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

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.