Direct Reprogramming Of Human Fibroblasts Into Sweat Gland-like Cells By Transcription Factors Combination: An Experimental Study

Objective:In the early experiment, our team has established a co-culture condition which could induce mesenchymal stem cell transdifferentiate into sweat gland like cells and tested in nude mice model test and preliminary human trials. There are some sweat gland-like structures in the area of wound after transplantation of autologous bone marrow mesenchymal stem cells and it has certain sweating function.The main purpose of this study is to further identify the effection and mediated signaling way of key reprogramming factors, NF-κB and Lef-1, in the process of differentiation into the sweat gland-like cells based on the previous work. To further clarify the mechanism of the possible differentiation of fibroblasts into sweat gland-like cells is another purpose, which may bring hopes to use other types of cells as seed sources for clinical treatment.Methods:Previous studies have indicated that NF-κB and Wnt are key signaling pathways in the development of sweat gland cells. Combined with the literature reports, we analyse the key transcription factors combinations, NF-κB and Lef-1, may positively involved in development of sweat gland cells. Our main tasks are to analyse of its involvement in the signaling network and to further verify its biological function.1. The experimental groups and operating procedures: When cells arrived at the logarithmic growth phase and growth density reached more than 80%, they were seeded at a density of 106 / well in 6-well plates and were divided into three groups (5 samples for each group) by completely randomized design (In completely randomized design, also known as group design, subjects were divided into several groups, with each group receiving an experimental treatment. Subjects from different groups are independent of each other, and thus this method can also be called "independent group" design).① Experinal genes group:The eukaryotic expression vector containing NF-κB and Lef-1 genes was transfected into the cells and screened out the stable recombinant plasmid for following researches.② Empty vector group:Empty pcDNA3.1 (+) vector was transfected into the cells and screened out the stable monoclonal, which were needed for following experiments.③ Blank control group:The target genes were not transfected and cultured under the same experimental conditions.2. Reprogramming of human fibroblasts by NF-κB and Lef-1 genes:2.1 Construction of eukaryotic expression vector:According to the sequence of NF-κB and Lef-1 found in the U.S. National Center of Biotechnology Information and experiment needs, we designed the PCR primer of open reading frame (ORF) of NF-κB and Lef-1. We extracted the total RNA of MG-63 cells with total RNA extraction kit and synthesized the cDNA by using reverse transcription enzyme. The cDNA and pcDNA3.1 (+) vector were respectively digested by HindⅢ/SalⅠ and HindⅢ/PstⅠ enzyme to connect and then transformed into Escherichia coli DH-5 alpha to amplify. In order to verify whether the mutation of NF-κB and Lef-1 genes sequence is happen, bacteria containing recombinant were sent to BGI. At last, we selected monoclonal bacteria liquid of no mutations for conservation, extracted the plasmid and detected the concentration.2.2 Transfection and selection:The cultured cells were seeded in 96-well plates at density of 5,000 / hole and incubated with different concentrations of the G418 medium. We chose concentration of 10-14 d killing all cells as the basic concentration and thus determined the screening concentration and maintaining concentration. Fibroblasts were seeded in 6-well plates at a density of 106/well the day before transfection and then transfected according to the standard procedure of liposome-mediated transfection. Medium containing G418 with the concentration of 800 mg/L was used to screen the successfully transfected cells 48 h after transfection. Screening pressure is maintained in a concentration of 200 mg/L after 2 weeks. We soon selected the positive monoclonal to expand for subsequent experiments. We used real-time PCR to detect the protein and mRNA expression level of NF-κB and Lef-1 in 3 groups respectively.3. The changes of reprogrammed fibroblasts:EDA-A1 can promote the development of the sweat glands and hair follicle of normal mice and mice with ectodermal-dysplasia. EGF can improve induction rate of the differentiation of MSCs into SGLCs. So auxiliary 1 μg/ml EDA-Al、50 ng/ml EGF were added into the culture medium to help induction of fibroblasts into SGLCs in our experiment.3.1 Detection of specific markers of sweat gland:Immunofluorescence assay was used for qualitative detection of specific markers of sweat gland, such as CEA, CK7, CK14 and CK19, for experimental gene group/empty vector/blank control group cells; Western blot was used for qualitative detection of specific markers of sweat gland, such as CEA, CK7, CK14 and CK19, for experimental gene group/empty vector/blank control group cells; Realtime-PCR was used for quantitative detection of specific markers of sweat gland, such as CEA, CK7, CK14 and CK19, for experimental gene group/empty vector/blank control group cells.3.2 Detection of downstream genes of NF-κB and Lef-1 