Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell-based therapy for skin regeneration. Aiming to establish human iPSCs as a potential cell source for skin tissue engineering, we expect to obtain an epidermal-like cell line with angiogenic and keratinogenic differentiation potential via inducing iPSC-derived mesenchymal stem cells (iPSC-MSCs) with basic fibroblast growth factor (bFGF) and/or keratinocyte growth factor (KGF). The results show that iPSC-MSCs were successfully induced with a positive FGFR/KGFR expression on the cell surface. BFGF/KGF induction could significantly increase the expression of vascularization marker CD31 and keratinization marker CK10, respectively, while when combined together, although CD31 and CK10 were still positively expressed, their expressions were lower than that of the single induction group, suggesting that the effects of the two growth factors interfered with each other. This cell line with angiogenic and keratinogenic differentiation potential provides a promising new source of cells for the construction of well vascularized and keratinized tissue engineered skin, furthermore establishing an effective strategy for iPSC-based therapy in skin tissue engineering.
Skin transplantation is one of the main methods to treat skin defects such as extensive burns and refractory skin ulcers. However, the source of skin grafts is limited and there is a greater damage to the donor area. In recent years, with the rise of tissue engineering, tissue engineered skin is considered to have the unique advantage of repairing damaged tissue and possibly exercising part of the skin function.
Mesenchymal stem cells (MSCs) are one of the most commonly used cell sources in tissue engineering due to their multipotential differentiation potential. Nevertheless, autologous MSCs are lack of in vitro expansion capacity, and their application is limited by patient age and some other health conditions [
IPSCs have been shown to differentiate into multiple cell types, providing attractive prospects for personalized therapy. However, one of the great limitations to the clinical application of iPSCs is their tumorigenicity. To limit the undesired tumorigenesis associated with iPSC pluripotency, in vitro differentiation of iPSCs into downstream cells before clinical application is considered to be necessary [
Vascularization and epidermal keratinization are important for ideal healing of the wound. Firstly, angiogenesis is a key step in the wound healing process, providing oxygen and nutrients for the defect area, while taking away metabolites to promote immune response and tissue regeneration. Meanwhile, keratinization of renascent skin is also critical: the keratin layer can resist external frictional stimuli and bacterial intrusion, which plays an important role in the quality of wound healing. Therefore, the construction of a cell line with the potential of vascularization and keratinization has an important significance for skin tissue engineering.
Cytokines are involved in cellular communication for regulating the differentiation and plasticity of MSCs. Fibroblast growth factor (FGFs) family is a kind of multifunctional growth factor; its effective application can restore blood perfusion in the wound area and promote wound healing [
Another member of the FGF family, basic fibroblast growth factor (bFGF), can accelerate angiogenesis and soft tissue regeneration and inhibit scar formation by regulating the degradation and rebuilding of the extracellular matrix, thus effectively promoting wound healing [
It has been proved that there is a possibility for iPSCs directly differentiated into endothelial cells [
The bFGF-treated MSCs could express higher vascularization markers, such as CD31, thereby facilitating vascularization of the injured area and promoting immune responses [
Plentiful researches about the induction of iPSC-MSCs into some cell lineages, such as osteocytes and cardiomyocytes, have already been reported [
Derivation of iPSC-MSCs from iPSCs and the angiogenic and/or keratinogenic differentiation via bFGF and/or KGF induction.
The human iPSC ATCC-DYR0100 cell line was purchased from ATCC (ATCC® ACS-1011™, USA). IPSCs were cultured as colonies on a feeder layer of mitotically inactivated murine embryonic fibroblasts (MEF) as previously reported [
IPSC-MSCs were obtained from iPSC according to previous study [
IPSC-MSCs were cultured until 70-80% confluence and the original medium was replaced with induction medium. BFGF induction was performed by exposure to 10 ng/ml bFGF, 100 U/ml penicillin, and streptomycin in DMEM medium containing 20% FBS for 2 weeks.
IPSC-MSCs were cultured until 70-80% confluence and the original medium was replaced with induction medium. KGF induction was performed by exposure to 40 ng/ml KGF, 100 U/ml penicillin, and streptomycin in DMEM medium containing 20% FBS for 2 weeks.
IPSC-MSCs were cultured until 70-80% confluence and the original medium was replaced with induction medium. Combined induction was performed were grown by exposure to 10 ng/ml KGF and 40 ng/ml bFGF, 100 U/ml penicillin, and streptomycin in DMEM medium containing 20% FBS for 2 weeks.
