Establishment of human hair follicle mesenchymal stem cells with overexpressed human hepatocyte growth factor

Abstract

Objectives: Chronic liver disease has become a major health problem that causes serious damage to human health. Since the existing treatment effect was not ideal, we need to seek new treatment methods.

Materials and methods: We utilized the gene recombination technology to obtain the human hair mesenchymal stem cells which overexpression of human hepatocyte growth factor (hHGF). Furthermore, we verified the property of transfected cells through detecting surface marker by flow cytometry.

Results: We show here establishment of the hHGF-overexpressing lentivirus vector, and successfully transfection to human hair follicle mesenchymal stem cells. The verified experiments could demonstrate the human hair follicle mesenchymal stem cells which have been transfected still have the properties of stem cells.

Conclusion: We successfully constructed human hair follicle mesenchymal stem cells which overexpression hHGF, and maintain the same properties compared with pro-transfected cells.

Keywords: HGF; Hair follicle; Lentivirus; Liver diseases; Stem cells.

Figure 1

Gel electrophoresis pattern of total RNA

Figure 2

Gel electrophoresis pattern for hepatocyte growth factor (HGF) gene obtained by PCR. Reverse transcription was performed to obtain the cDNA of HGF by using total RNA extracted from liver tissue as template, followed by PCR to obtain the HGF gene. M is marker 10000; 1 is negative control, 2 and 3 are positive controls with cDNA as the template Translation for embedded text:

Figure 3

Gel electrophoresis pattern for PCR identification after binding with hepatocyte growth factor (HGF)-T1 vector Escherichia coli plasmid infected with HGF-T1 was used as the template to perform PCR identification, verifying the presence of HGF gene in the E. coli. M is marker 10000; 1 is negative control;2 is the cDNA with HGF as the template; 3 is the plasmid extracted from infected E. coli

Figure 4

Gel electrophoresis pattern for digestion products after transduction and T1 vector binding. 1 and 2 are products of single digestion by BamHI and XbaI after T1 vector binding; 3 is the products of double digestion. M is marker 10000 Restriction Enzymes That Cleave Lv- cMyc -GFP Once

Figure 5

Existing Lenti- EF1a-cMyc-IRES-eGFP plasmid diagram and enzyme cleavage sites. According to the enzyme cleavage sites on the plasmid diagram, two common enzyme cleavage sites for BamH1 (4487) and XbaI (5825) were selected for double digestion

Figure 6

Gel electrophoresis pattern for identification of plasmid after binding with T4 ligase. 1 and 2 show bands after single digestion with XbaI while, 3 and 4 show bands after double digestion with XbaI and BamHI, 5 and 6 show bands after single digestion with BamHI, and 7 and 8 show PCR products. M1 and M2 represent marker 10000 and marker 2000, respectively

Figure 7

Gene sequencing data after hHGF insertion and positive expression. This indicates successful construction of lentivirus plasmid expressing hHGF

Figures 8

A and B are the images of 293T cells transfected with hHGF seen under a fluorescence microscope (A) and regular microscope (B) (40×)

Figure 9

Expression level of hepatocyte growth factor (HGF) protein after transfection of target plasmid into 293T cells

Figure 10

Expression of hepatocyte growth factor (HGF) mRNA after 24 hr and 48 hr following transfection of target plasmid into 293T cells

Figure 11

Lentivirus transfected 293 T cells that secreted the overexpressed hepatocyte growth factor (HGF) (48 hr after transfection, 40×)

Figure 12

Identification of adipogenesis and osteogenesis of hair follicle mesenchymal stem cells (HFMSCs). A: After 3 weeks of induction of HFMSCsin osteogenic differentiation medium, staining with alizarin red revealed mineralized cells in a reddish-brown color; C: After 2 weeks of induction in adipogenic differentiation medium, oil red O staining showed bright fat droplets in bright red color; B and D: Cells cultured for 2 weeks in basic medium instead of osteogenic and adipogenic differentiation media were used as the negative control. Alizarin red and oil red O staining revealed that cells did not change color. (200×)

Figure 13

Determination of phenotype characteristics of hair follicle mesenchymal stem cells by flowcytometry

Figure 14

The Growth status of cells under different culture conditions

Figure 15

A: Image of hair follicle mesenchymal stem cells after viral transfection seen under a fluorescence microscope, B: Image of cells after transfection as seen under an inverted microscope (40×)

Figure 16

Expression level of hHGF in human hair follicle mesenchymal stem cells. 1, 2, and 3 show the protein expression of the control group, only GFP transfection G-HFMSCs group, and human hepatocyte growth factor (hHGF) lentivirus transfection hHGF-HFMSCs group, respectively

Figure 17

Data from ELISA assay showing the amount of human hepatocyte growth factor (hHGF) secreted into the cell culture supernatant at different time points

Figure 19

A C:hHGF-HFMSCs group and GFP-HFMSCs guoup; alizarin red staining revealed red cells at the deposition sites of calcium salt after 4 weeks of induction(40×); B D: Cells cultured for 4 weeks in basic medium instead of osteogenic differentiation media were used as the negative control. Alizarin red staining revealed that cells did not change color (40×)

Figure 20

AC: hHGF-HFMSCs group and GFP-HFMSCs guoup change in cellular morphology was observed and oil red O staining revealed the presence of orange fat droplets inside cells after 2 weeks of induction; BD: Cells cultured for 2 weeks in basic medium instead of adipogenic differentiation media were used as the negative control. Oil red O staining revealed that cells did not change color (100×)

Figure 21

Detection of surface markers for hHGF-HFMSCs group and GFP-HFMSCs group by flow cytometry: A: Detection of surface markers for HGF-HFMSCs group; B: Detection of surface markers for GFP-HFMSCs group