Chemical Genetic Approach to Production of hESC-derived Cardiomyocytes

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Grant Award Details

Grant Type:

Comprehensive Grant

Grant Number:

RC1-00132

Investigator(s):

Name:

Dr. Mark Mercola

Institution:

Sanford Burnham Prebys Medical Discovery Institute

Type:

Disease Focus:

Heart Disease

Human Stem Cell Use:

Embryonic Stem Cell

Award Value:

$2,832,000

Status:

Closed

Progress Reports

Reporting Period:

Year 2

Reporting Period:

Year 3

This progress report covers FY3 of the project to identify and characterize novel small molecule probes of cardiomyocyte differentiation from stem cells. During FY3, we characterized 11 novel chemical entities that promote cardiomyocyte differentiation. The small, drug-like molecules affect distinct steps in cardiomyocyte differentiation – 5 compounds promote formation of uncommitted cardiac progenitors, 2 stimulated committed cardiac precursors, while 2 compounds act later to stimulate differentiation into cardiomyocytes. Thus, these compounds are novel probes of stem cell differentiation. Some of the compounds are characterized to act upon particular cellular target proteins while the targets of other compounds are unknown. Of the latter class, candidate targets have been characterized by biochemical studies; one of which has been confirmed by RNA interference, yielding a new pathway in cardiac cell formation from stem cells. Three of the chemical series have been described in a patent application. Additional primary hits are being characterized. For FY4, we will continue characterization of a novel compounds. Particular focus will be on 4 chemical entities that promote later stages of human stem cell cardiomyocyte differentiation and on characterizing and discovering additional candidates that act on late-stage differentiation. In addition, we will develop a new pathway screen for a cellular target involved in specifying cardiomyocyte progenitors that have recently been shown to form new myocytes in vivo. Our new compounds are valuable probes of the underlying mechanism(s) responsible for making cardiac cells from stem cells. Moreover, recent data has shown that endogenous stem cells that reside in the adult heart resemble progenitors in the hESC cultures, so certain of our compounds can be considered as targeting cellular proteins and signaling pathways that might be beneficial to stimulate endogenous regeneration. Towards this goal, we will optimize the drug-like properties of the compounds in anticipation of in vivo testing for regenerative potential.

Reporting Period:

Year 4

Grant Application Details

Application Title:

Chemical Genetic Approach to Production of hESC-derived Cardiomyocytes

Public Abstract:

Adult heart muscle cells retain negligible proliferative capacity and this underlies the inability of the heart to replace muscle cells that are lost to injury, such as infarct, and underlies progression to heart failure. To date, no stem cell therapiy has produced significant cardiomyocyte replacement. Instead, transplanted cells, if they persist at all, produce endothelial cells or fibroblasts and the ameliorating effects on heart function that have been reported have been achieved by improving contractility, perfusion or other processes that are impaired in the failing heart. This proposal is to develop specific reagents and ultimately drugs to stimulate regeneration. Our approach integrates advances in stem cell biology, high-throughput (HT) biology, informatics and proteomics to identify small molecules, proteins and signal transduction pathways that control heart muscle formation from human embryonic stem cells (hESCs). High throughput assays will be developed and implemented to identify genes, signaling proteins, and small molecules that that control important steps in the differentiation, proliferation, and maturation of cardiac cells. Computer modeling and informatics will be used to identify and validate the signaling pathways that direct these critical processes. The discovery of small molecules and pathways will lead to protocols for 1) efficient directed differentiation of cardiomyogenic precursors from hESCs for research into transplantation and toxicology, 2) biotech reagents to stimulate cardiomyocyte renewal through directed differentiation of hESCs, and 3) cellular targets or lead compounds to develop drugs that stimulate regeneration from endogenous cells.

