[C6TSEDRVAJZ, a combination of small-molecule compounds, induces differentiation of human placental fibroblasts into epithelioid cells in vitro].

Q3 Medicine

南方医科大学学报杂志 Pub Date : 2025-02-20 DOI:10.12122/j.issn.1673-4254.2025.02.13

Zhenjia Dai, Qunwei Gao, Mengjiao Ying, Ao Wang, Juan Hong, Chunjing Wang, Yu Guo, Changqing Liu, Gaofeng Liu

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引用次数: 0

Abstract

Objectives: To reprogram human placental fibroblasts (HPFs) into chemically induced epithelioid-like cells (ciEP-Ls) using a combination of small-molecule compounds.

Methods: HPFs cultured under normoxic conditions were identified using immunofluorescence assay, PCR and chromosomal karyotyping. Under hypoxic conditions (37 ℃, 5% O₂), HPFs were cultured in a medium containing small-molecule compounds C6TSEDRVAJZ (CHIR99021, 616452, TTNPB, SAG, EPZ5676, DZNep, Ruxolitinib, VTP50469, Afuresertib, JNK-IN-8, and EZM0414), and the cell morphology was observed daily. The expression levels of epithelial cell markers in the induced cells were detected by immunofluorescence, Western blotting and PCR. Chromosomal karyotyping of the induced cells was performed and the induction efficiency was calculated.

Results: Before induction, HPFs showed positive expressions of fibroblast surface markers CD34 and vimentin and were negative for epithelial surface markers. PCR results showed high expressions of fibroblast-specific genes S100A4 and COL1A1 in HPFs with a normal human diploid karyotype. After one day of induction, the HPFs underwent morphological changes from a multinodular spindle shape to a round or polygonal shape, which was morphologically characteristic of ciEP-Ls. On day 4 of induction, the cells exhibited high expressions of the epithelial cell markers E-cadherin and Lin28A. RT-qPCR results also showed that the cells expressed the epithelial markers Smad3, GLi3, PAX8, WT1, KRT19, and KRT18 with significantly down-regulated expressions of all the fibroblast surface markers and a normal human diploid karyotype. The reprogramming efficiency of HPFs into ciEP-Ls ranged from (64.53±2.8)% to (68.10±3.6)%.

Conclusions: The small-molecule compound combination C6TSEDRVAJZ is capable of inducing HPFs into ciEP-Ls under hypoxic conditions with a high induction efficiency.

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目的：利用小分子化合物组合将人胎盘成纤维细胞（HPFs）重编程为化学诱导上皮样细胞（ciEP-Ls）：方法：使用免疫荧光检测、PCR 和染色体核型鉴定在常氧条件下培养的 HPFs。在缺氧条件下（37 ℃，5% O2），用含有小分子化合物 C6TSEDRVAJZ（CHIR99021、616452、TTNPB、SAG、EPZ5676、DZNep、Ruxolitinib、VTP50469、Afuresertib、JNK-IN-8 和 EZM0414）的培养基培养 HPFs，每天观察细胞形态。通过免疫荧光、Western 印迹和 PCR 检测诱导细胞中上皮细胞标记物的表达水平。对诱导细胞进行染色体核型分析，并计算诱导效率：结果：诱导前，HPF 显示成纤维细胞表面标志物 CD34 和波形蛋白阳性表达，而上皮细胞表面标志物阴性。PCR 结果显示，正常人二倍体核型的 HPF 中成纤维细胞特异基因 S100A4 和 COL1A1 表达量较高。诱导一天后，HPF 的形态从多结节纺锤形变为圆形或多边形，这是 ciEP-Ls 的形态特征。在诱导的第 4 天，细胞表现出上皮细胞标记物 E-cadherin 和 Lin28A 的高表达。RT-qPCR结果还显示，细胞表达上皮细胞标志物Smad3、GLi3、PAX8、WT1、KRT19和KRT18，所有成纤维细胞表面标志物的表达均显著下调，核型为正常人类二倍体。HPFs重编程为ciEP-Ls的效率从（64.53±2.8）%到（68.10±3.6）%不等：小分子化合物组合 C6TSEDRVAJZ 能够在缺氧条件下高效诱导 HPFs 转化为 ciEP-Ls。

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