Funding opportunities

High-throughput label-free screening of stem cell differentiation

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01903
Funds requested: 
$1 449 443
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
A critical bottleneck in realizing stem cell-based therapies is the significant risk of tumor formation following treatment. Approaches used to direct pluripotent stem cells to medical-grade cells for these therapies may result in residual cells likely to form tumors once transplanted. Therefore, it is important to be able to recognize these residual unsafe cells or be able to predict if a batch of pluripotent stem cells is likely to yield many of these cells. Current approaches to identify these unsafe pluripotent cells have been developed for the research environment and are too costly and difficult to perform routinely. This has severely limited the practical realization of stem cell therapies. We have developed an automated and miniaturized approach to identify these unsafe cells based solely on their deformability (or ability to change shape when stretched) that promises to have significantly reduced costs. We propose to increase the accuracy of this approach through microfluidic and high-speed imaging technology development, and directly validate the approach for predicting tumor formation. Once guidelines have been developed for predicting tumor-forming potential we will implement this system to screen stem cell lines and their derivatives that are being developed for therapeutic purposes. This tool also promises to speed up the development of stem cell therapies by allowing high speed evaluation of many formulations that can be used to direct stem cell behavior.
Statement of Benefit to California: 
We propose to develop an instrument to determine the tumorigenic potential and improve the safety of stem cell therapies. We expect that this instrument will benefit Californians by (i) enabling stem cell therapies to enter the clinic by addressing safety concerns, (ii) reduce the cost of these therapies and the economic burden on our healthcare system, and (iii) provide jobs when the technology is commercialized through a California-based startup or industry partner. A major bottleneck in achieving stem cell therapies derived from embryonic stem cells or induced pluripotent stems cells is the significant risk of tumor formation if undifferentiated cells are carried through with a cell-based treatment. By providing a cost-effective solution to identify these cells prior to implantation we hope to increase the safety of these treatments and thus the likelihood that they will be made available to Californians with devastating, currently incurable diseases. Besides improving the chances that these treatments will be available, our approach would reduce the cost of these therapies when compared to currently used approaches to identify and sort unsafe cells. Cost may play a large role in whether insurance companies will reimburse for these therapies. A reduced cost will also reduce the tax-payer burden for providing these treatments through public healthcare. In addition to reducing the tax-payer burden in providing care that includes stem cell-based treatments, the development of our instrument will create many well-paying high technology jobs located in California as it is commercialized. Product development based on our instrument will provide high technology jobs in instrument engineering, product design, software development, and business development for commercialization through a startup or local industry partner. If successful these instruments will be used across the nation and world leading to a hundred million dollar market that will stimulate California's economy.
Review Summary: 
The goal of this application is to develop a new label-free assay and methodology to screen for the presence of human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) in populations of differentiated cells. This assay will allow the detection of residual pluripotent stem cells (PSC) or other abnormal cell populations after culture differentiation. Specifically, the applicant intends to detect PSCs by their deformability during exposure to fluidic forces and subsequently sort cells using a microfluidic platform. To accomplish this goal, the applicant will first improve a previously developed instrument, which assesses stem cell state using deformability measurements; these improvements will enhance the sensitivity and specificity of the cytometry analysis platform. Next, the applicant will examine correlations between human PSC deformability and their tumorigenic potential. Finally, a standardized deformability-based assay will be established for routine screening of human PSC lines. The reviewers praised the novel approach presented in the application. The proposed technology employs an innovative microfluidic strategy and avoids labeling of cells and the need for development of specific markers. However, reviewers had concerns about the significance and potential impact of the proposed research. They questioned the underlying premise that label-based flow cytometry, an alternative approach, is cost-prohibitive and expressed doubts that the proposed approach would be applicable to a large variety of differentiated cell types. Additionally, reviewers were concerned that the applicant did not consider the influence of cell cycle status or propose experiments that could adequately distinguish between various relevant cell types (e.g. normal undifferentiated PSCs, undifferentiated PSCs with impaired developmental potential, partially differentiated cells, and fully differentiated cells). Reviewers raised serious concerns about key aspects of the project’s feasibility and experimental design. Overall, the development of the proposed methodology appeared very preliminary; a large number of variables remain to be evaluated for determining specific flow conditions and the types of measurements most appropriate for the analyses. Additionally, reviewers considered the current process too slow and questioned the potential for scale-up to sorting speeds suitable for therapeutic manufacturing. There were also significant concerns about the potential of the method to distinguish with adequate fidelity and efficiency between relevant cell population, since preliminary data suggested that the difference in deformability between PSCs and differentiated progeny is not very large, and considerable overlap exists between the populations. Furthermore, no preliminary data were presented to support the contention that the developmental potential of undifferentiated cells is correlated with deformability or that residual PSCs (remaining after differentiation of the culture) will retain the deformability characteristics of the parental line. The Principal Investigator (PI) has excellent training, significant experience in microfluidics, and has developed many of the tools and theory underlying the proposed research. Although reviewers appreciated the inclusion of a stem cell biologist on the research team, they considered this individual’s 10% commitment to be inadequate, given the great need in the project for biological expertise. The budget was deemed appropriate considering the scope and timeline for the proposed investigations. In summary, this application proposes the development of a novel microfluidic platform to screen PSC-derived cell populations using a cell deformation assay. Despite the innovative approach, reviewer enthusiasm was diminished by their serious concerns regarding the project’s potential for impact and experimental design. Thus, the application was not recommended for funding.
Conflicts: 

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