Yes‐associated protein mediates human embryonic stem cell‐derived cardiomyocyte proliferation: Involvement of epidermal growth factor receptor signaling

Long

Baillargeon

et al. 2019

Sci Rep

Cardiac muscle cells lack regenerative capacity in postnatal mammals. A concerted effort has been made in the field to determine regulators of cardiomyocyte proliferation and identify therapeutic strategies to induce division, with the ultimate goal of regenerating heart tissue after a myocardial infarct. We sought to optimize a high throughput screening protocol to facilitate this effort. We developed a straight-forward high throughput screen with simple readouts to identify small molecules that modulate cardiomyocyte proliferation. We identify human induced pluripotent stem cell-derived cardiomyocytes (hiCMs) as a model system for such a screen, as a very small subset of hiCMs have the potential to proliferate. The ability of hiCMs to proliferate is density-dependent, and cell density has no effect on the outcome of proliferation: cytokinesis or binucleation. Screening a compound library revealed many regulators of proliferation and cell death. We provide a comprehensive and flexible screening procedure and cellular phenotype information for each compound. We then provide an example of steps to follow after this screen is performed, using three of the identified small molecules at various concentrations, further implicating their target kinases in cardiomyocyte proliferation. This screening platform is flexible and cost-effective, opening the field of cardiovascular cell biology to laboratories without substantial funding or specialized training, thus diversifying this scientific community.

Section: Resultsmentioning

confidence: 99%

A simple and flexible high-throughput method for the study of cardiomyocyte proliferation

Long

Baillargeon

et al. 2019

Sci Rep

“…We next wanted to identify an assay that could model a uniform reduction in contact inhibition across an entire population of hiCMs. A previous study used flow cytometry and Western blotting to show that embryonic stem cell-derived cardiac myocytes begin cycling when they are sparsely plated 20 . Thus, we plated hiCMs as a monolayer or plated them sparsely.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, we turned to in vitro models. It has previously been shown that plating low density human embryonic stem cell-derived CMs induces them to enter the cell cycle 20 . This was shown using flow cytometry to measure the expression of three cell cycle markers: BrdU, YAP, and cyclin D1.…”

Section: Introductionmentioning

confidence: 99%

The Hippo pathway regulates density-dependent proliferation of iPSC-derived cardiac myocytes

Dai

Liu

et al. 2021

Sci Rep

Inducing cardiac myocytes to proliferate is considered a potential therapy to target heart disease, however, modulating cardiac myocyte proliferation has proven to be a technical challenge. The Hippo pathway is a kinase signaling cascade that regulates cell proliferation during the growth of the heart. Inhibition of the Hippo pathway increases the activation of the transcription factors YAP/TAZ, which translocate to the nucleus and upregulate transcription of pro-proliferative genes. The Hippo pathway regulates the proliferation of cancer cells, pluripotent stem cells, and epithelial cells through a cell–cell contact-dependent manner, however, it is unclear if cell density-dependent cell proliferation is a consistent feature in cardiac myocytes. Here, we used cultured human iPSC-derived cardiac myocytes (hiCMs) as a model system to investigate this concept. hiCMs have a comparable transcriptome to the immature cardiac myocytes that proliferate during heart development in vivo. Our data indicate that a dense syncytium of hiCMs can regain cell cycle activity and YAP expression and activity when plated sparsely or when density is reduced through wounding. We found that combining two small molecules, XMU-MP-1 and S1P, increased YAP activity and further enhanced proliferation of low-density hiCMs. Importantly, these compounds had no effect on hiCMs within a dense syncytium. These data add to a growing body of literature that link Hippo pathway regulation with cardiac myocyte proliferation and demonstrate that regulation is restricted to cells with reduced contact inhibition.

“…We next wanted to identify an assay that could model a uniform reduction in contact inhibition across an entire population of hiCMs. A previous study used flow cytometry and Western blotting to show that embryonic stem cell-derived cardiac myocytes begin cycling when they are sparsely plated (Park et al, 2018). Thus, we plated hiCMs as a monolayer or plated them sparsely.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, we turned to in vitro models. It has previously been shown that plating low density human ESC-derived CMs induces them to enter the cell cycle (Park et al, 2018). This was shown using flow cytometry to measure the expression of three cell cycle markers: BrdU, YAP, and cyclin D1.…”

Section: Introductionmentioning

confidence: 99%

The Hippo pathway regulates density-dependent proliferation of iPSC-derived cardiac myocytes

Dai

Liu

et al. 2021

Preprint

Inducing cardiac myocytes to proliferate is considered a potential therapy to target heart disease, however, modulating cardiac myocyte proliferation has proven to be a technical challenge. The Hippo pathway is a kinase signaling cascade that regulates cell proliferation during the growth of the heart. Inhibition of the Hippo pathway increases the activation of the transcription factors YAP/TAZ, which translocate to the nucleus and upregulate transcription of pro-proliferative genes. The Hippo pathway regulates the proliferation of cancer cells, pluripotent stem cells, and epithelial cells through a cell-cell contact-dependent manner, however it is unclear if cell density-dependent cell proliferation is a consistent feature in cardiac myocytes. Here, we used cultured human iPSC-derived cardiac myocytes (hiCMs) as a model system to investigate this concept. hiCMs have a comparable transcriptome to the immature cardiac myocytes that proliferate during heart development in vivo. Our data indicate that a dense syncytium of hiCMs can regain cell cycle activity and YAP expression and activity when plated sparsely or when density is reduced through wounding. We found that combining two small molecules, XMU-MP-1 and S1P, increased YAP activity and further enhanced proliferation of low-density hiCMs. Importantly, these compounds had no effect on hiCMs within a dense syncytium. These data add to a growing body of literature that link the Hippo pathway regulation with cardiac myocyte proliferation and demonstrate that regulation is restricted to cells with reduced contact inhibition