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Amniotic fluid stem cells provide considerable advantages in epidermal regeneration: B7H4 creates a moderate inflammation microenvironment to promote wound repair.

Sun Q, Li F, Li H, Chen RH, Gu YZ, Chen Y, Liang HS, You XR, Ding SS, Gao L, Wang YL, Qin MD, Zhang XG - Sci Rep (2015)

Bottom Line: Co-localization of GFP/K14 and GFP/K10 in the repaired epidermis demonstrated that hAFS cells can differentiate into keratinocytes.During wound repair, hAFS cells did not directly secrete repair-related factors, such as bFGF, VEGF, CXCL12, TGF-β1 and KGF, and provided a moderate inflammation reaction with lower expression of IL-1β, IL-6, TNF-α, Cox2 and Mac3.Furthermore, with their uniquely high proliferation rate, hAFS cells offer a promising alternative for epidermal regeneration.

View Article: PubMed Central - PubMed

Affiliation: 1] The Stem Cell and Biomedical Material Key Laboratory of Jiangsu Province (the State Key Laboratory Incubation Base), Soochow University, Suzhou, Jiangsu Province, P.R. China [2] Department of Immunology, School of Biology and Basic Medical Sciences, Soochow University, Suzhou, Jiangsu Province, P.R. China.

ABSTRACT
The current treatments for severe skin injury all involve skin grafting. However, there is a worldwide shortage of donor skin tissue. In this study, we examined the advantages of using human amniotic fluid stem (hAFS) cells in skin wound healing. In vitro, hAFS cells differentiate into keratinocytes (termed hAFS-K). Like keratinocytes, hAFS-K cells express the markers K5, K14, K10 and involucrin; display typical cellular structure, including a tonofibril-rich cytoplasm; and construct a completely pluristratified epithelium in 3D culture. In vivo, in a mouse excisional wound model, GFP-positive hAFS cells participate in wound repair. Co-localization of GFP/K14 and GFP/K10 in the repaired epidermis demonstrated that hAFS cells can differentiate into keratinocytes. Real-time PCR results confirmed that hAFS cells can initiate and promote early-stage repair of skin damage. During wound repair, hAFS cells did not directly secrete repair-related factors, such as bFGF, VEGF, CXCL12, TGF-β1 and KGF, and provided a moderate inflammation reaction with lower expression of IL-1β, IL-6, TNF-α, Cox2 and Mac3. In hAFS cells, the negative co-stimulatory molecule B7H4 regulates low immunogenicity, which can provide a modest inflammatory reaction microenvironment for wound repair. Furthermore, with their uniquely high proliferation rate, hAFS cells offer a promising alternative for epidermal regeneration.

No MeSH data available.


Related in: MedlinePlus

Differentiation of hAFS cells and characterization of hAFS cell-derived keratinocytes(a) The morphology of hAFS cells, hAFS cell-derived keratinocytes (hAFS-K) and human keratinocytes. After induction, hAFS-K exhibited typical pavementous epithelial morphology and spontaneously formed colonies similar to keratinocytes. (b) mRNA expression of K5, K14, K19 in hAFS-K. hAFS cells were used as a negative control and keratinocytes as a positive control. Cropped gels were used, and the gels were run under the same experimental conditions. Full-length images are presented in Supplementary Figure S5. (c) hAFS-K (n = 4) were analysed by flow cytometry after staining with PE-conjugated control isotype IgG (grey peaks) or antibodies against cell surface proteins K5 and K14, showing the differentiation rates to be (40 ± 1.24)% and (35 ± 0.36)%, respectively. (d) Immunofluorescence staining of K5 and K14 (red) in hAFS cells, hAFS-K and keratinocytes. Nuclei (blue) were stained with DAPI. hAFS cells were used as the negative control and keratinocytes as the positive control.
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f2: Differentiation of hAFS cells and characterization of hAFS cell-derived keratinocytes(a) The morphology of hAFS cells, hAFS cell-derived keratinocytes (hAFS-K) and human keratinocytes. After induction, hAFS-K exhibited typical pavementous epithelial morphology and spontaneously formed colonies similar to keratinocytes. (b) mRNA expression of K5, K14, K19 in hAFS-K. hAFS cells were used as a negative control and keratinocytes as a positive control. Cropped gels were used, and the gels were run under the same experimental conditions. Full-length images are presented in Supplementary Figure S5. (c) hAFS-K (n = 4) were analysed by flow cytometry after staining with PE-conjugated control isotype IgG (grey peaks) or antibodies against cell surface proteins K5 and K14, showing the differentiation rates to be (40 ± 1.24)% and (35 ± 0.36)%, respectively. (d) Immunofluorescence staining of K5 and K14 (red) in hAFS cells, hAFS-K and keratinocytes. Nuclei (blue) were stained with DAPI. hAFS cells were used as the negative control and keratinocytes as the positive control.

