Paracrine effects of adipose-derived stem cells on keratinocytes and dermal fibroblasts

Abstract

Background: Adipose-derived stem cells (ASCs) are mesenchymal stem cells that have recently been applied to tissue repair and regeneration. Keratinocytes and dermal fibroblasts play key roles in cutaneous wound healing.

Objective: We investigated the paracrine effects of ASCs on HaCaT cells (i.e., immortalized human keratinocytes) and human dermal fibroblasts to explore the mechanism of the effects of ASCs on cutaneous wound healing.

Methods: HaCaT cells and primary cultured human dermal fibroblasts were treated with 50% conditioned medium of ASCs (ASC-CM). Viability, in vitro wound healing, and fibroblast-populated collagen lattice contraction assays were conducted, and reverse transcription-polymerase chain reaction (RT-PCR) for the type I procollagen α1 chain gene was performed.

Results: The proliferation of HaCaT cells and fibroblasts was increased by ASC-CM in the viability assay. ASC-CM promoted in vitro wound healing of HaCaT cells and increased the contraction of the fibroblast-populated collagen lattice. RT-PCR showed that the transcription of the type I procollagen α1 chain gene in fibroblasts was upregulated by ASC-CM.

Conclusion: The stimulatory effect of ASC on cutaneous wound healing may be partially mediated by paracrine effects of ASCs on other skin cells. Application of ASCs or ASC-derived molecules could be an innovative therapeutic approach in the treatment of chronic wounds and other conditions.

Keywords: Adipose tissue; Fibroblasts; Keratinocytes; Mesenchymal stem cells; Wound healing.

Fig. 1

Adipose-derived stem cells at passage 4 expressed CD29 and CD44, whereas hematopoietic cell marker CD31 and CD34 were not expressed (immunoperoxidase, ×100).

Fig. 2

Adipose-derived stem cell (ASCs) could differentiate toward the adipogenic and osteogenic lineages. ASCs at passage 4 were treated with adipogenic medium (A, B) or osteogenic medium (C~E) for 3 weeks. Oil red O staining (B), alkaline phosphatase staining (D), and Alizarin red S staining (E) were performed to confirm adipo- and osteogenesis.

Fig. 3

Proliferation of HaCaT cells and dermal fibroblasts was increased by the conditioned medium of adipose-derived stem cell (ASC-CM) in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. HaCaT cells and dermal fibroblasts were treated with 50% ASC-CM or Dulbecco's modified Eagle's medium (control) for 4 days. The values shown are the mean±standard error of the mean of six independent experiments performed in triplicate wells. ^*p<0.05 vs. the control group.

Fig. 4

The conditioned medium of adipose-derived stem cell (ASC-CM) promote in vitro wound healing of HaCaT cells. (A) Monolayers of confluent HaCaT cells were wounded with a micropipette tip, and then treated with 50% ASC-CM or Dulbecco's modified Eagle's medium (control). Photographs were taken at 24 hours and 48 hours. (B) The width of healed area was calculated with an image analysis program. The values shown are the mean±standard error of the mean of six independent experiments. ^*p<0.05 vs. the control group.

Fig. 5

The conditioned medium of adipose-derived stem cell (ASC-CM) increased the contraction of fibroblast-populated collagen lattice. (A) Collagen lattice was treated with 50% ASC-CM or Dulbecco's modified Eagle's medium (control) for 5 days. (B) The size of collagen lattice was measured with an image analysis program. The values shown are the mean±standard error of the mean of six independent experiments. ^*p<0.05 vs. the control group.

Fig. 6

Transcription of type I procollagen α1 chain gene is up-regulated in fibroblasts by the conditioned medium of adipose-derived stem cell (ASC-CM). (A) Fibroblasts were treated with 50% ASC-CM or Dulbecco's modified Eagle's medium (control) for 6 hours and then reverse transcription-polymerase chain reaction was performed. (B) The values of densitometric quantification are the mean±standard error of the mean of six independent experiments. ^*p<0.05 vs. the control group.