Direct isolation of myofibroblasts and fibroblasts from bleomycin-injured lungs reveals their functional similarities and differences

Abstract

BackgroundMyofibroblasts play a crucial role in tissue repair. The functional similarities and differences between myofibroblasts and fibroblasts are not fully understood because they have not been separately isolated from a living body. The purpose of this study was to establish a method for the direct isolation of myofibroblasts and fibroblasts from injured lungs by using fluorescence-activated cell sorting and to compare their functions.ResultsWe demonstrated that lineage-specific cell surface markers (lin), such as CD31, CD45, CD146, EpCAM (CD326), TER119, and Lyve-1 were not expressed in myofibroblasts or fibroblasts. Fibroblasts of bleomycin-injured lungs and saline-treated lungs were shown to be enriched in linneg Sca-1high, and myofibroblasts of bleomycin-injured lungs were shown to be enriched in linneg Sca-1low CD49ehigh. Results from in-vitro proliferation assays indicated in-vitro proliferation of fibroblasts but not myofibroblasts of bleomycin-injured lungs and of fibroblasts of saline-treated lungs. However, fibroblasts and myofibroblasts might have a low proliferative capacity in vivo. Analysis of genes for collagen and collagen synthesis enzymes by qRT-PCR showed that the expression levels of about half of the genes were significantly higher in fibroblasts and myofibroblasts of bleomycin-injured lungs than in fibroblasts of saline-treated lungs. By contrast, the expression levels of 8 of 11 chemokine genes of myofibroblasts were significantly lower than those of fibroblasts.ConclusionsThis is the first study showing a direct isolation method of myofibroblasts and fibroblasts from injured lungs. We demonstrated functional similarities and differences between myofibroblasts and fibroblasts in terms of both their proliferative capacity and the expression levels of genes for collagen, collagen synthesis enzymes, and chemokines. Thus, this direct isolation method has great potential for obtaining useful information from myofibroblasts and fibroblasts.

Glycolytic Reprogramming in Myofibroblast Differentiation and Lung Fibrosis

“…This definitely remains our focus of future interests. It is also worth noting that IPF fibroblasts are known to be heterogeneous, with cells being at different stages of differentiation (35). We found that IPF fibroblasts with lower PFKFB3 expression demonstrate less differentiation than those with greater PFKFB3 expression (see Figure E1B), suggesting that PFKFB3 promotes myo-Fbs differentiation.…”

“…IPF myo-Fbs are known to be heterogeneous, with cells being at different stages of differentiation (35). To determine the correlation of PFKFB3 expression and TGF-β1 6h con Figure 1.…”

A High-Fat Diet Promotes Mammary Gland Myofibroblast Differentiation through MicroRNA 140 Downregulation

“…While we observed a positive trend in ␣SMA expression in WT-HFD and miR-140 KO mouse SVF cells, primary SVF expression of ␣SMA was not a conclusive indicator of myofibroblast differentiation. Studies from our laboratory and others have established that murine myofibroblasts can be identified using fluorescence-activated cell sorting (FACS) for the markers SCA1 low CD49e high (29). Using these markers, we found that 23.73% of WT-RD mouse SVF cells were myofibroblasts, with an increase to 31.7% in the obese mouse SVF cell population and 48.94% in the miR-140 KO mouse SVF cells (Fig.…”

Sca-1+Cardiac Progenitor Cells and Heart-Making: A Critical Synopsis

“…Lin -Sca-1 + cells, in different organs, seem to overlap with the population of tissue-resident fibroblasts [101][102][103]. In the heart, Sca-1 expression has not been ascribed to a population of cardiac fibroblasts, although the evidence is that Sca-1-expressing cells are present in the myocardial interstitium.…”

“…The latter population expands postbirth and coexpress markers typical of mesenchymal affiliation, that is, PDGFRa, and CD90 [102]. Furthermore, after an injury stimulus, lung fibroblasts (Sca-1 high ) can differentiate into myofibroblasts (Sca-1 low ) and increase the ECM production toward a more efficient repair process [103].…”