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Vivek Singh, Flavia L. Barbosa, Vandana Agrawal, Steven E. Wilson; Optimization Of in vitro Conditions Of TGFβ And PDGF Modulation Of V+A+D+ Myofibroblast Development From Precursor Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2012.
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To test the hypotheses that: (a) development of mature vimentin+/alpha-smoothmuscle actin+/desmin+ (V+A+D+) myofibroblasts from corneal stromalor bone marrow-derived precursor cells are regulated by thecoordinated, sequential action of TGFβ and PDGF; (b) myofibroblastdevelopment in vitro follows a similar developmental pathwayof marker expression as it does in vivo.
Corneal keratocyte was isolated as per Yoshida et.al. (2005)protocol from the fresh eyes of swiss Webster mice (Pel freeze).The corneal fibroblast finally obtained was cultured in serumfree media ( augmented DMEM/F12) or low serum RSF media at differentdoses of TGFβ (0.1 to 2.0 ng/mL) with and w/o mice PDGF(2.0ng/mL) to determine optimal doses and time that promotetransition of the mouse corneal fibroblast to V+A+D+ myofibroblasts.At each time point (2 to 15 days), with each growth factor concentration,we determined the mean % of vimentin+, SMA+ and desmin+ cells(all from single IHC).
See Fig 1 & 2
We have established the optimal concentration and time requiredto convert the corneal fibroblast to myofibroblast (V+A-D- toV+A+D- to V+A+D+).Our results suggest that dose of 0.5ng/mland 1.0ng/mL of TGFβ along with 2.0ng/mL PDGF in DMEM/F12serum free media are the optimal concentrations to study theabove transformation in sequential manner to V+A+D+ myofibroblast( Fig-1 & 2). The present results will help in better understandingof the biology of myofibroblast development and will lead toprevent haze after PTK and other surface ablation procedures,perhaps through the modulation of cytokine effects on the myofibroblaststhat underlie haze.
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