High content screening for modulators of cardiac differentiation in human pluripotent stem cells.

Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology through screening of small molecules in in vitro assays of a biological phenotype of interest, followed by target identification. In the case of disease-specific assays, the cellular proteins identified might constitute novel drug targets, and the small molecules themselves might be developed as drug leads. In cardiovascular biology, a chemical genomics approach to study the formation of cardiomyocyte, vascular endothelial, and smooth muscle lineages might contribute to therapeutic regeneration. Here, we describe methods used to develop high content screening assays implementing multipotent cardiovascular progenitors derived from human pluripotent stem cells and have identified novel compounds that direct cardiac differentiation.

A Nodal-to-TGFß cascade exerts biphasic control over cardiopoiesis.

RATIONALE: The transforming growth factor-ß (TGFß) family member Nodal promotes cardiogenesis, but the mechanism is unclear despite the relevance of TGFß family proteins for myocardial remodeling and regeneration. OBJECTIVE: To determine the function(s) of TGFß family members during stem cell cardiogenesis. METHODS AND RESULTS: Murine embryonic stem cells were engineered with a constitutively active human type I Nodal receptor (caACVR1b) to mimic activation by Nodal and found to secrete a paracrine signal that promotes cardiogenesis. Transcriptome and gain- and loss-of-function studies identified the factor as TGFß2. Both Nodal and TGFß induced early cardiogenic progenitors in embryonic stem cell cultures at day 0 to 2 of differentiation. However, Nodal expression declines by day 4 due to feedback inhibition, whereas TGFß persists. At later stages (days 4-6), TGFß suppresses the formation of cardiomyocytes from multipotent Kdr(+) progenitors while promoting the differentiation of vascular smooth muscle and endothelial cells. CONCLUSIONS: Nodal induces TGFß, and both stimulate the formation of multipotent cardiovascular Kdr(+) progenitors. TGFß, however, becomes uniquely responsible for controlling subsequent lineage segregation by stimulating vascular smooth muscle and endothelial lineages and simultaneously blocking cardiomyocyte differentiation.

Small-molecule inhibitors of the Wnt pathway potently promote cardiomyocytes from human embryonic stem cell-derived mesoderm.

RATIONALE: Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes. OBJECTIVE: Our aim was to develop a human embryonic stem cell-based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis. METHODS AND RESULTS: Approximately 550 known pathway modulators were screened in a high-content screening assay, with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell, which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell-derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors were very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, which confirmed that Wnt inhibition was the relevant biological activity. CONCLUSIONS: Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications.