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RS1-00211-1: The Use of Microfluidic Chambers and Microtechnology to Study hESC-derived Neural Cells

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Human embryonic stem cells (hESCs) have the potential to revolutionize medical therapeutics by providing transplantable cells for future treatments of a variety of disorders, including diabetes, heart disease, and degenerative and traumatic nerve diseases, such as Multiple Sclerosis, Parkinson’s Disease, and spinal cord injury. It is imperative to determine which hESC lines are superior candidates for use in these treatments, prior to use in humans, since it is well accepted that there are subtle differences between the currently available cell lines. It is likely that these rather subjectively observed differences between commonly used cell lines will translate into variably successful cell-based therapies unless these differences are taken into account early in the course of stem cell research.

The goal of this research is to utilize microtechnology to objectively compare the function and health of differentiated cells derived from various hESC lines so that optimal choices of stem cells can be determined for cell-based therapies of neurologic diseases. It is our hypothesis that neurons derived from different stem cell lines will demonstrate subtle differences in their physiology when compared side by side with the use of microtechnology tools such as microfluidic chips. These chips use tiny grooves to isolate the neuron’s cell body from their axons that will grow across the grooves into a separate chamber for study.

These chips will be attached to arrays of microelectrodes and then used to isolate axons for electrical measurements. Once the axons grow across the grooved barrier and the multielectrode array into the isolation chamber, specific parameters will be recorded to determine the healthiest and most functional cell lines. In addition, the axon shape and appearance will be analyzed by optical and fluorescent microscopy.

We anticipate that the results will show that stem cell lines are not interchangeable for different purposes and that they can be objectively evaluated using this microfludic platform. This type of quality control is essential. The effects of variable agents that these transplanted cells might encounter in the body can also be evaluated, such as immune system factors and pharmacologic compounds. Additionally, the design of the microfluidic chip can be altered in the future to best accommodate and test different cell types from other organ systems.

Statement of Benefit to California (provided by applicant)

California led the nation in acknowledging the potential benefit of using human embryonic stem cells (hESCs) for medical research. More significantly, they committed the resources to explore these benefits by establishing CIRM. The research proposed in this application will use microtechnology tools to evaluate the quality of different hESC lines as a source for transplantable human neural cells. This type of quality assurance is essential prior to use of hESC cell-based therapies in human subjects. We are hopeful that this research will show that technology can provide the tools to adequately evaluate hESC lines, while minimizing the sacrifice of experimental animals for this purpose.

This chip can also be used to evaluate the effects of variable agents that these transplanted cells might encounter in vivo, such as cytokines and other inflammatory factors, and pharmacologic compounds. In addition, based on the small scale of these test chambers, massively parallel, automated, pre-programmed studies can be carried out allowing systematic trial of thousands of combinations of conditions at minimal financial expense for high throughput screening of the hESC progeny. This could lead to industrial development of this microtechnology for the study of hESCs. Finally, the design of the microfluidic chip can be altered to best accommodate different cell types (islet cells, cardiac muscle, etc.) for optimal testing of progeny for diseases of other organ systems.

We feel that applying this “cutting edge” microfluidic and MEA technology to the “cutting edge” biology of hESCs is very innovative. Although it might be considered risky by some, the results could help to change how cell-based therapies are developed and tested, and optimize the chances of success for this application of hESCs. We therefore believe that this research is well aligned with the goals of the CIRM Seed Grant Research Program, and proving the utility of this tool will be of great benefit to the State of California and its citizens.

Review

SYNOPSIS: The applicants will use microfluidic chips to assess neurons produced from hESC to see if there are subtle differences of the neurons produced by each stem cell line. These chips contain microgrooves that allow separate recordings from different parts of neurons grown on their surface. In addition to recording action potentials from the axons, they will examine them morphologically by optical fluorescence microscopy. They will redesign the chips to test hESC progeny intended for diseases of other organ systems. Both eligible and ineligible hESC lines will be studied.

SIGNIFICANCE AND INNOVATION: To the referee’s knowledge, this is the first proposal to assess the neurophysiology of hESC line-derived neurons in a system that is capable of high-throughput screening using microfluidics. Thus, this is quite novel and original.

This is a strong proposal merging two technologies to establish methods for quantitatively determining the clinical potential of different hESC lines. The innovation is a combination of (1) the idea of quantifying endpoint phenotype as a way of comparing the upstream starting cell populations, (2) bringing rigor to the evaluation of different stem cell lines, and (3) combining two relatively straightforward technologies in a justified manner.

