After culturing 4 days on gelatin, the shape of the ES cell colonies changed from spherical to planar and the population of cells expressing Flk1 was estimated at 10-20% using FACS analysis (Figures 1(a) and 1(b)).
Liuet al., "Molecular basis for FlK1 expression in hemato-cardiovascular progenitors in the mouse," Development, vol.
miR-142-3p overexpression did not significantly affect the expression of ectodermal (Fgf5, Nestin), endodermal (Fox2, Sox17, and Afp), and mesodermal (T, Eomes, and Flk1
) marker genes (Figures 5(a), 5(b), and 5(c)).
For immunocytochemistry, the following antisera were used: rat monoclonals against VE-cadherin (11D4.1, BD Biosciences), PECAM-1 (MEC 13.3, Santa Cruz), and E-Cadherin (DECMA-1, Santa Cruz), mouse monoclonals against cardiac Troponin T (CT3, Iowa Hybridoma Bank), Isl1 (39.4D5, Iowa Hybridoma Bank), Oct3/4 (C-10, Santa Cruz), SMA (Neomarkers), N-cadherin (clone 3B9, Invitrogen), MyHC (MF20, Iowa Hybridoma Bank), and aactinin (Clone BM-75.2, Sigma), goat polyclonals against GATA4 (C-20, Santa Cruz) and Isl1 (GT15051-100, Acris Antibodies), rabbit monoclonals against MEF2c (D80C1) and VEGF receptor 2 (Flk1) (55B11) from Cell Signaling, and rabbit polyclonals against EGFP (kindly provided from Dr.
cTnT, VE-cadherin, Isl1, GATA4, Nkx2.5, and Flk1 expression levels were quantified and compared in CEDPs and in CEDP-derived differentiation cells at day 12 by qPCR.
The rate of positivity for CD45 (hematopoietic cell marker), CD31 (endothelial cell marker), CD117 (C-kit, stem cell factor receptor), CD34 (hematopoietic progenitor cell antigen), CD166 (ALCAM, SB10), VEGFR2 (Flk1
), and NGFR was <2% in cells derived from both types of synovium (Figure 8).
In addition, in order to help understand the differentiation phase, mesodermal markers Flk1 and Mef2c were analyzed by quantitative real-time PCR.
To assess the differentiating capacity and commitment of differentiated cells in our experimental design, beside the expression of endodermal genes, we analyzed the expression of Brachyury and mesodermal genes Flk1 and Mef2c.