Expression And Function Of Kruppel Like Factor 4 (KLF4) In Mouse Epidermal Stem Cells

PARTâ… Expression of KLF4 in mouse epidermal stem cellsObjective:Examine the expression of KLF4 in mouse epidermal stem cells.Methods:Antibodies against CD34 and CD49f, two markers of skin bulge stem cells in this experiment were selected to enrich mouse skin bulge stem cells (CD34+CD49f+ cells). CD34+CD49f+ cells were purified by fluorescent activated cell sorting (FACS) from the skin of wild type C57BL/6 mice and quantitative RT-PCR analysis was performed to detect the expression of KLF4 in these purified cells. KLF4/EGFP mouse model was generated to label KLF4-expressing cells by EGFP. Flow cytometric assay and label retention cell assay were performed to examine whether if KLF4 was expressed in skin stem cells. Lineage tracing experiment was used to trace the KLF4-expressing stem cells and their progenies in KLF4/CreERTM/Rosa26RLacZ transgenic mouse.Results:Expression of KLF4 showed statistically significant increase in CD34+CD49f+ population when compared to CD34-CD49f+ cells. 12.3% CD34+CD49f+ cells and 50.4% Sca-1+/CD49f+ cells were observed in purified keratinocytes. EGFP-positive cells (58.6%) were among CD34+/CD49f+ cells. 73.0% of Sca-1+/CD49f+ cells were KLF4/EGFP-positive.14.6% BrdU-positive cells were observed in purified keratinocytes, and 4.1% were BrdU and KLF4/EGFP positive cells. In lineage tracing experiment, blue LacZ-positive cells (KLF4-expressing cells and their progenies) were observed in bulge area, sebaceous gland, and IFE.Conclution:Taken together, our results suggest that KLF4 is expressed in mouse epidermal stem cells. KLF4-expressing cells possess a label retaining property and give rise to differentiated cells.PARTâ…¡The function of KLF4 in epidermal stem cellsObjective:Detect the function of KLF4 in mouse epidermal stem cells.Methods:KLF4/CreERTM(+/-)/KLF4(flox+/+) mouse model was generated to knockout KLF4 gene by tamoxifen induction. Immunohistochemistry assay was performed to detect the potential change of skin structure upon KLF4 knockout. Hair follicle bulge stem cells (CD34+CD49f+ cells) and IFE stem cells (Sca-1+CD49f+ cells) were examined by flow cytometry in keratinocytes purified from control and KLF4 knockout mouse skin tissues. Self-renewal ability of keratinocytes or CD34+CD49f+ bulge stem cells purified from control and KLF4 knockout mice were detected by in vitro colony-formation assay.Results:Basal cell hyperplasia from a single layer to multilayer was observed in KLF4 knockout mice when compared to control mice. In addition, a significant increase of hair follicle density was observed upon KLF4 knockout. 12.8% CD34+/CD49f+ cells were observed in control mice detected by flow cytometric analysis. However, this population decreased to 6.26% in KLF4 knockout mice. Similarly, Sca-1+/CD49f+ population decreased from 68.73% in control mice to 52.6% in KLF4 knockout mice. In colony-formation assay, from 2000 keratinocytes, 22 colonies were formed in control mice, while this number decreased to 9 upon KLF4 knockout. When the same number of sorted CD34+/CD49f+ cells was used for the same assay, the number of colonies increased from 22 to 33 in control mice. However, the colony number was only 15 from KLF4 knockout keratinocytes.Conclution:Knockout of KLF4 decreases stem cell population and self-renewal potential, indicating that KLF4 plays an important role in maintenance of mouse epidermal stem cells. PARTâ…¢The function of KLF4-expressing epidermal stem cells in mouse acute cutaneous wound healingObjective:Test the contribution of KLF4-expressing stem cells to mouse acute cutaneous wound healing.Methods:To test the hypothesize that KLF4 is important for the process of mouse acute cutaneous wound healing, two full-thickness wounds in parallel were made on the back skin of KLF4 knockout mouse model as described above. In order to test the contribution of KLF4-expressing stem cells to wound healing, lineage tracing experiments was performed using KLF4/CreERTM/Rosa26RLacZ mice. In addition, to test the effect of KLF4 on cell migration in vitro, a process critical for wound healing, stable KLF4 knockdown HaCaT human keratinocytes were generated to perform scratch assay and detect potential molecular mechanism.Results:Wound healing assay shows that wounds started to heal within 2 days in control mice (KLF4+/+). Wound closure was observed 10 days after wound placement. However, in KLF4 knockout mice (KLF4-/-), the healing process was significantly delayed. At 10 days after wounding, healing was still in progress. KLF4/CreERTM/Rosa26RLacZ mice with tamoxifen injected upon wound placement, KLF4-expressing pluripotent cells and their progenies (blue cells and stripes) were clearly visible on the side of the wound and along the suprabasal layer of skin towards the wound area. Long term lineage tracing experiment shows that blue staining (long term survival KLF4-expressing stem cells) in intact skin tissues was barely detectable in mice without wound placement 8 months after tamoxifen induction. However, in mice with wound placement, blue cells were much more and clearly visible in hair follicles and basal layer of skin at the edge of wound area. At 12 h after in vitro scratch assay, the percentage of wound closure decreased from 40% in control cells to 12% in KLF4 knockdown cells, which is associated with decreased expression and phosphorylation level of epidermal growth factor receptor (EGFR).Conclution:KLF4-expressing pluripotent stem cells contribute to mouse acute cutaneous wound healing. Knockdown of KLF4 decreases cell migration in HaCaT cells accompanied by downregulation of EGFR.