pathway:Realtime-PCR was used for quantitative detection of downstream genes of NF-κB and Lef-1 pathway, such as Shh and Cyclin D1, for experimental gene group/empty vector/blank control group cells.3.3 Transplantation experiments in animals:Fibroblasts transfected with pcDNA3.1(+)-NF-κB and pcDNA3.1(+)-Lef-1 were implanted in an animal model. Fullthickness scald injuries were produced on both paws of the hind legs of 12 athymic BALB/c nude mice. Then, one scalded paw of each mouse received a subcutaneous injection of 1×106 reprogrammed fibroblasts suspended in 150 ml of medium. The contra-lateral (control) scalded paw received a subcutaneous injection of normal fibroblasts. Twenty days later, an iodine-starch perspiration test was performed on both paws of the mice. Skin biopsies from experimental and control paws were examined by histology. Implanted security was also evaluated.Results:1. Construction of NF-κB and Lef-1 eukaryotic expression vector:The ORF(open reading frame) of NF-κB and Lef-1 genes were cloned into the pcDNA3.1 (+) vector. Bands of molecular mass of 3000 bp and 1200 bp were separately shown in the result of agarose electrophoresis after HindⅢ/SalⅠ and HindⅢ/PstⅠ double enzyme digestion, which indicated that the recombinant plasmids pcDNA3.1(+)-NF-κB and pcDNA3.1(+)-Lef-1 eukaryotic expression vectors were successfully constructed.2.2. G418 selection and detection of NF-κB and Lef-1:After 2 weeks’ G418 screening with increasing concentration, untransfected cells all died. Positive monoclonals formed after the lasting 20d screening.Realtime-PCR results showed that the expression of NF-κB mRNA of experimental genes group, empty vector group, blank control group were 3.651 ± 0.062、0.987± 0.098、1.118 ± 0.024; the expression of Lef-1 mRNA of experimental genes group, empty vector group, blank control group were 2.451 ± 0.032,0.997 ± 0.078.1.158 ± 0.043. which exhibited that the expression of NF-κB and Lef-1 mRNA of stably transfected cells was remarkably increased. Compared with the blank control group, difference was statistically significant (P<0.01). Also compared with the empty vector group, difference was statistically significant (P<0.01), which means that recombinant vector in cells has been a higher level of transcription.3. Detection of specific sweat gland markers:Western-blot results showed that CEA, CK7, CK14 and CK19 proteins were obviously expressed in the group transfected with pcDNA3.1(+)-NF-KB and pcDNA3.1(+)-Lef-1, while CEA, CK7, CK14 and CK19 were not seen in the empty vector group, blank control group. Realtime-PCR results showed that CEA, CK7, CK.14 and CK19 mRNAs were obviously expressed in the group transfected with pcDNA3.1(+)-NF-κB and pcDNA3.1(+)-Lef-1, while CEA, CK7, CK14 and CK19 were not seen in the empty vector group, blank control group. Immunofluorescence staining showed that red and green fluorescence brightness of CEA, CK7, CK14 and CK19 of experimental genes group was very strong, while the same red and green fluorescence brightness were not seen in the empty vector group, blank control group.4. Detection of downstream genes of NF-κB and Lef-1 pathway:Realtime-PCR results showed that the expression of Shh mRNA of experimental genes group, empty vector group, blank control group were 3.151 ± 0.052,0.997 ± 0.038, 1.098 ±0.074; the expression of Cyclin D1 mRNA of experimental genes group, empty vector group, blank control group were 2.653 ± 0.045.0.997 ± 0.048,1.118 ± 0.053, which exhibited that the expression of Shh and Cyclin D1 mRNA of stably transfected fibroblasts were also remarkably increased. Compared with the blank control group, difference was statistically significant (P<0.01). Also compared with the empty vector group, difference was statistically significant(P<0.01), which means that the well-known key genes in the regeneration of sweat gland has been a higher level of transcription in the group transfected with pcDNA3.1(+)-NF-icB and pcDNA3.1(+)-Lef-1.5. Transplantation experiments in animals:Twenty days later, iodine-starch perspiration tests showed that 7 out of the 10 cell-treated paws were positive for perspiration, with a distinctive black point-like area appearing in the center of the paw. Contralateral paws tested negative. Histological examination of skin biopsies from experimental and control paws revealed that sweat glands 25 were fully reconstructed in the test paws, with integral, secretory and ductal portions, but were not present in the control paws.Conclusions:Gene combination of NF-κB and Lef-1 can directly reprogram human fibroblasts into sweat gland like-cells. The specific sweat gland markers, CK.7, CK.14, CK.19 and CEA, were significantly expressed in the process of direct reprogramming of fibroblasts into sweat gland-like cells. The key roles of EDA/NF-κB and Wnt/Lef-1 pathways in direct reprogramming are also confirmed by detection of Shh and Cyclin Dl. The biological function of reprogrammed fibroblasts was tested in an animal model. This is the first report of successful reprogramming of fibroblasts into sweat gland-like cells, which will provide a new cell source for sweat gland regeneration in patients with extensive deep burns. This study will provide us new insights into the regeneration of sweat gland-like cells by showing a new ideal cell source for cell therapy.