Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

Abstract

Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.

On-chip assay of the effect of topographical microenvironment on cell growth and cell-cell interactions during wound healing

“…For clear and comparative data analysis, we divided these tests into three groups: (i) HaCaT and ESF-1 mono-culture and their co-culture (Figures 6(a) and 6(c)), (ii) ESF-1 and HUVEC monoculture and their co-culture (Figures 6(b) and 6(d)), and (iii) HaCaT and HUVEC mono-culture and their co-culture (Figures 6(e) and 6(f)). For the first group, the migration distance of HaCaT cells during HaCaT and ESF-1 co-culture was much longer than that of HaCaT in mono-culture at each time point, confirming that the proliferation and migration of keratinocytes were enhanced when co-cultured with fibroblasts, 7 while the migration distance of ESF-1 cells during HaCaT and ESF-1 co-culture was close to that in the ESF-1 mono-culture except at 36 h (Figure 6(c)). With regard to the second group, ESF-1 cells during ESF-1 and HUVEC co-culture migrated slower than or equal to the mono-cultured ESF-1 cells (Figure 6(d)), while HUVEC cells in the ESF-1 and HUVEC-C co-culture migrated much faster than the monocultured HUVEC cells.…”

“…4 However, the complicated mechanism of interactions among these cells and each role in cutaneous wound healing have not yet been investigated thoroughly. Recently, on the basis of conventional in vivo (e.g., minipig 5 ) and in vitro (e.g., scratch assay 6 and transwell assay 7 ) models, a great deal of studies has greatly enhanced the understanding of cell interactions during wound healing process. For example, dermal fibroblasts would be activated and differentiated into myofibroblasts when co-cultured with dermal microvascular endothelial cells and then migrated to heal the wound.…”

“…6 Contacting with fibroblasts stimulated the migration and proliferation of keratinocytes during wound healing. 7 In return, the expression and synthesis of type I collagen (the predominant form of collagen in fibroblasts in human skin) was regulated by keratinocyte-releasable factors. 8 However, the conventional in vivo model is laborious and costly.…”

Models for the study of skin wound healing. The role of Nrf2 and NF-κB

“…The simplest co-cultures are constructed from primary dermal fibroblasts and primary epidermal keratinocytes 36 or HaCaT (ref.…”

Skin extracellular matrix components accelerate the regenerative potential of Lin− cells

“…In vitro models of cell proliferation and migration can be useful to obtain valuable information for the preparation of cells for transplantation in vivo [9]. The literature contains numerous studies of the regenerative capacities of different cells in vitro, including epithelial cells, fibroblasts, endothelial cells, and MSCs [10][11][12]. However, there are limited data on the behavior of Lin¯ cell population, although these cells play an important role in skin regeneration.…”