Fig 2: QKI is required for efficient cardiomyocyte cell differentiation. (A) Western blot of protein extracted from NKX2.5?GFP UD hESCs and day 8 cardiomyocyte (d8 c.myo) cells either wild-type (WT) or lacking QKI (polyclonal KO ‘pKO’) and probed with antibodies for tubulin (magenta) and panQKI (green). (B) Population-level EGFP abundance (relative to GAPDH) calculated per well, measured by high content imaging in WT, polyclonal QKI-KO (polyKO), and clone 4–2 QKI-KO (4–2KO) in UD, day 4 CM (d4 CM), and day 8 cardiomyocytes (d8 c.myo) NKX2.5?EGFP cells (n = 4 independent replicates; ns = not significant, *P < 0.05, and **P < 0.01 relative to WT, determined by Student's t-test). (C) Flow cytometry analysis of NKX2.5?EGFP UD (gray), WT, or polyKO day 8 c.myo (light or dark red, respectively); mean percent GFP positive is shown above each histogram (± standard deviation; biological replicate n = 3) and ****P < 0.0001 measured by Student's t-test; y-axis denotes number of events measured. (D) RT-qPCR measuring abundance of RNA extracted from UD hESCs, d4 CM cells, and d8 c.myo cells for endogenous NKX2.5 and TNNT2 (cTNT) mRNAs relative to HMBS mRNA in WT, polyKO or 4–2KO cells (biological replicate n = 2 or 3 each; ns = not significant, *P < 0.05, **P < 0.01, measured by Student's t-test). (E) Western blot of protein extracted from NKX2.5?EGFP UD hESCs and d8 c.myo cells either WT or pKO for QKI and probed with antibodies for tubulin (green, top) cTNT (magenta, middle (TNNT2)) and endogenous NKX2.5 (green, bottom). (F) Indirect immunofluorescence with high content imaging analysis of NKX2.5?EGFP WT (left) or QKI polyclonal KO (right) d8 cardiomyocyte cells showing QKI5 (magenta), cTNT (yellow), EGFP (green), brightfield (gray), and DAPI (blue) with each fluorescent channel overlaid (scale bar indicates 100 µm). On the right, the fluorescence intensity values per cell (relative to DAPI) are shown for QKI5 and cTNT with P values calculated by Mann–Whitney U test. (G) Real-time mitochondrial bioenergetic measurement of intact day 8 or 15 cardiac differentiated WT or QKI-KO 4–2 cells measuring basal respiration (BR), ATP production (ATP prod), proton leak (PL), maximal respiration (Max resp), spare respiratory capacity (SRC), and non-mitochondrial O2 consumption (NMOC); the y-axes denote oxygen consumption rate (OCR) normalized to total protein content in pMoles/min/µg protein (n = 9 or 10 independent replicates each; ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student's t-test). (H) RT-PCR and BioAnalyzer gel-like image showing quantification of RAI14, NF1, and BIN1 alternative exons using RNA extracted from WT or polyKO NKX2.5?EGFP cells either UD, or subjected to the following differentiation protocols: day 2 lateral mesoderm (d2 lat meso), day 4 CM, day 6 cardiomyocyte (d6 c.myo), and day 8 cardiomyocyte (d8 c.myo) (biological replicate n = 3; mean percent included ± is shown below by heatmap; see Supplementary Figure S4G for mean numerical values and standard deviation from the mean). See also Supplementary Figure S4.

Fig 3: 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; Tβ−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, α-MHC, α-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 4: Nkx2.5-expressing progenitors contribute significantly to endocardial ECs. (A,B) Flk1-GFP;Nkx2.5Cre;RosatdTom mouse embryos were isolated at E7.75 (A) and E8.5 (B), enabling specific identification of cells derived from Nkx2.5-expressing progenitors (red) and ECs (green). In B, arrows indicate myocardium and arrowheads indicate endocardium. (C,D) Cryosections of E11.5 heart with colocalization masking enabling quantification of the NkxEC:nEC ratio. (E) Organs isolated from Flk1-GFP;Nkx2.5Cre;RosatdTom embryos at E12.5 were dissociated and the distribution of NkxECs (green) and CD45+ cells (blue) was quantified within the tdTomato+ population (red). LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; Nuc, nuclei staining. Scale bars: 100 µm.

Fig 5: Multiphasic function of intersection of canonical and non-canonical Wnt signaling during cardiovascular lineage diversification. Wnt plays multiple roles during differentiation of pluripotent cells within the cardiovascular lineage. Wnt signaling initially directs pluripotent cells toward cardiac mesoderm, but is later inhibited during specification of cardiac progenitor cells expressing Nkx2.5. Subsequently, inhibition of Wnt signaling within the Nkx2.5+ pool via non-canonical Wnt5a promotes vascular specification.