Funding opportunities

Tracking of Human Embryonic Stem Cell Differentiation and Migration

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01130
Funds requested: 
$884 152
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
During normal embryonic development, cells follow a beautifully coordinated migration and differentiation program (to form tissues, then organs and whole animals); many details of which remain a mystery. Likewise, human embryonic stem cells (hESCs) differentiate into many tissue types and form rudimentary structures. In culture, we see differentiation of hESCs into, e.g., mesodermal cells that then differentiate into beating masses of heart cells, blood vessel-like structures and rudimentary neuro-vascula-like bundles; indicating the ability to form many of the tissues present in animals. Detailed analyses of the behaviors of human embryonic stem cells (hESCs) have not been possible because the huge volume of information is difficult to acquire and impossible to analyze with current tools. Needed automated computerized tools are proposed here to acquire, organize and analyze the huge quantity of information that will detail the movements, differentiation/development, cell division and interactions of hESC. In the embryo, key differentiation signaling events occur as a result, of cell-cell interactions as well other triggers. Our proposed development of biosensors that function in living cells will greatly aid automated identification of key developmental changes. Understanding these events is will help scientists control them, generate purified populations of appropriate progenitors, and learn to treat patients for a broad range of diseases. We propose to develop stable fluorescent protein (FP) labeled cell lines, automated microscopy and software tools for tracking hESC migration and cell lineages during differentiation into various cell types and structures. The Specific Aims are to: 1. 1. Develop and disseminate stable FP-reporter cell lines to label important markers of differentiation and drug selectable for isolation of pure populations of cardiomyocytes and endothelial cells. 2. Develop and disseminate automated microscopy and analysisstandard operating procedures (SOPs) for long-term (hours to weeks) time-lapse imaging in 3D. 3. Develop and disseminate software for fully automated tracking of the migration, differentiation/lineage, division, and intercellular interactions of hESCs. 4. Test and validate tracking of stable cell lines in SA1to track the appearance of endothelial cells and cardiomyocytes in EBs. The software tools will be disseminated by [REDACTED], and the stable biosensor hESC cell lines will be placed in [REDACTED] Ste Cell Center; both will be disseminated to stem cell scientists at large. These new tools will enable scientists to perform rapid, in-depth studies of hESC development and differentiation; enabling them to rapidly discover, test, improve and implement techniques for directing differentiation into appropriate cells, ultimately for therapeutic use.
Statement of Benefit to California: 
Discovering stem cell-derived treatments for important diseases is scientifically challenging. We propose to enable scientists to obtain the information needed to more rapidly overcome many of these challenges by developing and disseminating: 1) living human embryonic stem cell (hESC) lines containing fluorescent protein biosensors of key points of development/differentiation and 2) time-lapse automated microscopy image acquisition and software for organizing, collating and simplifying analyses of the complex movement, development, division and interplay of hESCs in culture. During normal embryonic development, cells follow a beautifully coordinated migration and differentiation program (to form tissues, then organs and whole animals); many details of which remain a mystery. Likewise, human embryonic stem cells (hESCs) differentiate into many tissue types and form rudimentary structures. In culture, we see differentiation of hESCs into, e.g., mesodermal cells that then differentiate into beating masses of heart cells, blood vessel-like structures and rudimentary neuro-vascular-like bundles; indicating the ability to form many of the tissues present in animals. Detailed analyses of the behaviors of human embryonic stem cells (hESCs) have not been possible because the huge volume of information is difficult to acquire and impossible to analyze with current tools. Needed automated computerized tools are proposed here to acquire, organize and analyze the huge quantity of information that will detail the movements, differentiation/development, cell division and interactions of hESCs. In the embryo, key differentiation signaling events occur as a result, of cell-cell interactions as well other triggers. Our proposed development of biosensors that function in living cells will greatly aid automated identification of key developmental changes. Understanding these events is will help scientists control them, generate purified populations of appropriate progenitors, and learn to treat patients for a broad range of diseases. We propose to develop stable fluorescent protein (FP) labeled cell lines, automated microscopy and software tools for tracking hESC migration and cell lineages during differentiation into various cell types and structures. The software tools will be disseminated commercially, and the stable biosensor hESC cell lines will be placed in a public stem cell center; both will be disseminated to stem cell scientists at large. These new tools will enable scientists to perform rapid, in-depth studies of hESC development and differentiation; enabling them to rapidly discover, test, improve and implement techniques for directing differentiation into appropriate cells, ultimately for therapeutic use. These new tools will enable California take/maintain its lead in stem cell research, rapidly develop stem cell-enabled treatments of important diseases, and coincidently contribute to a vibrant economy.
Review Summary: 
The overall goal of this project is to develop new reporter cell lines and advanced microscopy and software tools for tracking human embryonic stem cell (hESC) migration and differentiation into defined cell types. The Principal Investigator (PI) proposes to create genetically engineered reporter hESC lines harboring lineage-specific gene promoters directing fluorescent protein expression. The focus will be on the construction of reporter genes that allow for the detection of differentiation to endothelial cells and cardiomyocytes. The PI will use these lines to develop fully automated microscopic imaging systems, and software capable of tracking the differentiation and migration of fluorescent cells as they mature into contracting masses of cardiomyocytes and endothelial vessels in vitro. Reviewers were very positive about the technologies described in this application. The research addresses two fundamental bottlenecks in the stem cell field: differentiation of hESCs into mature somatic cell types, and the ability to monitor cell behavior and growth in culture. The proposed work has the potential to significantly enhance the ability to visualize hESC cellular and molecular characteristics at distinct time points in development and differentiation. The techniques using fluorescent protein biosensor design and time-lapse automated microscopy would increase the technical level to which the stem cell biology field could very efficiently analyze dynamic cell parameters. Each of these would independently have a positive impact on the field. The project was considered ambitious but achievable, and the proposal was well-organized and nicely presented. The PI and his/her team has already developed some of the fluorescent indicator cell lines proposed, and has considerable preliminary data demonstrating the potential of the new imaging technology. Reviewers were disappointed that the PI did not compare his/her proposed software to existing packages such as Metamorph, OpenLab, or Velocity, as it would have been useful to determine where the new software and microscopy system will exceed the others. While the microscopy system proposed can yield high-resolution images, it is usually computationally heavy and does not give images in real time. A discussion of and comparison to confocal or multi-photon microscopy would also have been useful. Another unanswered question is how the PI plans to disseminate the software. Will this be freeware, shareware, or will the software be sold? Finally, it is unclear how the software will be maintained and updated after development. In spite of these minor criticisms, reviewers felt that the project was feasible given the convincing preliminary data and the quality of the PI and the team. A major strength of the proposal is the expertise that the team brings to bear on the problem of in vitro live cell tracking and fate mapping. The PI of the project is a highly experienced cellular microscopy researcher who will devote 25% effort to the project. There are numerous Co-PIs with adequate time committed to the project, including software specialists and neuroscientists with expertise in human ESC biology. Reviewers commented that all of the tools and personnel are in place for this project to have a high likelihood of success. Project costs are modest and appropriate for the proposed project, although one reviewer commented that the subcontract seemed high. Overall, reviewers were highly enthusiastic about this technology and the applicant team. Although the project is high risk, this is mitigated by the fact that many of the reporter hESC lines have already been generated and characterized.
Conflicts: 

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