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. 2011 Jan 24;6(1):e16114.
doi: 10.1371/journal.pone.0016114.

Improved Survival, Vascular Differentiation and Wound Healing Potential of Stem Cells Co-Cultured With Endothelial Cells

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Free PMC article

Improved Survival, Vascular Differentiation and Wound Healing Potential of Stem Cells Co-Cultured With Endothelial Cells

Dora C S Pedroso et al. PLoS One. .
Free PMC article

Abstract

In this study, we developed a methodology to improve the survival, vascular differentiation and regenerative potential of umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34(+) cells), by co-culturing the stem cells in a 3D fibrin gel with CD34(+)-derived endothelial cells (ECs). ECs differentiated from CD34(+) cells appear to have superior angiogenic properties to fully differentiated ECs, such as human umbilical vein endothelial cells (HUVECs). Our results indicate that the pro-survival effect of CD34(+)-derived ECs on CD34(+) cells is mediated, at least in part, by bioactive factors released from ECs. This effect likely involves the secretion of novel cytokines, including interleukin-17 (IL-17) and interleukin-10 (IL-10), and the activation of the ERK 1/2 pathway in CD34(+) cells. We also show that the endothelial differentiation of CD34(+) cells in co-culture with CD34(+)-derived ECs is mediated by a combination of soluble and insoluble factors. The regenerative potential of this co-culture system was demonstrated in a chronic wound diabetic animal model. The co-transplantation of CD34(+) cells with CD34(+)-derived ECs improved the wound healing relatively to controls, by decreasing the inflammatory reaction and increasing the neovascularization of the wound.

Conflict of interest statement

Competing Interests: João Duarte is an employee of Crioestaminal, a stem cell company. This does not alter the adherence of authors to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Protein expression in undifferentiated CD34+ cells and their endothelial progenies.
A) Immunofluorescence results for undifferentiated CD34+ cells. Bar corresponds to 20 µm. B, C) FACS analysis of (B) undifferentiated CD34+ cells and (C) CD34+-derived ECs at passage 1 (1st P) and 3 (3rd P). Percentages of positive cells were calculated based on the isotype controls (grey plot) and are shown in the histogram plots.
Figure 2
Figure 2. Characterization of CD34+-derived ECs.
A) Immunofluorescence analysis. Bar corresponds to 40 µm. B) Quantitative RT-PCR analysis. Results are average ± SD, n = 4. C) Quantification of cord length and number of sprouts at 15 h and 48 h. Counts were performed using a magnification of ×100. Results are average ± SD, n = 3. *, **, *** denote statistical significance (P<0.05, P<0.01, P<0.001, respectively).
Figure 3
Figure 3. Adhesion of CD34+ cells to different substrates.
CD34+ cells were seeded on 24-well plates coated with fibrin gel, type I collagen gel (2.5 mg/mL) and fibronectin (50 µg/mL), and incubated for 3 h at 37°C. Polystyrene culture wells were used as control. After that time, the cells were washed and the attached ones were counted in six random microscope fields (×200 magnification) for each replicate. Results are average ± SD, n = 6. * and ** denote statistical significance (P<0.05 and P<0.01, respectively).
Figure 4
Figure 4. Viability of encapsulated cells.
A) Viability of encapsulated cells, as assessed by a LIVE/DEAD assay. Results are average ± SD, n = 3. B) Mitochondrial metabolic activity of encapsulated cells. Results are average ± SD, n = 6. Each absorbance at 540 nm was normalized by day 2 absorbance. C) Viability of encapsulated cells, as quantified by a LIVE/DEAD assay. Results are average ± SD, n = 3. In all figures, * denote statistical significance within time group: * P<0.05, **P<0.01, *** P<0.001. # denotes statistical significance between time groups comparing the respective control/treatment groups: # P<0.05, ## P<0.01. D) Phosphorylated Akt/total Akt and phosphorylated ERK/total ERK ratios assessed by a Bio-Plex phosphoprotein detection assay. Results are average ± SD, n = 3.
Figure 5
Figure 5. Multiplex cytokine analysis.
Analysis of cytokines secreted by (A) CD34+-derived ECs grown on tissue culture polystyrene or encapsulated in fibrin gels for 2 or 10 days, (B) CD34+ cells grown on tissue culture polystyrene or encapsulated in fibrin gels for 2 or 10 days and (C) co-culture of CD34+ and CD34+-derived ECs encapsulated in fibrin gels for 2 or 10 days. Cell media were collected after being in contact with the cells for 2 days. Results are average ± SD, n = 3.
Figure 6
Figure 6. Viability and differentiation of CD34+ cells cultured on top of fibrin gels and gene expression analysis of encapsulated cells.
A) Apoptosis and necrosis of CD34+ cells cultured for 7 days on top of fibrin gels, in EGM-2 medium or EGM-2 medium conditioned by CD34+-derived ECs. Results are average ± SD, n = 3 (magnification of ×100). B) CD34+ cell differentiation on top of fibrin gels for 7 days in the absence (B.1) or in the presence (B.2) of CD34+-derived ECs. B.3 shows the percentage of cells able to uptake Ac-LDL. Results are average ± SD, n = 18. C) Quantitative RT-PCR analysis of CD34+, CD34+-derived ECs, or a co-culture of both cells, encapsulated in fibrin gels for 10 days. Results are average ± SD, n = 4. In all figures, * denote statistical significance: * P<0.05, **P<0.01, *** P<0.001.
Figure 7
Figure 7. Regenerative effect of encapsulated CD34+ cells and CD34+-derived ECs on diabetic wounds.
A) Wound closure (relatively to initial wound area) in diabetic mice treated by topical application of 1×105 CD34+ cells and 0.35×105 CD34+-derived ECs (A.1), CD34+ cells (A.2) or CD34+-derived ECs (A.3), encapsulated in fibrin gels. Control wounds received a saline solution (PBS) only. Results are average ± SEM, n = 6. * denotes statistical significance (P<0.05). B) Representative images of vWF immunostaining of wounds at day 3. Scale bar represents 50 µm. C) Quantification of wound capillary density (number of capillaries per mm2), based on the vWF immunostaining results, for 3 and 10 day-old wounds in the different experimental groups. Results are average ± SEM, n = 3. ** denotes statistical significance (P<0.001). D) Cytokine expression on mouse wound samples excised 3 days post-wounding, as determined by a Bio-Plex mouse cytokine assay. Wounds had been treated by topical application of CD34+ cells or CD34+ cells plus CD34+-derived ECs encapsulated in fibrin gels. Control wounds received fibrin gel alone. Results are average ± SD, n = 3.

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