Mentions: To stimulate differentiation into keratinocytes, hAFS cells were grown in inducing medium for approximately 30 days and then maintained in KGM2 without serum. After the third passage, colonies of cells formed spontaneously with typical pavementous epithelial morphology; we named these cells hAFS-K (keratinocytes derived from hAFS cells). The phenotypic characteristics of hAFS-K were similar to those of human primary keratinocytes but clearly distinct from those of hAFS cells (Fig. 2a). RT-PCR results identified hAFS-K as K5 and K14 positive, like basal keratinocytes, with comparable gene expression levels in three independent cell lines (Fig. 2b). Flow cytometry analysis of K5- and K14-positive hAFS-K cells showed the differentiation rate to be (40 ± 1.24% and 35 ± 0.36%, respectively (Fig. 2c), and immunostaining confirmed that the cells express the K5 and K14 proteins (Fig. 2d). Taken together, these results demonstrate that hAFS-K cells have similar biological characteristics to keratinocytes.


Amniotic fluid stem cells provide considerable advantages in epidermal regeneration: B7H4 creates a moderate inflammation microenvironment to promote wound repair.

Sun Q, Li F, Li H, Chen RH, Gu YZ, Chen Y, Liang HS, You XR, Ding SS, Gao L, Wang YL, Qin MD, Zhang XG - Sci Rep (2015)

Differentiation of hAFS cells and characterization of hAFS cell-derived keratinocytes(a) The morphology of hAFS cells, hAFS cell-derived keratinocytes (hAFS-K) and human keratinocytes. After induction, hAFS-K exhibited typical pavementous epithelial morphology and spontaneously formed colonies similar to keratinocytes. (b) mRNA expression of K5, K14, K19 in hAFS-K. hAFS cells were used as a negative control and keratinocytes as a positive control. Cropped gels were used, and the gels were run under the same experimental conditions. Full-length images are presented in Supplementary Figure S5. (c) hAFS-K (n = 4) were analysed by flow cytometry after staining with PE-conjugated control isotype IgG (grey peaks) or antibodies against cell surface proteins K5 and K14, showing the differentiation rates to be (40 ± 1.24)% and (35 ± 0.36)%, respectively. (d) Immunofluorescence staining of K5 and K14 (red) in hAFS cells, hAFS-K and keratinocytes. Nuclei (blue) were stained with DAPI. hAFS cells were used as the negative control and keratinocytes as the positive control.
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Related In: Results  -  Collection

