Int J Stem Cells.  2020 Mar;13(1):93-103. 10.15283/ijsc18146.

The Overexpression of Epidermal Growth Factor (EGF) in HaCaT Cells Promotes the Proliferation, Migration, Invasion and Transdifferentiation to Epidermal Stem Cell Immunophenotyping of Adipose-Derived Stem Cells (ADSCs)

• Mao Y
• Ma J
• Xia Y
• Xie X

• KMID: 2500569
• DOI: http://doi.org/10.15283/ijsc18146

Abstract

Background and Objectives
The application of adipose derived stem cells (ADSCs) in skin repair has attracted much attention nowadays. Epidermal growth factor (EGF) participates in the progress of skin proliferation, differentiation and so forth. We aimed to explore the role of EGF in the proliferation, invasion, migration and transdifferentiation into epidermal cell phenotypes of ADSCs.
Methods and Results
ADSCs were extracted from adipose tissues from patient. Immunophenotyping was determined by flow cytometry. Overexpressed EGF or siEGF was transfected by lentiviruses. EGF was determined by enzyme linked immunosorbent assay (ELISA) or western blot. ADSCs and HaCaT cells were co-cultured by Transwell chambers. Conditioned medium (CM) was obtained from cultured HaCaT cells and used for the culturing of ADSCs. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Invasion rate was measured by Transwell invasion assay and migration rate by wound healing test. mRNA and protein levels were measured by qPCR and western blot respectively. The extracted cells from adipose tissues were identified as ADSCs by morphology and immunophenotyping. The expression of EGF was up or down regulated constantly in HaCaT cell line after transfection. EGF overexpression upregulated the proliferation, migration and invasion rates of ADSCs, and EGF expression regulated the expression of cytokeratin-19 (CK19) and integrin-β as well.
Conclusions
EGF could be served as a stimulus to promote the proliferation, migration, and invasion as well as the transdifferentiation into epidermal stem cell immunophenotyping of ADSCs. The results showed that EGF had a promising effect on the repair of skin wound.

Keyword

EGF; ADSCs; Repair; Transdifferentiation; Tissue engineering

Figure

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  Fig. 1 ADSCs were identified by morphology and immunophenotyping. (A) The representative images of ADSCs (×100, 200). (B) ADSCs were identified by immunophenotyping, with the detection of CD13, CD14, CD34, CD44, CD90, and CD105.

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  Fig. 2 The expression of EGF after EGF or siEGF transfection into HaCaT cells was detected. (A) Relative mRNA levels of EGF (of control) in each group were detected using RT-qPCR. (B) Relative protein levels of EGF (of β-actin) in each group were detected using Western blot. (C) Protein levels of excretive EGF (pg/ml) within 3 days in each group were detected using ELISA. Bars indicated means±SD. **p<0.01 vs. Mock group; ^^p<0.01 vs. NC group.

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  Fig. 3 Effect of EGF expression in HaCat on the viability of ADSCs. (A) Cell viability of ADSCs was detected using MTT assay. (B) Protein levels of excretive EGF (pg/ml) were detected using ELISA. (C) Relative mRNA levels (of control) of CK19 and integrin-β were detected using RT-qPCR. (D, E) Relative protein levels (of β-actin) of CK-19 and integrin-β were detected using Western blot. Bars indicated means±SD. **p<0.01 and *p<0.05 vs. HaCaT-Mock group; ##p<0.01 and #p<0.05 vs. control group; ^^p<0.01 and ^p<0.05 vs. HaCat-siEGF group.

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  Fig. 4 Effect of EGF-containing medium of EGF-expressed HaCat cells on the viability of ADSCs. (A) Cell viabilities of ADSCs after 24, 48, 72 h of culturing were detected using MTT assay. (B) Protein levels of excretive EGF (pg/ml) were detected using ELISA. (C) Relative protein levels (of β-actin) of EGF were detected using Western blot. (D) Relative mRNA levels (of control) of CK19 and integrin-β were detected using RT-qPCR. (E, F) Relative protein levels (of β-actin) of CK-19 and integrin-β were detected using Western blot. Bars indicated means±SD. **p<0.01 and *p<0.05 vs. Mock-CM group; ##p<0.01 vs. control group; ^^p<0.01 and ^p<0.05 vs. siEGF-CM group.

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  Fig. 5 Effect of EGF-containing medium of EGF-expressed HaCat cells on invasion and migration rates of ADSCs. (A) Cell invasion rates were detected using transwell assay. (B) Cell migration rates were were detected using wound healing assay. Bars indicated means±SD. *p<0.05 vs. Mock-CM group.

Reference

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