Funding opportunities

Depletion of Teratoma-Forming Cells for hESC-Based Transplantation Technologies

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02033
Funds requested: 
$1 819 101
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human embryonic stem cells (hESCs) have the potential to differentiate into all the types of cells in our bodies. They therefore represent a highly promising source for replacing cells and tissues in countless diseases and conditions (e.g., Parkinson's disease and diabetes). Many laboratories are working on controlled methods to differentiate hESCs into the cell types of their choice. To date, however, no method has been able to reach a 100 % differentiation efficiency. This issue poses a major roadblock to stem-cell based therapies. Specifically, hESCs that are only partially differentiated or remain non-differentiated (due to the inefficiencies in these methods) have the potential to cause harmful tumors or prevent proper integration of the differentiated cells into the body. We therefore propose a novel method to eliminate these potentially-harmful cells from the beneficial ones, thus enabling these cells to be used for therapy. The current, conventional methods that are used to purify one type of cell from another – either fluorescent-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) – suffer from several inadequacies. Cells that are purified using both FACS and MACS undergo direct modification by outside chemicals and reagents and cells that are purified using FACS do not survive. While MACS is gentler on cells than FACS, it still is not efficient enough to remove all of the potentially-harmful cells. Our device will allow us to efficiently and gently purify out the potentially-harmful cells from a mixed population of both beneficial and harmful cells, without modifying or damaging the cells. Our device will also be easily scalable so that we can rapidly purify enough cells to be used in a clinically-relevant application. Specifically, our device is made up of a microfluidic channel that will be attached to a glass surface that is modified with antibodies. These antibodies identify and bind to receptors that are present on the surface of the potentially-harmful cells and trap those cells in the channel. While FACS and MACS use a similar method of antibody-recognition of cell-surface receptors, they directly label the cells by attaching the antibodies to the cell, whereas in our method, we do not modify the cells in any way. Also, our device will allow us to screen several different surface receptors in one device by attaching different antibodies in each channel. In summary, our novel microfluidic device will provide a new benchmark for purifying out potentially-harmful cells from beneficial cells (derived from hESCs) thus allowing them to be safely used for therapy.
Statement of Benefit to California: 
The proposed research will benefit the state of California and its citizens for a number of reasons, both clinical and economic. The ability to implant hESC-derived cells for clinical therapy without the risk of contamination of teratoma-forming cells is of extreme clinical importance. Our technology—a highly efficient, high-throughput cell depletion device that will remove all the teratoma-forming cells—will eliminate this critical bottleneck that has thwarted the safe and effective implementation of stem-cell therapies. This will lead to a significant advancement in the field of regenerative medicine and lead to the treatment of diseases that have thus far been untreatable. Aside from the clinical impact, the development of our technology with broad clinical application will likely lead to commercialization. This would generate jobs and spur economic development in the state of California. Furthermore, the development and utilization of our technology would educate and train new interdisciplinary scientists, who will be at the forefront of the Medical Sciences and Engineering, thereby providing a strong technological workforce in California. Overall, the successful development of our technology will enable the implementation of novel stem-cell therapies, improve human health, spur economic development within the state of California, and contribute to the scientific knowledge and technological resources of the state.
Review Summary: 
This proposal is focused on the development of a novel microfluidic device to separate undifferentiated, potentially tumorigenic human embryonic stem cells (hESCs) from their differentiated progeny. The applicant identifies the lack of efficient methods to eliminate undifferentiated hESCs from cell populations intended for transplant as a bottleneck to clinical translation. There are three Specific Aims: (1) to develop and optimize a scalable microfluidic device to deplete undifferentiated hESCs from a heterogeneous cell population; (2) to further develop this device to also deplete cells of intermediate differentiation status; and (3) to validate the device by transplanting purified cells into animal models and monitoring for teratoma formation. The reviewers agreed that this proposal addresses a significant translational bottleneck, as currently available cell sorting technologies are limited in throughput and efficiency. However, they did not find the proposal to be particularly innovative, describing it as essentially an updated version of column-based panning, methodology that has been abandoned due to poor performance. Reviewers also raised concerns about the scientific rationale for key aspects of the proposal. The applicant proposes to rely on negative selection to deplete residual hESCs, but, for the large cell numbers that will be required for therapies, this approach demands extremely high efficiency. One reviewer estimated, using an optimistic scenario, that purification efficiency will need to be greater than 99.9%, and cautioned that this will be very difficult to achieve with a single process. Reviewers also questioned whether the goal of producing a low-cost device is reasonable, given that the applicant’s cost estimate does not include clinical grade protein and antibodies. Reviewers raised significant concerns about the research plan and ultimately were not convinced of its feasibility. They appreciated the use of microfluidics combined with parallel processing as well as the applicant’s recognition that large numbers of cells will need to be processed for the device to be therapeutically relevant. Reviewers agreed that a comparison of the applicant’s device to fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) would be valuable, but would have liked more detail about these experiments. One reviewer described Aims 2 & 3 as exciting, but dependent on Aim 1. This reviewer especially appreciated the use of animal models in Aim 3 to validate their device. The reviewers generally found the preliminary data to be weak, demonstrating less than 50% hESC removal with single-stage sorting relative to a control. In addition, they noted that the throughput presented in an ideal model system is approximately 30 times slower than FACS, which would necessitate hundreds of parallel channels. Reviewers also described a number of technical challenges that were not addressed by the applicant. For example, each antibody will likely have different fluid-flow characteristics for optimal capture. Cells may aggregate in the microfluidic device and the first cells entering may clog the channel, subjecting subsequent cells to altered flow conditions. Certain hESC differentiation protocols may result in final cell populations that are not amenable to single cell manipulations (e.g. epithelial monolayers) and so removal of residual undifferentiated cells would have to occur earlier in the differentiation process. Finally, reviewers cautioned that the entire proposal relies on the development of specific high-affinity reagents that recognize hESCs but not their differentiated progeny. They noted that the applicant does not address how this specificity will be achieved, or the cost of developing such reagents in a Good Manufacturing Process (GMP)-compliant manner. The reviewers noted that the Principal Investigator’s (PI) has significant expertise in microfluidics and cell sorting and their application to diverse biological systems. They appreciated the Co-Investigators complementary expertise in stem cell biology and animal models. In general, they found the team to be quite strong but were concerned about the large number of personnel to be hired. Overall, while reviewers agreed that this proposal addresses a significant bottleneck, they raised serious concerns about the scientific approach and research plan. In light of these concerns, reviewers did not judge the project to be feasible.
Conflicts: 

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