Funding opportunities

Optimizing Sorting Flow Cytometry for Translational Stem Cell Therapies

Funding Type: 
Early Translational I
Grant Number: 
TR1-01243
Funds requested: 
$3 476 476
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
When stem cells are grown in the laboratory, they often grow into a variety of cell types, not just the type that would ultimately be useful in cell-based therapies. Our goal is to achieve nearly pure collections of stem cells from these cultures. To achieve this goal, we will improve and customize cell sorting using a technique called fluorescence activated cell sorting (FACS). This technique allows a researcher to separate cells based on measurements of shape, size and proteins present on their surfaces. This separation is done rapidly and with high precision, but has not been optimized for routine use with stem cells. Improvements in this technology can produce higher and more consistent yields of specific stem cells by purifying them from undesired types in a scalable and economical way. We will define conditions and optimize methods for purification to the level necessary to ensure transplant safety. Using FACS for stem cell sorting is a natural fit, as stem cell cultures produce closely related sub-populations that require careful analysis to separate. Stem cell sorting has remained an niche technology for purifying stem cell populations for a variety of reasons. FACS instruments are made to accommodate a wide variety of cell types, and are therefore complex, and FACS has not been thoroughly optimized for stem cells. Success with FACS can also be cell type dependent, with stem cells being highly variable and sensitive to stress. Among the significant challenges in the use of current FACS instruments for purifying stem cells for patient therapy, perhaps the most important is the cumbersome implementation of clinically suitable sorting due to concerns over sterility and patient-to-patient contamination. Improvements achieved in this proposal will address these issues and be made available as a central resource to guide researchers in successfully preparing and sorting stem cells. By combining novel instrument capabilities with FACS expertise, we seek to address the needs of the stem cell community to allow the routine separation of stem cell populations for research and clinical use.
Statement of Benefit to California: 
The work outlined in this proposal would benefit California by supporting its position as a world leader in stem cell related knowledge and technologies. This is a collaborative effort between a privately held California company and a local research institution to provide techniques, tools and technology for advanced stem cell research. The work will employ local researchers to carry out the daily experiments, it will require many locally produced reagents and supplies, and include major equipment purchases from California companies to complete the proposed aims. This California based spending would contribute to the $2.2 to $4.4 billion dollars in additional income and sales tax revenue predicted from Proposition 71*. In addition to boosting the California economy, this work will depend on local scientific expertise at both academic institutes and local companies, ensuring productive work and advances in the scientific fields of some of California’s brightest minds. In addition to providing valuable gains in scientific knowledge for the company and collaborating institute, the work will be made widely available to the larger California research community. This will include online web-based availability, and a plan for an in-depth training course hosted at a local institute for hands-on dissemination of the knowledge gained through this work. In turn, this training would ensure future generations of California scientists benefit from this work and allow them to rapidly advance future stem cell therapies. If stem cell therapies are to be realized, there is a need for rapid and effective methods for separating the cells of interest from the background. There is currently no clear solution to this problem, and that is what this proposal will address. Producing safe, effective cell purifications will more rapidly allow these promising therapies to enter into clinical use. These therapies could provide relief for a host of conditions from arthritis to heart disease, and are predicted to save the State of California between $3.4 and $6.9 billion health-care related dollars*. *Analysis Group Report, Economic Impact Analysis, Proposition 71 California Stem Cell Research and Cures Initiative, 2004.
Review Summary: 
This proposal is focused on the use of cell sorting technology to address two perceived bottlenecks in the development of human embryonic stem cells (hESCs) for clinical use. The first aim is to improve the yield and purity of specific stem cell subpopulations sorted from mixed populations of cells. The second aim is to optimize sorting methods for the pre-transplant purification of differentiated cells derived from hESCs to prevent teratoma formation. The applicant proposes two approaches to address these aims: 1) the development of standardized methods and process improvements for existing cell sorting technology; and 2) the development and optimization of a novel, proprietary microfluidics-based cell sorting system. The reviewers had significant doubts about the scientific rationale for this proposal and its potential impact. One reviewer felt that the first proposed bottleneck is of debatable significance, given that existing hESC lines have been generated, maintained and successfully differentiated without a critical need for fluorescence-activated cell sorting (FACS). This reviewer found the second bottleneck to be more important. Methods to completely eliminate residual pluripotent, potentially tumorigenic hESCs could be of tremendous value, both for pre-clinical research and therapeutic applications. FACS, with its ability to isolate cell subpopulations based on multiple simultaneous parameters, provides an advantage over batch processing systems such as magnetic beads. Unfortunately, FACS measures and sorts one cell at a time, which, while useful in certain settings, is too slow for most clinical applications. Traditional FACS has other inherent disadvantages: 1) it’s an open system, allowing aerosol formation; 2) it’s difficult to design single-use disposable materials; and 3) it can be a relatively harsh procedure, generating high shear forces for certain cell types. The applicant addresses these limitations by also proposing to optimize a proprietary microfluidics-based sorting device that incorporates low shear forces and a cartridge, single-use, disposable design into a closed system. Unfortunately, both traditional and microfluidics based FACS rely on fluorescent signal, which either requires genetic labeling of cells or a specific and reversible antibody. A reviewer noted that the former is not likely to be a viable clinical option and that the applicant did not adequately discuss the latter. This reviewer felt that the proposal described a technology in search of an application to stem cell research rather than one ready to address a potential clinical need in the next 3-5 years. The reviewers felt that the research plan lacked detail and had poorly defined milestones and metrics of success. They noted that one of the proposed advantages of the proprietary microfluidics-based sorting device may in fact be a significant limitation. The applicant emphasized that this device can deal with small starting cell numbers (1,000-300,000 cells), which might be important in the initial stages of hESC derivation, but is clearly insufficient for certain research applications and completely underpowered for clinical use. One reviewer estimated that most cell therapies will require one to ten million cells yet the applicant does not discuss scalability to these numbers. Another reviewer noted that the applicant proposes >90% purity as a target milestone, but this again is insufficient for most research studies, let alone clinical applications. Reviewers also criticized a lack of pertinent preliminary data. One reviewer pointed out that the data presented from the novel sorting device did not indicate a significant advantage over traditional FACS. The device only achieved 80% purity sorting a small number of cells using an idealized model system. In addition, Table 1 indicates cell capture rates of 150 cells/second which is >100 times slower than current FACS technology and >10,000 times slower than magnetic beads. Another reviewer noted that there is a general lack of detail regarding quantitative output criteria and how analyses will be conducted. For example, the applicant mentions monitoring teratoma formation but doesn’t specify time points or means of analysis. There is also very little discussion of possible limitations or viable alternative approaches. The reviewers noted that while the applicant has had excellent training, s/he does not have significant management or leadership experience, as evidenced by a lack of senior author publications and the relatively short time elapsed since completion of a post-doctoral fellowship in 2006. One reviewer also pointed out that the applicant has no publications listed that relate to cell sorting. Reviewers appreciated the expertise brought to the research team by a collaborator with experience with stem cells and high-throughput screening but noted the lack of a supporting letter. One reviewer did mention the strong letter of support from an industry collaborator that uses the applicant’s proprietary cell sorting technology. Overall, the reviewers were not convinced that this proposal addresses critical bottlenecks or that the proposed approaches would advance cell therapies to the clinic. They also found the research plan lacking both sufficient detail and supporting preliminary data.
Conflicts: 

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