Transforming growth factor-beta-induced transition of fibroblasts: a model for myofibroblast procurement in tissue valve engineering.
OBJECTIVE
The selection of a suitable cell type for scaffold seeding, its isolation and adequate expansion in vitro remains a major challenge in tissue valve engineering. The study aim was to establish a model for efficient procurement of myofibroblasts for in-vitro seeding using fibroblasts as progenitor cells.


METHODS
Dermal and arterial mesenchymal cells from human (hDMC1.1 and hAMC1.1) and sheep (sDMC1.1 and sAMC1.1) were isolated by sequential culture. Cell isolates were characterized by stringent criteria based on morphology, immunocytochemistry using antibodies to vimentin, cytokeratin, prolyl 4-hydroxylase, smooth muscle alpha-actin (alpha-SMA) and smooth muscle myosin, and by Western blotting for alpha-SMA and N-cadherin. Cultures with less than 10-20% alpha-SMA-positive cells were considered to be fibroblastic. Cells were subsequently transdifferentiated with the cytokine transforming growth factor-beta1 (TGF-beta1) during five days, and then evaluated morphotypically, by immunocytochemistry, and by Western blotting. The metabolic and functional properties of TGF-beta1-treated and untreated colonies were compared by measuring the expression of extracellular proteins (collagen type 1 and tenascin-C) and by a collagen matrix contraction assay.


RESULTS
TGF-beta1 successfully transformed both human and sheep fibroblasts to metabolically active and functional myofibroblasts based on stringent criteria for myofibroblast characterization. Alpha-SMA positivity of 100% was obtained in all cases (hDMC1.1, hAMC1.1, sDMC1.1, and sAMC1.1) after transformation compared to less than 50% in the non-transformed state (hAMC1.1, 17%; hDMC1.1, 10%; sAMC1.1, 43%; and sDMC1.1, 30%). This observation was further supported by increased contractility and an up-regulation of extracellular protein production in transdifferentiated cells.


CONCLUSIONS
Untreated arterial cell isolates were, at best, less than 50% alpha-SMA-positive. By allowing procurement of high densities of myofibroblasts in a relatively short time, the model was seen to be a potentially useful tool in tissue valve engineering, at least in investigations using autologous cells in the sheep model.
Narine
Kishan
K
Department of Cardiac Surgery, University Hospital Ghent, Ghent, Belgium. kishan.narine@ugent.be
DeWever
Olivier
O
Cathenis
Koen
K
Mareel
Marc
M
Van Belleghem
Yves
Y
Van Nooten
Guido
G
D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001021:Aortic Valve / Q000166:cytology / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000187:drug effects / Q000378:metabolism; D008955:Models, Cardiovascular; D011393:Procollagen-Proline Dioxygenase / Q000187:drug effects / Q000378:metabolism; D012756:Sheep; D024745:Smooth Muscle Myosins / Q000187:drug effects / Q000378:metabolism; D019063:Tenascin / Q000096:biosynthesis / Q000187:drug effects; D023822:Tissue Engineering; D009927:Tissue and Organ Procurement; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D014746:Vimentin / Q000187:drug effects / Q000378:metabolism
D000199:Actins; D024042:Collagen Type I; C508960:TGFB1 protein, human; D019063:Tenascin; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014746:Vimentin; D007633:Keratins; D011393:Procollagen-Proline Dioxygenase; D024745:Smooth Muscle Myosins