STRENGTHS: Numerous strengths of this proposal were cited by the reviewers. This project brings together numerous collaborators with experience including one who has the clean room and device fabrication facilities, one who is president of an institute which includes the microfluidic foundry, one who is experienced and maintaining and differentiating hESC, one who will be providing equipment and expertise for the electrophysiology experiments, and one who will provide access to the stem cell lines and expertise in culturing the cells. In addition, the background of the Principal Investigator (PI) in biomedical engineering is relevant.

The proposal describes a novel approach to characterizing cells grown from hESC lines and the reviewers believe that it is feasible to grow the cells on microfluidic chips and to record from them.

Preliminary results showing succesful differentiation in the microfluidic device is considered a strength, and the experimental plan is well thought out and appropriate, with care taken to anticipate possible complications

WEAKNESSES: There are several weaknesses to this application. The applicant exhibits low productivity having few publications (one publication from his/her dissertation and two reviews on microfluidics that are in press), despite now working full-time as a researcher. Another weakness is the inexperience of the PI with electrophysiology of neurons. This shows up clearly in the lack of discussion of this subject and the criteria that will be used to evaluate the cells grown on the microfluidic chips. The applicant’s primary concern was growth of the cells on the chips but the pitfalls of extracellular field potential recordings were not considered, nor was the use of powerful pharmacological tools available to assess sodium and potassium channels. Additional useful tools would include patch-clamping of the neurons, sucrose gap recordings, and current source-sink analyses to obtain data for comparison with the microfluidic chip recorded data. There is no use of pharmacological means to assess the neurophysiology of the cells. An experienced neurophysiologist would be proposing blockade of sodium and potassium channels, stimulated versus spontaneous activity, threshold, activation rate, current sink and source analyses, use of drugs to assess presence of neurotransmitter channels, documentation of the regeneration rates of the neurons, and other approaches to assess the neurons.

Culturing of mixed populations of cells on the microfluidic chips will present significant challenges that the applicant seems not to know. For example, not all the cells will be excitable and there may be myelination, multicellular clusters of axons and other cellular processes, and other complications that will dramatically alter the neurophysiology of axonal conduction in the chips. There is no consideration of how the applicant will characterize the types of cells that will be growing on the chips, the statistical analyses of the types of cells and culture conditions, and how the neurophysiological characteristics of the cells will be correlated with the morphology of the cells and axons. The PI does not provide details on how exactly he/she will evalute the different cell lines. Beyond generally looking at morphology, electrical activity, and protein labeling, what specific features of those assays will he/she use to ascertain function, how will he/she quantify those, and how does one go from those outputs to the notion that one hESC line is more suitable than another. If one cell line creates non-electrically excitable “neurons”, then the assessment is easy. However, what if all 5 cell lines give neurons that stain for the appropriate proteins, launch action potentials, etc. What factors does one use, then, to distinguish? More fundamentally, if one cell line is deemed suboptimal, is that instrinsic to the cell line, or are the differentiation conditions not tailored for that cell line. Could it be that with the proper differentiation conditions, a poorly performing cell line would end up being the best? In other words, perhaps the differentiation/culture conditions are to blame, not the cells. While these issues deserve extensive thought, this platform could be used to address them, speaking to its strengths.

In sum, this is an interesting proposal from an investigator who has published relatively little on human stem cells and neurophysiological investigations. The applicant needs to consider other major pitfalls including the presence of multiple cell types and multicellular recordings. Finally, the culture and recording conditions must be standardized.

DISCUSSION: There was general concern over this proposal based on the lack of an underlying hypothesis. What will we learn from the electrophysiological readings? Cells will likely have different electrophysiological characters, but how will those effects be related to differentiation? One reviewer was quite enthusiastic about the proposal, and found the screen based on endpoint phenotypes to be exciting and the focus on growth using microfluidics to be a strength. In addition, it was pointed out that a major career change may be the reason the PI has very few publications. Applying microfluidics to growth and differentiation of hESC is thought to be challenging, but the applicant’s approach is thought to be good. A reviewer was concerned that a major weakness is the lack of clarity about how to derive quantitiative analysis of different lines but noted that if it were possible, the information could be of use. It was pointed out that these studies can and should be done using the mouse system.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None