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:
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.
SYNOPSIS: The applicants will use microfluidic chips to assess neurons produced from human embryonic stem cells 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 human embryonic stem cell progeny intended for diseases of other organ systems. Both eligible (ESI, Wicell) and ineligible (Harvard, Monash) 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: • Experienced collaborators: Armand Tanquay (USC) who has the clean room and device fabrication facilities, Axel Scherer (Caltech) who is President of the Kavil Institute which includes the microfluidic foundry, Leslie Weiner (USC) who is experienced and maintaining and differentiating hESC, Theodore Berger (USC) who will be providing equipment and expertise for the electrophysiology experiments, and Martin Pera (USC) who will provide access to the stem cell lines and expertise in culturing the cells. • Novel approach to characterizing cells grown from hESC liines. • Feasible to grow the cells on microfluidic chips and to record from them. • Background of the investigator in biomedical engineering (a PhD and dissertation in acoustic apnea monitoring) • Preliminary results showing succesful differentiation in the microfluidic device • The experimental plan is well thought out and appropriate, with care taken to anticipate possible complications WEAKNESSES: • Low Productivity of the applicant. The principal investigator has very few publications (one from her dissertation in 1987 and two reviews on microfluidics that are in press), despite now working full-time since 2003 as a research associate in the Computer Science Department (2003-2005) and in the Neurology Department (2005-2006) of USC. • Inexperience 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, use of powerful pharmacological tools available to assess sodium and potassium channels, neurotransmitter receptor presence, etc. • Lack of consideration of other powerful techniques, such as 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 neurophysiologists would be proposing blockade of sodium (TTX) and potassium channels (4-AP), 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 assessing the neurons. • Culturing of mixed populations of cells on the microfluidic chips will present significant challenges that the applicant seems to be unaware of. 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 she will evalute the different cell lines. Beyond generally looking at morphology, electrical activity, and protein labeling, what specific features of those assays will she use to ascertain function, how will 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 assesment 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. This is an interesting proposal from an investigator who has published relatively little on human stem cells and neurophysiological investigations. Although the applicant will be supported by excellent collaborators, the application does not discuss several important challenges of the proposed technology. These include standardization of the culture and recording conditions, use of electrical and pharmacological tools, and statistical analyses of morphological and physiological data. The applicant needs to consider other powerful techniques such as patch clamping, sucrose gap, current source-sink analyses (i.e. Lorente de No), and other approaches to obtaining data to validate data collected from the microfluidic chips. 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 effect/be related to differentiation? Reviewer 2 was more 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, Reviewer 2 pointed out that a major career change may be the reason the PI has very few publications. A discussant remained concern about the track record of the PI. A reviewer noted that applying microfluidics to growth and differentiation of hESC is challenging but thinks applicant's approach is good. Reviewer 2 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. Reviewer 1 responded that these studies can and should be done using the mouse system.