion were screened. Among candidates TWIST1-knockdown enhanced efficiency of conversion. Rosiglitazone, an inhibitor of epithelial-mesenchymal transition (EMT), also improved the conversion efficiency. Moreover, a 2nd second-stage conversion process, in which VE-cadherin (+) cells were incubated for additional two weeks, further enhanced the efficiency. The final protocol for 6 weeks yielded a conversion rate of 19.6 ± 3.0% iECs, defined by DP cells depicting the nature of mature ECs in various analyses. Further analyses revealed that the genetic and epigenetic profiles of iECs resembled those of functional ECs. Collectively, aHDFs can be converted into functional ECs through the transduction of ER71, KLF2, and TAL1, combined with two EMT inhibitors (siTWIST1 and rosiglitazone), followed by 2nd stage conversion.
我们旨在直接将成人皮肤成纤维细胞（aHDF）转化为功能性内皮细胞（EC）。构建了编码内皮转录因子（TF）的慢病毒载体。我们研究了用于生成小鼠诱导性 EC（iEC）的五个 TF（FOXO1、ER71、KLF2、TAL1 和 LMO2）是否可以将 aHDF 转化为人类 iEC。慢病毒构建体转导 28 天后，32.1±5.1%的细胞表达血管内皮（VE）-钙黏蛋白。因子筛选显示，只有三个因子（3F：ER71、KLF2 和 TAL1）是诱导 VE-钙黏蛋白（+）细胞（49.4±3.5%）所必需的。然而，全转录组测序表明，VE-钙黏蛋白（+）细胞并未完全重编程。仅获得具有鹅卵石外观的 VE-钙黏蛋白/PECAM1（DP 细胞）双阳性的成熟 iEC 频率为 5.1±0.6%。使用全转录组分析筛选可能阻止转化的潜在因子。在候选物中，TWIST1 敲低增强了转化效率。上皮-间充质转化（EMT）抑制剂罗格列酮也提高了转化效率。此外，第二个第二阶段转化过程，即在另外两周内孵育 VE-钙黏蛋白（+）细胞，进一步提高了效率。最终的 6 周方案产生了 19.6±3.0%的 iEC，定义为 DP 细胞在各种分析中描绘成熟 EC 的性质。进一步的分析表明，iEC 的遗传和表观遗传特征与功能性 EC 相似。总之，通过转导 ER71、KLF2 和 TAL1，结合两种 EMT 抑制剂（siTWIST1 和罗格列酮），然后进行第二阶段转化，可以将 aHDF 转化为功能性 EC。