To confirm the derivation of iPSC-MSCs, surface antigen expression of iPSC-MSCs was characterized via flow cytometry. IPSC-MSCs (passage 5) were harvested by trypsin-ethylenediaminetetraacetic acid (EDTA) and washed with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and then resuspended to ~5×105 cells in 50
IPSC-MSCs were plated on glass coverslips in 24-well plates at 5×105/well. The following day, cells were serum starved overnight. iPSC-MSCs were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 0.5% BSA prior to antibody addition. Indirect immunofluorescence experiments were performed using the KGFR, FGFR, CD31, and K10 monoclonal antibodies (Abcam) overnight at 4°C. Coverslips were then incubated for 60 minutes at room temperature with biotinylated goat anti-rabbit IgG (Abcam) diluted 1:250 in 0.5% BSA. Nuclei were counterstained with 300 nM 4’,6-diamidino-2-phenylindole (DAPI, Suobaolai, Beijing). The stained cells were observed with a confocal laser scanning microscope with 100 times magnification (CLSM Nikon, A1),
All results are presented as mean value ± standard deviation. Differences between groups were analyzed by analysis of variance (one-way ANOVA) by statistic software SPSS 22.0.
The shape of the iPSCs was round before induction, and after three-day induction, the cell morphology was changed into a fibroblast-like long spindle type (Figure
(a) Morphology of iPSCs and iPSC-MSCs observed by a light microscope (×100). The morphology of the iPSC-MSCs resembled elongated spindle shaped cells and differed significantly from the undifferentiated iPSCs. The yellow arrow showed that cell morphology exhibited a round shape, and the red arrows showed that cell morphology changed to a long spindle shape. Scale bar 200
FGFRs have been shown to be expressed on the surface of MSCs and has an important role for tissue development, such as chondrogenesis and osteogenesis [
Immunofluorescence staining of KGF receptor and bFGF receptor on iPSCs and iPSC-MSCs (×100). The results showed that the expression of FGFR/KGFR on the cell surface was significantly enhanced after MSC induction.
CD31 and CK10 are markers of vascularization and keratinization, respectively [
Immunofluorescence staining of DAPI (blue) and CD31/CK10 (green) in iPSC-MSCs after bFGF/KGF induction (×100). After bFGF/KGF induction, CD31/CK10 was significantly expressed in iPSC-MSCs. The yellow arrows indicated that cell morphology still exhibited a MSC-like long spindle type, and the red arrow showed that cell morphology gradually changed from long spindle shape to oblate or irregular shape.
At the same time, we also found that the morphology of iPSC-MSCs induced by KGF changed: the cell morphology gradually changed from long spindle shape to oblate or irregular shape, which was close to the morphology of epithelial cells. Changes in cell morphology indicate iPSC-MSCs differentiation towards epidermal-like cells. However, no obvious morphological change was observed in bFGF-induced iPSC-MSCs, suggesting that bFGF had no significant effect on promoting the differentiation towards epidermal-like cells.
In order to construct a cell lineage that is more suitable for skin tissue engineering, we further combined the two growth factors together for inducing iPSC-MSCs into a cell lineage with both angiogenic and keratinogenic differentiation potential. Immunofluorescence staining showed that both CD31 and K10 were significantly expressed after bFGF and KGF coinduction.
However, unlike in Figure
(a) Immunofluorescence staining of DAPI, CD31, and CK10 of iPSC-MSCs after combined induction (×100). (b) Results of Western blot assay, indicating that bFGF or KGF induction could enhance the expression of CD31 or CK10, whereas in the combined induction group, the expression of CD31 and K10 was lower than that of the single induction group.
Similarly, western blot results (Figure
In this study, iPSC-MSCs were successfully differentiated into cells with angiogenic and/or keratinogenic potential via the induction of bFGF and/or KGF. This cell line with angiogenic and keratinogenic differentiation potential provides a promising new source of cells for the construction of well vascularized and keratinized tissue engineered skin. At the same time, we also found that although the induction of bFGF and KGF could increase the expression of vascularization and keratinization markers, respectively, the effects of inducing cell differentiation by the two conflicted with each other, thus more appropriate concentration of growth factors still remained to be explored.
The pictures used to support the findings of this study are included within the article.
The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
Weimin Lin and Miao Chen contributed equally to this work. Weimin Lin, Yili Qu, and Yi Man designed this study. Weimin Lin, Miao Chen, Chen Hu, and Siyu Qin completed the experiments. Weimin Lin and Miao Chen wrote this manuscript. Miao Chen and Chenyu Chu edited the figures. Lin Xiang reviewed and checked the manuscript.
This work was supported by grants from the National Natural Science Foundation of China (no. 81671023) and the Miaozi Project in Science and Technology Innovation Program of Sichuan Province (no. 17-YCG053).