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f2: Differentiation of hAFS cells and characterization of hAFS cell-derived keratinocytes(a) The morphology of hAFS cells, hAFS cell-derived keratinocytes (hAFS-K) and human keratinocytes. After induction, hAFS-K exhibited typical pavementous epithelial morphology and spontaneously formed colonies similar to keratinocytes. (b) mRNA expression of K5, K14, K19 in hAFS-K. hAFS cells were used as a negative control and keratinocytes as a positive control. Cropped gels were used, and the gels were run under the same experimental conditions. Full-length images are presented in Supplementary Figure S5. (c) hAFS-K (n = 4) were analysed by flow cytometry after staining with PE-conjugated control isotype IgG (grey peaks) or antibodies against cell surface proteins K5 and K14, showing the differentiation rates to be (40 ± 1.24)% and (35 ± 0.36)%, respectively. (d) Immunofluorescence staining of K5 and K14 (red) in hAFS cells, hAFS-K and keratinocytes. Nuclei (blue) were stained with DAPI. hAFS cells were used as the negative control and keratinocytes as the positive control.
Mentions: To stimulate differentiation into keratinocytes, hAFS cells were grown in inducing medium for approximately 30 days and then maintained in KGM2 without serum. After the third passage, colonies of cells formed spontaneously with typical pavementous epithelial morphology; we named these cells hAFS-K (keratinocytes derived from hAFS cells). The phenotypic characteristics of hAFS-K were similar to those of human primary keratinocytes but clearly distinct from those of hAFS cells (Fig. 2a). RT-PCR results identified hAFS-K as K5 and K14 positive, like basal keratinocytes, with comparable gene expression levels in three independent cell lines (Fig. 2b). Flow cytometry analysis of K5- and K14-positive hAFS-K cells showed the differentiation rate to be (40 ± 1.24% and 35 ± 0.36%, respectively (Fig. 2c), and immunostaining confirmed that the cells express the K5 and K14 proteins (Fig. 2d). Taken together, these results demonstrate that hAFS-K cells have similar biological characteristics to keratinocytes.

Bottom Line: Co-localization of GFP/K14 and GFP/K10 in the repaired epidermis demonstrated that hAFS cells can differentiate into keratinocytes.During wound repair, hAFS cells did not directly secrete repair-related factors, such as bFGF, VEGF, CXCL12, TGF-β1 and KGF, and provided a moderate inflammation reaction with lower expression of IL-1β, IL-6, TNF-α, Cox2 and Mac3.Furthermore, with their uniquely high proliferation rate, hAFS cells offer a promising alternative for epidermal regeneration.

View Article: PubMed Central - PubMed

Affiliation: 1] The Stem Cell and Biomedical Material Key Laboratory of Jiangsu Province (the State Key Laboratory Incubation Base), Soochow University, Suzhou, Jiangsu Province, P.R. China [2] Department of Immunology, School of Biology and Basic Medical Sciences, Soochow University, Suzhou, Jiangsu Province, P.R. China.

ABSTRACT
The current treatments for severe skin injury all involve skin grafting. However, there is a worldwide shortage of donor skin tissue. In this study, we examined the advantages of using human amniotic fluid stem (hAFS) cells in skin wound healing. In vitro, hAFS cells differentiate into keratinocytes (termed hAFS-K). Like keratinocytes, hAFS-K cells express the markers K5, K14, K10 and involucrin; display typical cellular structure, including a tonofibril-rich cytoplasm; and construct a completely pluristratified epithelium in 3D culture. In vivo, in a mouse excisional wound model, GFP-positive hAFS cells participate in wound repair. Co-localization of GFP/K14 and GFP/K10 in the repaired epidermis demonstrated that hAFS cells can differentiate into keratinocytes. Real-time PCR results confirmed that hAFS cells can initiate and promote early-stage repair of skin damage. During wound repair, hAFS cells did not directly secrete repair-related factors, such as bFGF, VEGF, CXCL12, TGF-β1 and KGF, and provided a moderate inflammation reaction with lower expression of IL-1β, IL-6, TNF-α, Cox2 and Mac3. In hAFS cells, the negative co-stimulatory molecule B7H4 regulates low immunogenicity, which can provide a modest inflammatory reaction microenvironment for wound repair. Furthermore, with their uniquely high proliferation rate, hAFS cells offer a promising alternative for epidermal regeneration.

No MeSH data available.


Related in: MedlinePlus