Microfluidic Platform for Screening Chemically Defined Conditions that Facilitate Clonal Expansion of Human Pluripotent Stem Cells
Tools and Technologies I
Statement of Benefit to California:
The investigators propose to develop high-throughput microfluidic-based technology that allows for acquisition of high-content phenotypic information based upon fluorescent reporters and immunofluorescence assays to assess self-renewal, differentiation, and signaling. The ability to screen stem cells for improved self-renewal, clonal expansion, and directed differentiation is critical for developing the culture conditions needed to further our understanding of basic stem cell biology, to manipulate stem cells in vitro, and ultimately to apply them therapeutically. The applicants will screen various defined culture conditions for ability to support human pluripotent stem cell (hPSC) self-renewal, and then use the optimized culture conditions to screen two libraries of small molecules for improvements in hPSC clonal expansion. Finally, they propose methods to help disseminate the technology by improving manufacturing, user interface, and packaged assays. A major strength of this proposal is its potential impact. Reviewers were enthusiastic about the focus on the particular problem of clonal expansion, and this focus makes it likely the research will yield important novel information and have a high impact. Their system will have the ability to assay cells during culture through an integrated, on-board imaging system. Such a system would be very useful and find many important applications in the field. The goal of making this complex system more user-friendly was also applauded. The research plan has many strong features. The use of a diversity of cell lines, including unmodified human embryonic stem cells (hESCs), hESC reporter lines, and human induced pluripotent stem cell (hiPSC) lines should adequately address concerns regarding robustness of the system and reproducibility of the data. The logical progression through studies--to first determine reproducible culture conditions and then use those conditions to screen the libraries, is reasonable, and the investigators demonstrate understanding of the critical technological aspects that they need to address (e.g., substrate coating, comparisons of defined media, etc.). Reviewers pointed out that the screens, though described as high throughput, were based on fairly small libraries. Combining microfluidics with microscopy to phenotype the cells is also exciting, especially when combined with reporter cell lines, allowing ongoing analysis of live cells for endpoint analyses. Together, reviewers considered the combination of microfluidics and imaging and reporter cell lines to be quite powerful. Reviewers were concerned, however, that the full advantages of microfluidics were not exploited in the study design and noted that some of the studies, as described, could be more easily performed in simple multiwell plates. The advantages of microfluidics, to deliver specific time courses and interrogate multiplex conditions, were not captured in the design. For example, commercial multiwell incubated microscopy systems are available, and the applicants did not make a strong case for the superiority of their system. Reviewers were also concerned that the inherent complexities of the proposed experiments were not addressed, such as the challenges associated with five-color immunofluorescence and all the controls that would be necessary to verify cross-reactivity. (No information was provided on light source or means of wavelength separation, all important in evaluating this kind of imaging.) Other important issues were glossed over including measurement of total protein vs. phosphorylated protein, or the number of samples needed to, for instance, assay 9 endpoint markers across multiple time points and 6 cell lines. Some reviewers noted that the need for medium changes was not addressed in the proposal, or at least was unclear. Further, reviewers felt that the description of the work as ‘continuous fate mapping’ was misguided, as neither continuous (every 24 hours is not continuous) studies nor mapping of fate were proposed. The team generated considerable enthusiasm, the principal investigator provides a strong link between engineering and the life sciences, and the team as a whole has all the expertise needed to succeed with microfluidics, chemistry, imaging, screening and stem cell biology represented.