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Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation.

Park IS, Chung PS, Ahn JC - PLoS ONE (2015)

Bottom Line: The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF).Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+.On the other hand, monolayer-cultured hASCs were negative for these markers.

View Article: PubMed Central - PubMed

Affiliation: Beckman Laser Institute Korea, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 330-714, Korea.

ABSTRACT
We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.

No MeSH data available.


Related in: MedlinePlus

Enhanced expression of hypoxia-induced survival factors and angiogenic growth factors in hASC L-spheroids.(A) The light source used was LED (660 nm) designed to fit over a microplate (12.5 × 8.5 cm) for cell culture. (B) Formation of hASC L-spheroids. hASCs morphology on non–tissue culture–treated 24-well plates at day 3. Scale bar = 500 μm. (C) Western blot analysis and quantification of HIF1-α in hASCs cultured as spheroids, L-spheroids and monolayers (*p < 0.01, compared to the L-spheroid group). (D) Angiogenesis-related protein analysis of L-spheroids (*, p < 0.05, compared to the spheroid group, t-test, n = 3 in each group). (E) ELISA measurement of spheroids cultured for 3 days. Concentrations of VEGF are presented as pg-corrected for 104 cells. (*, p < 0.05, compared with spheroid 6J/cm2 group, t-test, n = 3 in each group).
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pone.0122776.g001: Enhanced expression of hypoxia-induced survival factors and angiogenic growth factors in hASC L-spheroids.(A) The light source used was LED (660 nm) designed to fit over a microplate (12.5 × 8.5 cm) for cell culture. (B) Formation of hASC L-spheroids. hASCs morphology on non–tissue culture–treated 24-well plates at day 3. Scale bar = 500 μm. (C) Western blot analysis and quantification of HIF1-α in hASCs cultured as spheroids, L-spheroids and monolayers (*p < 0.01, compared to the L-spheroid group). (D) Angiogenesis-related protein analysis of L-spheroids (*, p < 0.05, compared to the spheroid group, t-test, n = 3 in each group). (E) ELISA measurement of spheroids cultured for 3 days. Concentrations of VEGF are presented as pg-corrected for 104 cells. (*, p < 0.05, compared with spheroid 6J/cm2 group, t-test, n = 3 in each group).

Mentions: hASCs were cultured on non-tissue culture-treated 24-well plates in the presence of FBS and formed a floating spheroid after irradiation with low-level light (Fig 1A) 3 days after seeding. The diameter of most L-spheroids ranged from 1.2 to 1.5 mm (Fig 1B). L-spheroid cultures showed a dramatic increase in the expression of hypoxia-induced survival factors, such as hypoxia-inducible factor (HIF)-1α, relative to cells in a monolayer culture (Fig 1C). Therefore, L-spheroid hASCs seemed to be more adaptable and more resistant to hypoxia compared to hASCs in monolayer cultures. HIF-1α is known to upregulate the expression of angiogenic growth factors [19, 20], and L-spheroid hASCs showed considerable expression of angiogenic growth factors, i.e., hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2). The expression of angiogenic growth factors in L-spheroid hASCs was much greater than that of hASCs cultured in a monolayer culture (Fig 1D and 1E).


Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation.

Park IS, Chung PS, Ahn JC - PLoS ONE (2015)

Enhanced expression of hypoxia-induced survival factors and angiogenic growth factors in hASC L-spheroids.(A) The light source used was LED (660 nm) designed to fit over a microplate (12.5 × 8.5 cm) for cell culture. (B) Formation of hASC L-spheroids. hASCs morphology on non–tissue culture–treated 24-well plates at day 3. Scale bar = 500 μm. (C) Western blot analysis and quantification of HIF1-α in hASCs cultured as spheroids, L-spheroids and monolayers (*p < 0.01, compared to the L-spheroid group). (D) Angiogenesis-related protein analysis of L-spheroids (*, p < 0.05, compared to the spheroid group, t-test, n = 3 in each group). (E) ELISA measurement of spheroids cultured for 3 days. Concentrations of VEGF are presented as pg-corrected for 104 cells. (*, p < 0.05, compared with spheroid 6J/cm2 group, t-test, n = 3 in each group).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465903&req=5

pone.0122776.g001: Enhanced expression of hypoxia-induced survival factors and angiogenic growth factors in hASC L-spheroids.(A) The light source used was LED (660 nm) designed to fit over a microplate (12.5 × 8.5 cm) for cell culture. (B) Formation of hASC L-spheroids. hASCs morphology on non–tissue culture–treated 24-well plates at day 3. Scale bar = 500 μm. (C) Western blot analysis and quantification of HIF1-α in hASCs cultured as spheroids, L-spheroids and monolayers (*p < 0.01, compared to the L-spheroid group). (D) Angiogenesis-related protein analysis of L-spheroids (*, p < 0.05, compared to the spheroid group, t-test, n = 3 in each group). (E) ELISA measurement of spheroids cultured for 3 days. Concentrations of VEGF are presented as pg-corrected for 104 cells. (*, p < 0.05, compared with spheroid 6J/cm2 group, t-test, n = 3 in each group).
Mentions: hASCs were cultured on non-tissue culture-treated 24-well plates in the presence of FBS and formed a floating spheroid after irradiation with low-level light (Fig 1A) 3 days after seeding. The diameter of most L-spheroids ranged from 1.2 to 1.5 mm (Fig 1B). L-spheroid cultures showed a dramatic increase in the expression of hypoxia-induced survival factors, such as hypoxia-inducible factor (HIF)-1α, relative to cells in a monolayer culture (Fig 1C). Therefore, L-spheroid hASCs seemed to be more adaptable and more resistant to hypoxia compared to hASCs in monolayer cultures. HIF-1α is known to upregulate the expression of angiogenic growth factors [19, 20], and L-spheroid hASCs showed considerable expression of angiogenic growth factors, i.e., hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2). The expression of angiogenic growth factors in L-spheroid hASCs was much greater than that of hASCs cultured in a monolayer culture (Fig 1D and 1E).

Bottom Line: The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF).Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+.On the other hand, monolayer-cultured hASCs were negative for these markers.

View Article: PubMed Central - PubMed

Affiliation: Beckman Laser Institute Korea, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 330-714, Korea.

ABSTRACT
We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.

No MeSH data available.


Related in: MedlinePlus