Fig 2: Fibroblasts can be directly reprogrammed to spontaneously contracting patches with chemical cocktails. (A) The scheme of direct cardiac reprogramming with small molecule cocktails. Fibroblasts were plated in fibroblast growth medium for one day and then the medium was changed into cardiac reprogramming medium (CRM) containing the small molecule cocktails (first stage). At day 16, the medium was changed into cardiomyocyte maintaining medium (CMM, second stage). The first beating clusters could be observed on day 6–8. (B) Representative morphologies of various MEF-derived beating clusters induced by the small molecule cocktail CRFVPT. See also Supplementary information, Movie S2. (C) Number of the beating clusters induced from MEFs with CRFVPT at various time points. Results are presented as means ± SEM, n = 3. (D) Screening for compounds essential for cardiomyocyte induction. Numbers of beating clusters at day 20 are shown. (E) Morphology of TTF-derived beating cells by small molecule cocktail CRFVPT at day 14. See also Supplementary information, Movie S3. (F) Induction of TTF-derived beating cells with CMM at the second stage supplemented with various growth factors (NRG1, 100 ng/ml; G-CSF, 20 ng/ml; Tß-4, 100 ng/ml; GDF11, 100 ng/ml). (G) Induction of TTF-derived beating cells with CRFVPT plus Rolipram (3 µM) in the first stage, and the growth factors (100 ng/ml NRG-1 and 20 ng/ml G-CSF) in the second stage. Data are means ± SEM, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. (H) Immunostaining of cardiac markers Mef2c, Gata4, Nkx2.5, a-MHC, a-actinin, cTnT, cTnI, N-cad, and Cx43 in beating clusters generated from MEFs on day 24. Nuclei were stained with Hoechst. Scale bars represent 50 µm in B and E, 20 µm in H.

Fig 3: The Dll4in3 enhancer requires MEF2 binding to direct expression during adult neovascularization. (A) Representative images of Dll4in3:LacZ transgene expression during neovascularization into Matrigel plugs and B16F10 melanoma tumors demonstrate robust vascular X-gal staining. (B) Representative images of the mutant Dll4in3mutMEF:LacZ transgene demonstrate that the mutated enhancer was unable to direct any detectable reporter gene expression during adult neovascularization. See also Supplemental Figure 3.

Fig 4: MEF2C deficient mice show attenuated pathological bone resorption in the K/BxN serum transfer arthritis model. a Schematic of experiments. b Time course of joint swelling and clinical score of K/BxN serum transfer arthritis in WT and MEF2C?MX KO mice. WT; n = 5, KO; n = 6. c TRAP staining of histological sections of hind paw and histomorphometric analysis of tarsal bones. Arrow, osteoclasts. Scale bars, 400 µm. Data are shown as mean ± SD. Statistics used: c Welch’s t-test *P < 0.05

Fig 5: The function of histamine in the cardiac differentiation is mediated by the histamine 1 receptor. a The expression of H1R and H2R during cardiac differentiation was confirmed by immunoblot. Representative images and the densitometric analysis are shown. b Immunofluorescence staining for H1R, H2R, and NKX2.5 in hiPSCs and hiPSC-CPCs. Scale bar 100 µM. c Percentages of a-actinin+ cardiomyocytes on day 10 in the total population derived from iPSCs with or without pyrilamine and cimetidine treatment analyzed by FACS. d The mRNA levels of cardiogenic markers in hiPSC-CMs on day 10 with histamine and pyrilamine treatment. e The protein levels of a-actinin and cTnT in hiPSC-CMs on day 10 of differentiation. f Representative immunofluorescence images of ?-actinin+ hiPSC-CMs. Scale bar 100 µM. g Representative immunostaining images of MEF2C (red), NKX2.5 (green), and nuclei (blue), in hiPSC-CPCs at day 5 of differentiation. Scale bar 200 µM. h The protein levels of GATA4, MEF2C, and NKX2.5 in hiPSC-CPCs on day 5. i Percentages of NKX2.5+ cells on day 5 in the total population derived from both hiPSC lines with or without histamine and pyrilamine treatment. Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 vs control, #p < 0.05 vs His (n = 3 for each group)