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Characterization of Dermal Stem Cells of Diabetic Patients

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Characterization of Dermal Stem Cells of Diabetic Patients

Letizia Ferroni et al. Cells.

Abstract

Diabetic foot ulcers (DFUs) are lesions that involve loss of epithelium and dermis, sometimes involving deep structures, compartments, and bones. The aim of this work is to investigate the innate regenerative properties of dermal tissue around ulcers by the identification and analysis of resident dermal stem cells (DSCs). Dermal samples were taken at the edge of DFUs, and genes related to the wound healing process were analyzed by the real-time PCR array. The DSCs were isolated and analyzed by immunofluorescence, flow cytometry, and real-time PCR array to define their stemness properties. The gene expression profile of dermal tissue showed a dysregulation in growth factors, metalloproteinases, collagens, and integrins involved in the wound healing process. In the basal condition, diabetic DSCs adhered on the culture plate with spindle-shaped fibroblast-like morphology. They were positive to the mesenchymal stem cells markers CD44, CD73, CD90, and CD105, but negative for the hematopoietic markers CD14, CD34, CD45, and HLA-DR. In diabetic DSCs, the transcription of genes related to self-renewal and cell division were equivalent to that in normal DSCs. However, the expression of CCNA2, CCND2, CDK1, ALDH1A1, and ABCG2 was downregulated compared with that of normal DSCs. These genes are also related to cell cycle progression and stem cell maintenance. Further investigation will improve the understanding of the molecular mechanisms by which these genes together govern cell proliferation, revealing new strategies useful for future treatment of DFUs.

Keywords: ALDH1A1; dermal stem cells; diabetic foot ulcers; gene expression array.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Volcano plot of the wound healing real-time PCR array results. The vertical black line indicates a 1.0-fold change in gene expression. The vertical dashed lines indicate the desired threshold of a 2.0-fold change in gene expression. The horizontal black line indicates the desired 0.05 threshold for the p-value of the t-test.
Figure 2
Figure 2
Morphology of cells isolated from (a) diabetic dermis and (b) normal dermis. The immunostaining of actin filaments with phalloidin (red) shows a spindle-shaped fibroblast-like morphology. Cell nuclei are counterstained in blue with DAPI (magnification 40×).
Figure 3
Figure 3
Characterization of cells isolated from diabetic dermis. Immunofluorescent staining of: (a) CD44 (in green), (b) CD73 (in red), (c) CD90 (in red), (d) CD105 (in green). Nuclei are stained with DAPI in blue (magnification 40×). (e) Detection of cell surface markers by flow cytometry: cells are positive to CD44, CD73, CD90, and CD105, and negative to CD14, CD34, CD45, and HLA-DR.
Figure 3
Figure 3
Characterization of cells isolated from diabetic dermis. Immunofluorescent staining of: (a) CD44 (in green), (b) CD73 (in red), (c) CD90 (in red), (d) CD105 (in green). Nuclei are stained with DAPI in blue (magnification 40×). (e) Detection of cell surface markers by flow cytometry: cells are positive to CD44, CD73, CD90, and CD105, and negative to CD14, CD34, CD45, and HLA-DR.
Figure 4
Figure 4
Growth and migration of diabetic dermal cells compared with those of normal dermal cells. (a) Cumulative population doubling (CPD) of diabetic cells (square indicator) and normal cells (round indicator). (be) In vitro wound healing assay: representative images (10x magnification) of the whole wound area taken in the scratch assay for (b) diabetic cells at 0, (c) diabetic cells at 24 h, (d) normal cells at 0, (e) normal cells at 24 h. (f) The migration percentages (%) are expressed as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
Volcano plot of the stem cell real-time PCR array results. The vertical black line indicates a 1.0-fold change in gene expression. The vertical dashed lines indicate the desired threshold of a 2.0-fold change in gene expression. The horizontal black line indicates the desired 0.05 threshold for the p-value of the t-test.

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