SMAD4 Is Essential for Human Cardiac Mesodermal Precursor Cell Formation

Abstract

Understanding stage-specific molecular mechanisms of human cardiomyocyte (CM) progenitor formation and subsequent differentiation are critical to identify pathways that might lead to congenital cardiovascular defects and malformations. In particular, gene mutations in the transforming growth factor (TGF)β superfamily signaling pathways can cause human congenital heart defects, and murine loss of function studies of a central component in this pathway, Smad4, leads to early embryonic lethality. To define the role of SMAD4 at the earliest stages of human cardiogenesis, we generated SMAD4 mutant human embryonic stem cells (hESCs). Herein, we show that the loss of SMAD4 has no effect on hESC self-renewal, or neuroectoderm formation, but is essential for the formation of cardiac mesoderm, with a subsequent complete loss of CM formation during human ES cell cardiogenesis. Via transcriptional profiling, we show that SMAD4 mutant cell lines fail to generate cardiac mesodermal precursors, clarifying a role of NODAL/SMAD4 signaling in cardiac mesodermal precursor formation via enhancing the expression of primitive streak genes. Since SMAD4 relative pathways have been linked to congenital malformations, it will become of interest to determine whether these may due, in part, to defective cell fate decision during cardiac mesodermal precursor formation. Stem Cells 2018 Stem Cells 2019;37:216-225.

Keywords: Heart; Human embryonic stem cells; Mesoderm; SMAD4.

Figure 1

SMAD4 is not required for the maintenance of human embryonic stem cells (hESCs). (A): Mutated sequences in the SMAD4 loci. Clone 1, two patterns: 12 deletion/1 insertion‐1 deletion; clone 2, two patterns: 4 deletion/18 deletion. (B): Western blot demonstrates absence of detectable SMAD4 protein in mutant clones SMAD4.C1 and SMAD4.C2. (C): Immunostaining demonstrates absence of detectable SMAD4 protein in mutant clones SMAD4.C1 and SMAD4.C2 (scale bar = 100 μm). (D): Bright field images demonstrate normal hESC morphology of SMAD4 mutant clones compared with wild‐type hESC (scale bar: left = 400 μm; right = 200 μm). (E): Immunostaining images demonstrate both SMAD4 mutant and the wild‐type hESCs express the pluripotency markers POU5F1 in green and NANOG in red (scale bar = 100 μm).

Figure 2

SMAD4 is required for human cardiomyocyte differentiation. (A): Flow cytometry analysis demonstrates SMAD4 mutant human embryonic stem cells (hESCs) fail to form TNNT2+ cardiomyocytes. Compare left panel hESC with middle and right panel mutant SMAD4 clones C1 and C2, respectively. (B): SMAD4 rescue construct used to overexpress/rescue SMAD4 in wild‐type and the mutant clones contains a UbC promoter driving V5‐tagged SMAD4. (C): Flow cytometry analysis demonstrates rescued clones recue cardiac differentiation to became TNNT2+ cardiomyocytes. (D): Flow cytometry analysis demonstrates SMAD4 mutated hESCs failed to become TNNT2+ cardiomyocyte using growth factor‐based differentiation method. (E): Summary graph of TNNT2 flow cytometry of SMAD4 WT, mutant clones, rescue clones, and growth factor‐based differentiation method. *, p < .0001; compared with ES03 (n ≥ 3). ^#, p < .0001; compared with SMAD4.C1 (n = 4). ⁺, p < .0001; compared with SMAD4.C2 (n = 4). ES03 + Rescue.C2 with similar level SMAD4v5 expression as the mutant rescue clones. Ns, p = .2664 (n = 4). ^&, p < .0001; compared with ES03.AB (n = 3). AB indicates Activin a/BMP4 differentiation protocol.

Figure 3

RNA‐Seq reveals stage‐specific difference between the wild‐type and SMAD4 mutants. (A): MDS plot demonstrates the similarities in transcriptional profiles between samples. (B): Increasing numbers of differentially expressed genes comparing SMAD4 wild‐type and mutants at 0, 1, 3, and 6 days. (C): Heat map demonstrates the dynamic expression of ligands from WNT, TGFβ superfamily, and FGF signaling pathways. (D): Heat map demonstrates representative marker genes expression. Abbreviations: PN, proneuroectoderm; PS, primitive streak; MESEND, mesendoderm; CPC, cardiac progenitor cells; CM, cardiomyocyte; NE, neuroectoderm; MDS, multidimensional scaling.

Figure 4

SMAD4 mutant fail to form cardiac mesoderm precursor cells and defaults to neuroectoderm fate under conditions for cardiac differentiation. (A): Immunostaining demonstrates expression of the cardiac progenitor marker ISL1, cardiac mesoderm marker MESP1 and cardiomyocyte marker NKX2‐5 after 3 days cardiac differentiation. Scale bar = 100 μm. (B): Immunostaining demonstrates expression of the pluripotent marker POU5F1, cardiac marker NKX2‐5, and neuroectoderm marker PAX6 after 6 days cardiac differentiation. Scale bar = 100 μm.

Figure 5

Model summarizing the essential roles of SMAD4 in human cardiomyocyte differentiation. SMAD4 is essential for human cardiac mesodermal precursor cell formation.