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SAN DIEGO, CA, United States

Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.07M | Year: 2014

Not Available


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase II | Award Amount: 1.23M | Year: 2013

DESCRIPTION (provided by applicant): Monitoring physiology of individual cardiomyocytes in high throughput has not been reported. The inability to perform high throughput physiological measurements limits many basic and applied studies, including the use of stem cell derived cardiomycoytes in cardiotoxicity testing. Current automated cardiotoxicity tests have poor predictive value because they use tumor cell lines engineered with single channels (e.g. hERG), and physiologically relevant tests are reserved for few candidates during the relatively late stages of development. The poor biological relevance of these models contributes to the high failure rate of drug candidates before FDA approval and even after commercialization. We have automated recording frommyocytes for Calcium Transients, but are still limited by use of electrode devices for pacing that prevents miniaturization beyond 96- well format. Furthermore, Action Potential measurement, the most relevant physiological parameter in excitable cells, isstill reserved to low throughput analysis. We propose several conceptual advances to solve these problems by developing a miniaturized, cell-based optogenetic pacing device for high throughput analysis of human Induced Pluripotent Stem Cell (hIPSC)-derived cardiomyocytes in an automated platform for cell-by-cell cytometric analysis of cardiomyocyte physiology. We will also develop automatic segmentation/analysis of Action Potentials (AP) through fluorescent voltage probes and post-recording tracking to identify the same cells after fixation and immunostaining analysis. Calcium Transient (CT) analysis, already developed in a previous SBIR contract, will converge with AP and post-recording tracking to generate single cell multiparametric measurement of all these endpoints conducted in High Throughput. Extensive evaluation will be conducted with drugs that alter AP through different mechanisms to validate the platform. Preliminary data show that stable cell lines expressing the light-triggered protein Channelrhodopsin-2 (ChR2) will electrically couple to cardiomyocytes, allowing optically controlled stimulation of AP without disruption of normal cardiomyocyte physiology. Membrane AP can be recorded in cardiomyocytes through voltage probes and are suitable to image segmentation analysis. Automatic CT measurement and hIPSC-derived cardiomyocytes are an effective model to test cardiotoxic effects of reference drugs. The Aims will advance the use of fluorescent probes to measure action potential, calcium flux and cell characteristics in response to the stimulation. Cardiomyocyte physiology will be quantified by image analysis software that records and analyzes single-cell AP and CT in relation to cardiac subtype or specific protein expression. The software will segment the images into single cell recordings, thus all measurements and data analysis will be on a cell-by- cell basis. The format will be evaluated for 384- and 1536-well to conduct screening on hundreds of cells per individual data point (e.g. compound tested), allowing throughput of tens to hundreds of thousands of datapoints in a single screen by the end of the funding period. Channel openers and blockers will be tested to validate the platform. The platform will find applications in basic and applied research, including regenerative medicine research and drug development/safety testing. PUBLIC HEALTH RELEVANCE Large-scale studies of heart safety early during the drug development process are not currently possible or have low value because physiological testing is too arduous to perform on more than a few cells in a single experiment. Here we propose a conceptual advance of a previous design, where we have automated some of the steps enabling moderate throughput but that still rely on traditional methods to stimulate cardiomyocytes and analyze only one parameter relevant to cardiac physiology. We propose to develop a cell-based miniaturized pacing device that is stimulated by light and then activates the cardiomyocytes. We will also automate the process of Action Potential measurement and will link to Calcium Transient and cardiomyocytes subtypes to generate a comprehensive platform that analyze several aspect of cardiomyocyte physiology. The device will enable many applications, including large physiological screening for new cardioactive drugs and early testing of drug candidates for adverse cardiac toxicity, which is a major reason for drug failure during development, costing 2.5 billion annually.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.55K | Year: 2013

Congenital cardiac defects (CCDs) are among the most common birth defects and fetal cardiac defects likely contribute to miscarriages, which terminate up to 25% of all pregnancies. Environmental factors may influence up to 80% of all CCDs, but little is known about how chemicals from the environment may affect formation of the heart. We propose to develop a cardiopoiesis (formation of the heart) assay to screen chemicals using stem cells, which recapitulate early event in heart formation, in which cardiac myocytes, vascular endothelial cells, and vascular smooth muscle cells differentiate from mesodermal multipotent cardiac precursors. The assay is performed in 384 well plates and uses murine embryonic stem cells (mESCs) bioengineered with reporter genes toreport the proportion of cardiac myocytes, vascular endothelial cells, and vascular smooth that emerge from the cultures. In preliminary experiments, 550 known chemical pathway modulators were screened with the cardiopoiesis assay, and inhibitors of the Wnt pathway were found to strongly upregulate production of cardiac myocytes. This is consistent with the known influence of the Wnt pathway on cardiac development and the suspected role of dysregulated Wnt activity in producing CCDs. Phase I goals will be to further improve the assay by identifying compounds or genomic constructs that can serve as reliable positive or negative effectors in the system, to develop methods to maximize the information that can be garnered from each screening run, and to developmethods to improve the consistency and further miniaturize the assay. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Defects of the heart are among the most common birth defects and might be caused, in part, by toxic compounds from the environment interfering with heart development before the baby is born. However, there are few methods to test chemicals for such effects. We propose to develop a test to screen chemicals for dangerous effects on heart formation, by using approved stem cells, which can becultured in a manner so that heart cells form within the cultures. In fact, heart cells derived from stem cells, beat, rhythmically, jst like a human heart. If a chemical interferes with the development of heart cells from stem cells, it will likely have harmful effects on heart development in human embryos, as well.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 223.05K | Year: 2014

Not Available


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 761.59K | Year: 2016

DESCRIPTION provided by applicant Defects in heart formation during embryonic and fetal development likely contribute to miscarriages which terminate up to of all pregnancies and Congential Heart Defects CHDs which are among the most common birth defects Environmental factors may influence miscarriages and up to of all CHDs but little is known about how such chemicals affect formation of the heart during early embryonic differentiation The goal of this SBIR project is to develop a Cardiopoiesis Assay heart formation assay to enable testing of compounds to determine if they influence the emergence of cardiac myocytes from multipotent mesodermal progenitors a critical early decision point in heart formation During the previous Phase I portion of the project a version of the assay was developed using mouse embryonic stem cells and this assay has proven useful in identifying chemicals siRNAs and miRs that modify cardiac differentiation For Phase II we propose to develop the assay using human induced pluripotent stem cells hiPSCs which will be derived from multiple donors to increase the genetic diversity represented by the assay The Cardiopoiesis Assay will be useful in predicting whether chemical compounds affect heart formation in the human embryo which is of critical importance to human health The assay will be of interest to government agencies in the US such as the Environmental Protection Agency and worldwide concerned with environmental toxicology PUBLIC HEALTH RELEVANCE Defects of the heart are the most common birth defects and may be caused in part by toxic compounds from the environment that interfere with heart formation prior to birth We propose to develop a test system to screen chemicals for dangerous effects on heart formation We propose to use human stem cells which we will derive from skin samples obtained from volunteer donors the skin samples are obtained by a painless procedure that is risk free Such stem cells can be cultured in a manner that they form beating heart cells in laboratory dishes We will develop a procedure to test if chemicals prevent formation of heart cells from stem cells as such chemicals are likely to cause heart birth defects

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