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.
• 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
• 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.