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Enhanced keratinocyte proliferation and migration in co-culture with fibroblasts.

Wang Z, Wang Y, Farhangfar F, Zimmer M, Zhang Y - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic and Aesthetic Surgery, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China.

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 (11(th) to 15(th) 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.

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Effects of different cultures on the growth of keratinocytes (2A) and fibroblasts(2B). 2A: Keratinocytes were cultured with fibroblasts in common 6-well plates (Kera/Fibr), with Fibr in transwell toseparate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or keratinocytes alone (Kera). Compared to Kera or Fibr//Kera, *P<0.01. Four cultures in each condition. 2B: Fibroblasts were cultured with keratinocytes in common 6-well plates (Kera/Fibr), with keratinocytes in transwell to separate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or fibroblasts alone (Fibr). Compared to the groups without star, *P<0.01. Four cultures in each condition.
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pone-0040951-g002: Effects of different cultures on the growth of keratinocytes (2A) and fibroblasts(2B). 2A: Keratinocytes were cultured with fibroblasts in common 6-well plates (Kera/Fibr), with Fibr in transwell toseparate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or keratinocytes alone (Kera). Compared to Kera or Fibr//Kera, *P<0.01. Four cultures in each condition. 2B: Fibroblasts were cultured with keratinocytes in common 6-well plates (Kera/Fibr), with keratinocytes in transwell to separate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or fibroblasts alone (Fibr). Compared to the groups without star, *P<0.01. Four cultures in each condition.

Mentions: Keratinocytes were cultured in the following conditions: with fibroblasts in common 6-well plates (Kera/Fibr), with fibroblasts in transwell plates (Kera//Fibr), with varying concentrations of EGF (Ker + EGF), or cultured alone (Kera). On day 5 after the experiment was initiated, the cells were trypsinized and counted by Trypan blue exclusion and flow cytometry. The keratinocyte cell count in the Kera/Fibr was significantly higher (P<0.05) than Kera//Fibr and Kera, suggesting that the increase in the number of keratinocytes in the early stage of the culture is secondary to the contact between keratinocytes and fibroblasts, although such diverges cannot be seen at later time points. To mimic the effects of fibroblasts on the keratinocytes, different concentrations of EGF were added to the keratinocyte cultures. Similar to the fibroblasts, EGF promoted keratinocyte growth and exhibited a dose-dependent effect (fig. 2a).


Enhanced keratinocyte proliferation and migration in co-culture with fibroblasts.

Wang Z, Wang Y, Farhangfar F, Zimmer M, Zhang Y - PLoS ONE (2012)

Effects of different cultures on the growth of keratinocytes (2A) and fibroblasts(2B). 2A: Keratinocytes were cultured with fibroblasts in common 6-well plates (Kera/Fibr), with Fibr in transwell toseparate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or keratinocytes alone (Kera). Compared to Kera or Fibr//Kera, *P<0.01. Four cultures in each condition. 2B: Fibroblasts were cultured with keratinocytes in common 6-well plates (Kera/Fibr), with keratinocytes in transwell to separate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or fibroblasts alone (Fibr). Compared to the groups without star, *P<0.01. Four cultures in each condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401236&req=5

pone-0040951-g002: Effects of different cultures on the growth of keratinocytes (2A) and fibroblasts(2B). 2A: Keratinocytes were cultured with fibroblasts in common 6-well plates (Kera/Fibr), with Fibr in transwell toseparate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or keratinocytes alone (Kera). Compared to Kera or Fibr//Kera, *P<0.01. Four cultures in each condition. 2B: Fibroblasts were cultured with keratinocytes in common 6-well plates (Kera/Fibr), with keratinocytes in transwell to separate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or fibroblasts alone (Fibr). Compared to the groups without star, *P<0.01. Four cultures in each condition.
Mentions: Keratinocytes were cultured in the following conditions: with fibroblasts in common 6-well plates (Kera/Fibr), with fibroblasts in transwell plates (Kera//Fibr), with varying concentrations of EGF (Ker + EGF), or cultured alone (Kera). On day 5 after the experiment was initiated, the cells were trypsinized and counted by Trypan blue exclusion and flow cytometry. The keratinocyte cell count in the Kera/Fibr was significantly higher (P<0.05) than Kera//Fibr and Kera, suggesting that the increase in the number of keratinocytes in the early stage of the culture is secondary to the contact between keratinocytes and fibroblasts, although such diverges cannot be seen at later time points. To mimic the effects of fibroblasts on the keratinocytes, different concentrations of EGF were added to the keratinocyte cultures. Similar to the fibroblasts, EGF promoted keratinocyte growth and exhibited a dose-dependent effect (fig. 2a).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic and Aesthetic Surgery, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China.

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 (11(th) to 15(th) 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.

Show MeSH