Optimizing stem cell differentiation platforms
As stem cells move from the research laboratory toward commercial and clinical applications, the ability to control stem cell fates in an efficient and scalable manner becomes paramount. Stem cell differentiation protocols strive to transition cells through several developmental stages in vitro by applying chemical or physical cues at the appropriate temporal phase. Selekman and colleagues provide an integrated experimental and computational approach for analyzing cell fate transitions during stem cell differentiation. Employing kinetic modeling and flow cytometric analysis of population kinetics, they determined that the rate-limiting steps for differentiating human pluripotent stem cells to epithelial cells occur at different differentiation stages in two distinct protocols. They used the results of this analysis to improve epithelial cell yield by specifically targeting expansion of an epithelial progenitor predicted to be a bottleneck in the differentiation process. This study provides a framework for optimizing and scaling stem cell differentiation platforms.
Selekman et al., Biotechnol Bioeng 2013;110:3024–3037.
Emulsion PCR opens perspectives for the massive identification of paired TCR chains
The analysis of diversity and function of T-cell adaptive immunity has long been limited to the separate profiling of alpha and beta TCR repertoires. Turchaninova et al. demonstrate the potential for the high-throughput identification of paired TCR chains using emulsion RT-PCR technologies that start from living cells. Each T cell is isolated in a separate emulsion droplet – individual reaction volume – thus allowing for the multiple parallel reverse transcription, PCR amplification and overlap extension reactions resulting in a library of linked native TCR chain pairs. Post-emulsion amplification required to produce the library suitable for the deep paired-end sequencing analysis is protected from the non-specific pairing of chains by the new PCR-suppression technology that was developed to specifically block overlap-extension of and mega-priming by the unpaired molecules outside emulsion. The proposed approach opens perspectives for the massive paired analysis of TCR chains, being of great demand both in clinical and research settings.
Turchaninova et al., Eur J Immunol 2013;43:2507-2515.