Human pluripotent stem cells (hPSCs) hold a great potential to treat many devastating injuries and diseases. However, current hPSC cloning still faces challenges in creating animal product-free culture conditions for performing genetic manipulation and induced differentiation of hPSCs for cell-based therapy. In order to obtain the ideal culture conditions for hPSC cloning, microfluidic technology can be applied as a powerful tool. Microfluidic systems handle and manipulate tiny amounts of fluids at volumes a thousand times smaller than a tear drop. The goals of our proposal are to develop and validate a robotic microfluidic platform, composed of a robotic liquid dispensing system, a fluorescence microscope, cell culture chips and an operation interface. We will apply such a robotic platform to (i) perform chemical screening in search of culture conditions and small molecules that facilitate single-cell expansion of hPSCs and (ii) achieve a better understanding on how chip-based culture environments and the molecules identified in the screens affect the hPSC fate. Compared to the macroscopic setting employed for the conventional hPSC research, the advantages of the robotic microfluidic technology are sample/reagent economy, precise fluidic delivery, scalability and automated operation.
As the proposed project unfolds, we anticipate making contributions in the following four areas.
First, the successful demonstration of the microfluidic platform will provide a powerful technology for contemporary hPSC research. Since the size of each cell culture chamber is very small, the consumption of hPSC samples and the associated reagents will be significantly reduced (2 to 3-orders lower than the conventional setting).
Second, two quantitative assays will be developed: (i) a phenotype assay for parallel detection of pluripotency, apoptosis, proliferation and differentiation, and (ii) a cell signaling assay for parallel monitoring of four signaling nodes, which are potential targets of the molecules identified in the screens. Since the regulation of hPSC survival is not well understood, the resulting phenotypic/signaling signatures can help to identify which cell signaling events and surrounding environments are responsible for hPSC fate. Most importantly, the improved understanding of the mechanisms regulating hPSC survival can help to guide the optimization of hPSC culture conditions.
Third, this microfluidic platform promises to improve the screening process in search of culture conditions for hPSC self renewal and differentiation, as well as identify small molecules that facilitate single-hPSC expansion.
Fourth, we will test this microfluidic technology for everyday use in other hPSC laboratories. A user friendly operation interface will be developed and then tested in the other hPSC groups. The final versions of the chip design and control programs will be freely available for download from the PI’s research group web site.
As the proposed project unfolds, we hope to benefit the state of California and its citizens on seven fronts:
Human pluripotent stem cells (hPSCs) hold a great potential for regenerative medicine to treat many devastating injuries and diseases, such as Alzheimer’s disease, Parkinson’s disease, diabetes, Lou Gehrig’s disease, cancer, cardiovascular disease, rheumatoid arthritis and spinal cord injuries.
The proposed robotic microfluidic platform allows us to create animal product-free culture conditions for performing genetic manipulation and induced differentiation of clonal hPSCs. In addition, animal product-free culture conditions will eliminate immuno-rejection challenges for regenerative medicine.
The successful demonstration of the proposed new-generation microfluidic platform, composed of a robotic liquid dispensing system and a fluorescence microscope, will present a new technology for far-reaching application to all types of stem cell research with significantly improved operation efficiency. In contrast, conventional stem cell research is plagued by the use of macroscopic setting, resulting in several constraints: high sample/reagent consumption, poor precision to control the environments of hPSC experiments and the lack of integrated platforms for accurate measurements.
Using the proposed microfluidic platform, the costs for hPSC research will be significantly reduced. We estimate that each hPSC experiment in the microfluidic cell array chip consumes 0.9 microliters of cell culture reagents/media, which are 2 to 3 orders of magnitude lower than commonly used 384-well plates (requiring 100 microliters of reagents/media for a single study). Therefore, many hPSC studies that require large-scale hPSC experiments can utilize the proposed microfluidic platform in a cost-efficient manner.
The proposed robotic microfluidic platform allows greater precision of measurements. By using a robotic pipette for dispensing hPSC samples and screening solutions, critical parameters for hPSC experiments can be monitored and controlled with superior precision, which is unattainable in conventional macroscopic conditions.
The robotic microfluidic platform promises to understand the mechanisms behind hPSC survival and to speed up the drug screening process in search of chemically defined conditions for hPSC clonal expansion. In our design, the microfluidic cell array chip will contain a 4 x 10 cell array. The average throughput is 1600 to 8000 screens/day.
User-friendly interfaces of the proposed technology will be created for everyday use in other hPSC laboratories. The final versions of the chip design and control programs will be freely available for download from the PI’s research group web site.
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.