Rationale
Human embryonic stem cells (hESCs) can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that enrich for cardiac lineages are poorly understood and limit the yield of cardiomyocytes.
Objective
Our aim was to develop a hESC-based high content screening (HCS) assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology. 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
About 550 known pathway modulators were screened in a HCS 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, demonstrating that Wnt inhibition alone is sufficient for deriving cardiomyocytes from hESC originating mesoderm cells. Transcriptional profiling of inhibitor-treated compared to vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors are very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, confirming Wnt inhibition as the relevant biological activity.
Conclusions
Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes, yielding novel tools for the benefit of pharmaceutical and clinical applications.
Rationale
The Transforming Growth Factor–β (TGFβ) family member Nodal promotes cardiogenesis, but the mechanism is unclear despite the relevance of TGFβ family proteins for myocardial remodeling and regeneration.
Objective
Determine the function(s) of TGFβ family members during stem cell cardiogenesis.
Methods and Results
Murine embryonic stem cells (mESCs) 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 TGFβ2. Both Nodal and TGFβ induced early cardiogenic progenitors in ESC cultures at day 0–2 of differentiation. However, Nodal expression declines by day 4 due to feedback inhibition whereas TGFβ persists. At later stages (day 4–6), TGFβ suppresses the formation of cardiomyocytes from multipotent Kdr+ progenitors, while promoting the differentiation of vascular smooth muscle and endothelial cells.
Conclusions
Nodal induces TGFβ, and both stimulate the formation of multipotent cardiovascular Kdr+ progenitors. TGFβ, however, becomes uniquely responsible for controlling subsequent lineage segregation by stimulating vascular smooth muscle and endothelial lineages and simultaneously blocking cardiomyocyte differentiation.
We have identified and assessed a new pathological subgroup of rectal cancer patients who had residual microscopic disease after neoadjuvant therapy. The survival analysis aligned them closely with pCR patients.
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