Our video protocol has been published in JoVE online.

This experiment was based on our publication published in Small, 2017.

Please take a look for the video to carry out our experiments!

ABSTRACT

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

Our collaborative achievement with Prof. Inoue-Murayama and Mr. Endo (WRC, Kyoto Univ.) got published in Journal of Evolutionary Biology. “Publications” page in our web was also updated accordingly.

Title: Genetic signatures of lipid metabolism evolution in Cetacea since the divergence from terrestrial ancestor

Abstract: In mammalian evolutionary history, Cetacea (whales, dolphins, and porpoises) achieved astonishing success by adapting to an aquatic environment. One unique characteristic of cetaceans, contributing to this adaptive success, is efficient lipid utilization. Here we report a comparative genetic analysis of five aquatic and five terrestrial Cetartiodactyla species using 144 genes associated with lipid metabolism. Mutation ratio (d_N/d_S), amino acid substitution in functional domains, and metabolic pathways were evaluated using branch‐site model in PAML, Pfam, and KEGG, respectively. Our tests detected 20 positively selected genes in Cetacea compared to 11 in Bovidae with little overlap between the lineages. We identified lineage‐specific patterns of amino acid substitutions and functional domains that were mutually exclusive between cetaceans and bovids, supporting divergent evolution of lipid metabolism since the divergence of these taxa from a common ancestor. Moreover, a pathway analysis showed that the identified genes in cetaceans were associated with lipid digestion, lipid storage, and energy producing pathways. This study emphasizes the evolutionary context of lipid metabolism modification of cetaceans and provides a foundation for future studies of elucidating the adapted biological mechanisms of cetacean lipid metabolism and a framework for incorporating ecological context into studies aimed at investigating adaptive evolution.

DOI: 10.1111/jeb.13361