Statement of Benefit to California:

This proposal is a multidisciplinary collaboration among stem cell biologists, chemists, and engineers to address a critical problem that limits the widespread use of hESC for cardiology applications. Developing the multidisciplinary technology and overcoming the hurdles to application of hESCs to biotech and clinic will benefit California in many ways, including: Research to discover novel tools to stimulate heart muscle regeneration from hESCs is clinically important. Cardiovascular disease is the single largest cause of death in the U.S. and the assays we will develop and the reagents themselves will be useful tools to direct cardiomyocyte regeneration. This will speed the translation of hESCs to the clinic, specifically by stimulating production of cardiomyocytes and potentially by enhancing their integration and function after engraftment. Heart regeneration from hESCs probably uses similar cellular proteins and signaling pathways as regeneration of cardiomyocytes from other sources, thus, this research might be broadly applicable to heart muscle repair. Regeneration from endogenous cells remains controversial but these tools should be useful reagents to study and hopefully stimulate endogenous repair. Bringing the diverse people together (chemists, cell biologists, and engineers) to address a stem cell problem forges new links in the academic community that should be capable of opening new areas of research. These new areas of research will be a important legacy of the stem cell initiative and promises to invigorate academic research. The technology that we are developing applies the new discipline of chemical biology to stem cell biology, and the merger promises to spin off new areas of investigation and biotech products with the potential to benefit the practice of medicine and the local economy. Lastly, supporting the leading edge technology and the collaboration will build the California infrastructure of high throughput chemical library screening so that it can be focused on other areas of biomedical research, both stem cell and non-stem cell.

Publications

PLoS Comput Biol (2009): Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors. (PubMed: 19893621)
Proc Natl Acad Sci U S A (2010): Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation. (PubMed: 20498046)
Ann N Y Acad Sci (2010): Cardiac myocyte force development during differentiation and maturation. (PubMed: 20201894)
J Cardiovasc Transl Res (2011): A chemical biology approach to myocardial regeneration. (PubMed: 21424858)
Assay Drug Dev Technol (2008): A comparative analysis of standard microtiter plate reading versus imaging in cellular assays. (PubMed: 18795873)
PLoS One (2008): Contrasting expression of keratins in mouse and human embryonic stem cells. (PubMed: 18941637)
Circulation (2009): Electrophysiological challenges of cell-based myocardial repair. (PubMed: 20008740)
Mol Cell (2011): Fine-tuning of Drp1/Fis1 availability by AKAP121/Siah2 regulates mitochondrial adaptation to hypoxia. (PubMed: 22099302)
Nature (2014): Inhibition of miR-25 improves cardiac contractility in the failing heart. (PubMed: 24670661)
PLoS One (2009): Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes. (PubMed: 19352491)
Pediatr Cardiol (2009): Natural and synthetic regulators of embryonic stem cell cardiogenesis. (PubMed: 19319460)
Stem Cells Dev (2010): Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation. (PubMed: 20001453)
Dev Biol (2008): A novel activity of the Dickkopf-1 amino terminal domain promotes axial and heart development independently of canonical Wnt inhibition. (PubMed: 18840423)
Curr Protoc Stem Cell Biol (2012): Serum-free generation of multipotent mesoderm (kdr(+)) progenitor cells in mouse embryonic stem cells for functional genomics screening. (PubMed: 23154934)
Circ Res (2011): Small-Molecule Inhibitors of the Wnt Pathway Potently Promote Cardiomyocytes From Human Embryonic Stem Cell-Derived Mesoderm. (PubMed: 21737789)
Cell Stem Cell (2012): Small Molecule-Mediated TGF-beta Type II Receptor Degradation Promotes Cardiomyogenesis in Embryonic Stem Cells. (PubMed: 22862949)
J Med Chem (2012): Synthesis and SAR of b-Annulated 1,4-Dihydropyridines Define Cardiomyogenic Compounds as Novel Inhibitors of TGFbeta Signaling. (PubMed: 23130626)
Genes Dev (2012): Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis. (PubMed: 23152446)
J Med Chem (2012): Wnt Inhibition Correlates with Human Embryonic Stem Cell Cardiomyogenesis: A Structure-Activity Relationship Study Based on Inhibitors for the Wnt Response. (PubMed: 22191557)