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.
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.
In the 2nd-year funding period, our joint team at UCLA devoted our research endeavors on the development of (i) the combined use of phenotype and pluripotent assays in conjunction with a bioinformatic approach, (ii) MIC-based signaling assay, (iii) performing large-scale screening using a small chemical library in search of chemical defined conditions (CDCs) for clonal expansion of hPSCs. Here, the three MIC-based phenotype, pluripotent and signaling assays were employed to monitor dynamic changes in the hPSCs. Currently, the two research papers that summarize the development of the MIC-based phenotype, pluripotent and signaling assays and their application on studying clonal expansion of hPSCs under CDCs are under preparation. In parallel with the originally proposed research activities, our research group has recently created a convenient, flexible and modular approach for preparing nanoscale vectors that exhibit superb transfection/transduction performance. We have been exploring the use of these vectors for replacing viral vectors that carry reprogramming factors or cell-penetrating peptide-fused reprogramming transcription factors to generate human induced pluripotent stem cells (hiPSCs). A paper summarize the development of DNA⊂SNPs was published in ACS Nano in 2010. We conclude our progress as the follows:
First, on the basis of the previously established robotic microfluidic platform, we have been working on establishing three type of stem cell assays capable of (i) detecting cell growth rates, viability and death, (ii) differentiating stem cells and the differentiated cells and (iii) monitoring signaling events that pay critical roles in controlling the cell fates during clonal expansion of hPSCs. In order to correlate the single-cell MIC data obtained from these stem cell measurements, we adapt bioinformatic methods to stratify different hPSC lines in the presence of different culture conditions into early-to-read indexes/charts. We found that these indexes/charts correlate with the pluripotent/differentiation status of these hPSCs.
Second, we optimized cell handling approaches and parameters using two cancer cell lines (U87, brain tumor line and M229 melanoma line) that exhibit stable signaling events in order to show reproducible quantification two signaling molecules (i.e., pAKT and pERK). We then established the dynamic ranges of MIC measurement for pAKT and pERK using the same cell lines w/wo their respective inhibiters. Subsequently, the optimized approaches and parameters was employed for measuring a smaller collection of hPSC lines (H1, HSF6, iPS2, iPSA1 and iPSB2) cultured in different cell culture conditions. We have also applied the bioinformatic methods to stratify different hPSC lines under different culture conditions into clusters.
Third, we conducted screening on a small molecule library (composed of ca. 500 compounds) using the robotic microfluidic platform in order to identify a number of small molecules that can rescue dissociated hESCs in chemical defined media. Among the 500 molecules studies, we could NOT discover any molecules, which exhibit significant improved performance to rescue dissociated hESCs from apoptosis. Further, a smaller collection of hPSC lines (H1, HSF6, iPS2, and iPSA1) was cultured in the chemically defined culture media with HA1077 and/or Y-27632. The MIC-based phenotype/signaling assays were performed to obtain the single-cell molecular signatures for HA1077 and Y-27632. Together with cell survival rates, the phenotypic signatures provide an extensive assessment of the hit culture conditions, covering pluripotency, apoptosis, proliferation and differentiation of the chip-cultured hPSCs.
Forth, our research group has recently created a convenient, flexible and modular approach for preparing transcription factors (TFs)-encapsulated supramolecular nanoparticles (TFs⊂SNPs) that exhibit superb transfection/transduction performance and low toxicity, compared to the conventional artificial transaction reagents. Our joint research team is exploring the use of SNP vectors for highly efficient delivery of the four reprogramming transcription factors (i.e., OCT4, SOX2, KLF4 and c-MYC) in their intact forms in order to generate hiPSCs. Reprogramming process in the TFs⊂SNPs-treated cells will be studied using the MIC-based phenotype, puripotent and signaling assays for monitoring (i) pluripotency/differentiation status (OCT4, NANOG, TRA-1-60 and SSEA1), and (ii) a phenotype assay for parallel detection of Hoechst (cell cycle), pHistone H3 (M-Phase), EdU (S-Phase) and Caspase-3/-7 (apoptosis) over the reprogramming process, respectively.