Postnatal Dermal Fibroblasts Research Articles

692 Stable cell line of postnatal dermal fibroblasts as a new experimental tool for dermatological research

692 Stable cell line of postnatal dermal fibroblasts as a new experimental tool for dermatological research

Direct Reprogramming of Human Dermal Fibroblasts Into Endothelial Cells Using ER71/ETV2.

Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored. We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR+ (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks' transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia. This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process.

Wnt signal increases during postnatal skin repair and Wnt3a treatment increases pro-regenerative TGF-b3 in fetal but not in postnatal dermal fibroblasts

INTRODUCTION: Postnatal acute skin injury results in scar formation, while fetal injury does not. Multiple cellular differences occur between dermal fibroblasts derived from fetal and postnatal skin. In the present study, we localize canonical Wingless (Wnt) expression during postnatal repair and test the effects of Wnt treatment on fetal and postnatal fibroblasts. The effect of (r)Wnt-3a was compared to treatment with these wound-related cytokines:TGF-b1 and -b3,IGF-1,and PDGF-AA. METHODS: Genetically modified (BAT-gal) mice that localize Wnt signal underwent excisional wounding. Spatial and temporal expression profiles of canonical Wnt,IGF-1,PDGF,TGF-B 1&3 were demonstrated. Fetal and postnatal dermal fibroblasts were isolated and exposed to recombinant (r)Wnt-3a, IGF-1, TGF-b1,b3 & PDGFAA; Target gene (Axin2, HAS1, HAS2, Tg-fb1, Tgf-b3, FGF2, CTGF) modulation was examined by qRT-PCR and protein expression by western blot.

Contractile capacity of fibroblasts from different sources in the model of living skin equivalent

Human fibroblasts contract collagen gel in vitro and produce a connective tissue-like structure termed the living skin equivalent. In this study, the contractile capacity of postnatal dermal fibroblasts, bone marrow mesenchymal cells and mesenchymal cells derived from the fat tissue has been compared to that of fetal dermal fibroblasts in the model of living skin equivalent. The results show that fetal fibroblasts contract the collagen gel approximately six times stronger than do all other fibroblast cell types, with the numbers of all these cells being equal. A deeper insight into the behavior of fibroblasts differing in their origin will help to develop new approaches to the treatment and regulation of wound healing and fibrosis formation.

Differential gene expression in response to transforming growth factor‐β1 by fetal and postnatal dermal fibroblasts

The multipotent growth factor transforming growth factor (TGF)-beta1 is consistently linked with fibrosis and scarring. The perfect (scarless) healing of cutaneous wounds in early gestational age fetuses is proposed to be due to this tissue's predominance of the TGF-beta3 isoform over the profibrotic TGF-beta1 and 2. Nevertheless, TGF-beta1 is present during wound healing in the early fetus and recently we demonstrated that relevant intracellular signaling pathways are activated (albeit transiently) on TGF-beta1 stimulation. This study aimed to determine whether TGF-beta1 has different effects on gene transcription in human fetal (<14 weeks) vs. human postnatal dermal fibroblasts, using real-time polymerase chain reaction. The regulation pattern of a number of TGF-beta response genes differed dramatically between the two cell sources. The typical autocrine loop of TGF-beta1 autoinduction did not occur in fetal fibroblasts and genes that are normally up-regulated, connective tissue growth factor and collagen type I were actually down-regulated. Furthermore, other response genes responded in a delayed fashion (TGF-beta3) compared with that seen in the more developmentally mature postnatal fibroblasts. Finally, genes unaltered by TGF-beta stimulation in postnatal cells, TGF-beta2 and collagen III, were up-regulated in fetal cells. These developmentally related differences in fibroblast response to TGF-beta1 may influence wound-healing outcome, i.e., perfect regeneration or fibrosis.

Dermal fibroblasts derived from fetal and postnatal humans exhibit distinct responses to insulin like growth factors

BackgroundIt has been well established that human fetuses will heal cutaneous wounds with perfect regeneration. Insulin-like growth factors are pro-fibrotic fibroblast mitogens that have important roles in both adult wound healing and during development, although their relative contribution towards fetal wound healing is currently unknown. We have compared responses to IGF-I and -II in human dermal fibroblast strains derived from early gestational age fetal (<14 weeks) and developmentally mature postnatal skin to identify any differences that might relate to their respective wound healing responses of regeneration or fibrosis.ResultsWe have established that the mitogenic response of fetal cells to both IGF-I and -II is much lower than that seen in postnatal dermal fibroblasts. Further, unlike postnatal cells, fetal cells fail to synthesise collagen in response to IGF-I, whereas they do increase synthesis in response to IGF-II. This apparent developmentally regulated difference in response to these related growth factors is also reflected in changes in the tyrosine phosphorylation pattern of a number of proteins. Postnatal cells exhibit a significant increase in phosphorylation of ERK 1 (p44) in response to IGF-I and conversely the p46 isoform of Shc on IGF-II stimulation. Fetal cells however only show a significant increase in an unidentified 100 kDa tyrosine-phosphorylated protein on stimulation with IGF-II.ConclusionDermal fibroblasts exhibit different responses to the two forms of IGF depending on their developmental maturity. This may relate to the developmental transition in cutaneous wound healing from regeneration to fibrosis.

Fetal Cutaneous Myofibroblasts — Fact or Fallacy?

It has been known for several decades that early gestational human fetuses will regenerate cutaneous wounds resulting in a scarless repair. In postnatal humans, myofibroblasts are known to play a central role in wound healing and in the pathology of fibrosis. The role of the profibrotic growth factor TGF‐β1 in human fetal fibroblasts remains unclear, with recent data now showing that TGF‐β1 is present in fetal wounds but for shorter periods. We have therefore examined the effects of TGF‐β1 on fibroblasts isolated from early human fetuses (&lt;14week EGA). We have identified that stimulation with 5 ng/ml TGF‐β1 induces a significant proportion of human fetal fibroblasts to differentiate into myofibroblasts (approximately 40%), as identified by staining of α‐smooth muscle actin (n = 5). Interestingly, this response was earlier (peaking at day 2–3) and more transitory (over by day 4–5) than that seen with postnatal dermal fibroblasts (peaking at day 6–8 and dropping at day 10). We examined the effects of blocking various intracellular pathways involved in TGF‐β1 signalling. The early and transitory myofibroblast induction seen in fetal cells is blocked by an inhibitor of p‐JNK (n = 4; p =&lt; 0.05). In contrast, the later and more prolonged induction of myofibroblasts seen in postnatal cells was not affected by inhibition of p‐JNK. Despite the fact that fetal fibroblasts do indeed differentiate into myofibroblasts they were further shown to exhibit differential behavior to postnatal myofibroblasts with respect to TGF‐β1 stimulation of collagen expression, producing significantly less soluble collagen (approximately 50%, n = 4) after 24 or 48 hours of treatment with TGF‐β1 in serum‐free media. These results appear to indicate a role for TGF‐β1 during cutaneous wound healing in the early fetus and show that the response of fetal cells to this important profibrotic cytokine is rapid and more controlled, being short lived. It is possible that a prolonged response to this cytokine may lead to the overblown nature of postnatal wound healing, resulting in fibrosis.