Both adult and embryonic stem cells hold profound therapeutic promise. However, a major bottleneck thwarting the effective use of both adult and embryonic stem cells in the clinic is the lack of rapid and accurate flow-based sorting strategies that are compatible with Good Manufacturing Practices (GMP). With embryonic stem cells, the current inability to rapidly and cleanly sort out and eliminate the primitive, pluripotent cells that remain in differentiated cultures is a serious hurdle. These cells can cause the formation of encapsulated tumors, called teratomas, as well as other possible adverse consequences when used therapeutically. Until these primitive, pluripotent cells can be removed, it will remain difficult to obtain regulatory approval for the use of human embryonic stem cells (hESC) in clinical trials. Current proposed hESC clinical protocols consist of cellular products differentiated from hESCs without the use of sorting to definitively exclude primitive, pluripotent hESC that could potentially form teratomas or undergo uncontrolled differentiation in vivo. Trials done without appropriate separation strategies could be very detrimental to the field.
Flow-based cell sorters that use fluorescent cellular markers have been in use for many years in research applications. While excellent research tools, these sorters are ill-suited for use in a clinical setting. In particular, they rely on aerosolization of the patient sample, resulting in the risk of sample contamination and potential safety risks to the operator of the machine. While they have been used in clinical trials, they are complex, difficult to sterilize between patients, and in the context of cellular therapy, relatively slow. Therefore, as trials progress it would be beneficial to have sorters that are improved upon in their ease of use, sort speed and incorporating a closed system which can readily be exchanged for single patient use.
The goal of the proposed research is to eliminate this bottleneck by providing an ultra-high speed cell sorter and appropriate methodology for use in clinical applications. The key features of such a system are: 1) speed and yield sufficient to process clinically relevant cell populations, 2) the ability to provide a very high purity sort, and 3) the ability to perform this sorting in a completely sterile, disposable fluid path that can be manufactured using GMP-compliant practices. The system makes use of micro-electromechanical systems (MEMS) technology to fabricate a silicon chip with integrated sorter mechanisms. This chip is then assembled into a sterile and disposable fluidic system that includes blood bags and appropriate tubing to connect the bags and chip. Sorting of adult stem cell samples has been demonstrated, and the system is now at a beta test level. However, significant development and testing is necessary to progress to demonstrated performance of fully functional clinical machines.
Statement of Benefit to California:
From the California Institute for Regenerative Medicine website ,
“In theory, there’s no disease that is exempt from a possible treatment that comes out of stem cell research. Given that researchers may be able to study all cell types via embryonic stem cells, they have the potential to make breakthroughs in any disease. …The promise of embryonic stem cells is that they can form any type of cell in the body. The trouble is that when implanted into an animal they do just that, forming all tissue types in the form of tumors called teratomas. …Even when researchers have learned to mature cells into a single cell type, getting those cells pure enough to eliminate the risk of remaining immature cells forming teratomas has been extremely difficult.”
This project is the development of a faster, more efficient cell sorting system that will remove the bottleneck of stem cell purification. Besides enabling advances in hESC research and clinical development, this will also accelerate the development and commercialization of adult stem cell therapies. The proposed project dramatically benefits the state of California in 3 ways:
Health of Californians: Because removal of this bottleneck may result in a profound positive impact on numerous envisioned therapies, quantification of that impact can be difficult. Consider just one application: cell therapies for Peripheral Vascular Disease (PVD). PVD, also known as arteriosclerosis of the extremities, is a disease of the blood vessels characterized by hardening of the arteries that supply the legs and feet. It is estimated that 1 to 1.4 million people suffer from PVD in California alone. The size of the affected populations poses a real issue in delivering therapies, especially if they depend on conventional cell sorting. The system that will arise from this project permits ready scale-up so these therapies can actually be delivered to Californians.
Pace of Medical Research: The inability to purify stem cell populations has seriously hindered the fundamental research of many disease states and the development of clinical treatments derived from that research. By providing a means of stem cell purification, researchers will be able to focus their attention on this much needed basic research.
Economic benefit: The overriding economic benefit to the state is in fact in the form of improved health for Californians. This directly translates into reduced medical expenses that result from secondary care of many diseases. The proposed research also results in the development of a cell isolation technology that relies on the manufacture of machines and disposable cell therapy kits. A substantial portion of these will be manufactured in California, thereby creating additional jobs and tax revenue. Furthermore, the availability of a technology that enables readily scalable manufacture of clinical grade cell therapies will encourage the development of companies that make cellular products to treat Californians.
The primary focus of this proposal is on the development of a high-speed cell sorter capable of efficiently eliminating residual pluripotent stem cells from large numbers of differentiated cells in a therapeutic product derived from embryonic stem cells (ESCs). The applicant’s technology is based on a micro-electro-mechanical system (MEMS) that allows parallel processing. This system should achieve a much higher speed of processing than that obtained with conventional fluorescence activated cell sorting (FACS). Upon completion of the specific aims, the applicant hopes to have progressed from beta test versions to an improved product, one that is clinically applicable.
Cell sorting represents one solution to the bottleneck of obtaining pure cell populations for transplantation. The technical limitations of currently available methods necessitate the need for greatly improved or novel technologies. The applicant’s chip-based approach represents an engineering advance that appears scientifically justifiable and could make a major impact on the advancement of cell therapies to the clinic. However, reviewers found that a significant deficiency in this proposal was the lack of supportive scientific and technical evidence and data validating the technology. Beyond some meager preliminary results and the applicant’s assurances, the provided data related only to detection, actuation speed and preliminary sorting results. Importantly, a quantitative comparison with other (established) cell sorting systems was lacking. The evidence presented did not indicate that a truly functional multichannel FACS system has been achieved but suggests that the current instrument is a prototype in need of much in-depth development. This application, however, is focused on making the instrument compatible with clinical use, and the remaining technological hurdles and ways to overcome them are not adequately addressed.
Reviewers uniformly praised the proposed global milestones and timelines as reasonable and quantitative. However, these strengths did not overcome substantial weaknesses of the proposal. Reviewers felt that the application was focused primarily on achieving product development milestones rather than addressing directly the issues of overcoming the specified bottleneck and underlying biological issues. The applicant failed to address the key issue of how he/she will demonstrate the elimination of teratoma-forming cells from a therapeutic preparation. The model system used for technology development utilizes positive selection of the desired cells, yet negative selection of unwanted cells will likely be required for obtaining hESC-derived products for clinical use. In addition, the applicant proposed to work with a model cell line in which the promoter of a key regulatory gene drives a fluorescent reporter, but this model is not appropriate for actual clinical development. Reviewers were also concerned that the current instrument has yet to achieve high cell purities even though the applicant has spent considerable time and effort working and developing this sorter. The slow development pace called into question whether this group could meet their proposed timeline goals.
The applicant and research team have considerable expertise with MEMS platforms and are well qualified to handle the engineering aspects of the proposal. The commitment of time from the research team is adequate. Reviewers were concerned that the principal investigator lacked sufficient experience and knowledge of the relevant biological issues. Although this deficiency could be addressed via collaboration with a group noted for its expertise in stem cell biology, there was concern about whether this collaborative arrangement was appropriately organized.
In summary, the applicant proposes to resolve a bottleneck in sorting desired stem cells for cell-based therapies. Although the idea is noteworthy, significant issues exist in the team’s ability to meet proposed milestones, to screen cells using a single biomarker, and to demonstrate robust preliminary data.