Human pluripotent stem cells (hPSCs) hold a great potential to treat 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 is an idea operation platform. Microfluidics handles and manipulates 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.
Over the 1st-year funding period, our joint team at UCLA has successfully developed (i) a robotic microfluidic platform for robust hPSC culture in a high-throughput and automated fashion, and (ii) an associated microfluidic image cytometry (MIC) approach capable of and phenotypic analysis of hPSCs. Two papers that summarize the development of the microfluidic platforms for reproducible culture and analysis of hPSCs have been published in Lab on a Chip in 2009 and 2010. Besides, a manuscript that concludes how our MIC approach can be applied to distinguish pluripotent and differentiated phenotypes of the chip-cultured hPSCs is currently under preparation. Our research team has started to perform large-scale screening of chemically defined culture conditions that allow hPSC clonal expansion using a small chemical library provided by the UCLA Molecular Screening Shared Resource. We conclude our progress as the follows:
First, we developed a robotic microfluidic platform to expand the capability of our experiments with a high-throughput fashion. Since the sizes of these hPSC culture chambers are very small, the consumptions of hPSC samples and the associated reagents will be significantly reduced (3 to 4 orders of magnitude lower than the conventional setting). By using this robotic pipette system, critical parameters for hPSC experiments can be monitored and controlled in the hPSC-Chips with superior precision, which is unattainable using the conventional culture setting. For the 2nd-year funding period, we will be able to perform thousands screens/day.
Second, we optimized the chemically defined culture conditions in hPSC-Chips. To study the key mechanisms of hPSC fate decisions, chemically defined hPSC culture conditions are critical in order to evaluate the influence of the extrinsic factors. By screening a collection of media and substrates, we were able to identify the optimal conditions that enable hPSCs to maintain their stemness in the hPSC-Chips for a week.
Third, in order to characterize hPSCs, we developed quantitative phenotypic assays for parallel detections of phenotypic readouts using the microfluidic image cytometry (MIC) technology developed by our research laboratory. The MIC technology is a combination of microfluidic technology and microscopic-image-based cell analysis and capable of quantitative single-cell phenotypic profiling of hPSCs. In proof-of-concept studies, we assessed various phenotypic characteristics across different hPSC lines in several chemically defined hPSC culture conditions. Using biostatistical analysis, we were able to systematically compare the characteristics of various hPSC lines in these conditions.
In parallel with the originally proposed research activities, our research group has recently developed a convenient, flexible and modular approach for preparing plasmid DNA-encapsulated supramolecular nanoparticles (DNA-SNPs) that exhibit super transfection performance and low toxicity compared to the conventional artificial transaction reagents. We envision SNPs could serve as a new type of gene delivery reagents to replace viral vectors (e.g., retrovirus and lentivirus) that carry reprogramming factors for reprogramming of somatic cells. We would like to ask for a permission to broaden the scope our research project, so that we can test a feasibility to apply reprogramming factors-encapsulated SNPs for generation of human induced pluripotent stem cells (hiPSCs). We will also utilize the above-mentioned MIC technology for continuously monitoring of the reprogramming process of the DNA-SNPs-treated somatic cells. The resulting single-cell phenotypic signatures will provide feedback for optimization of the structure and functional properties of DNA-SNPs to generate hiPSCs. Developing non-viral transfection reagents for the reprogramming of cellular samples is one of the most crucial topics in the field of stem cell biology. Our research team would like to contribute to the research community by leveraging and integrating the power of the two technologies platforms (i.e., microfluidic image cytometry (MIC) platform and supramolecular nanoparticles (SNPs)-based gene delivery system) developed in our research laboratory.