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.