High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

published_or_final_versio

Organic wastes management in Reunion Island: Bio-methane tests and results

International audienc

A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N -glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.link_to_subscribed_fulltex

Metabolic rates, growth phase, and mRNA levels influence cell-specific antibody production levels from in vitro-cultured mammalian cells at sub-physiological temperatures

Previous work has shown that recombinant protein yield can be improved from in vitro-cultured mammalian cells by culturing at sub-physiological temperatures, although this effect is cell line and product dependent. The mechanism(s) by which low temperature leads to enhanced product yield are currently unknown; however, recent reports suggest that increased mRNA levels at sub-physiological temperatures may be largely responsible for this. Here, we have investigated whether low-temperature cultivation of cell lines selected for antibody production at 37C leads to changes in heavy- and light-chain mRNA levels and if this is reflected in antibody yields. Low-temperature in vitro culturing resulted in reduced viable cell concentration, prolonged cell viability, a reduction in metabolite consumption and production, cell cycle arrest in both CHO and NS0 cells, and changes in the levels of heavy- and light-chain mRNA. Despite increases in the level of heavy- and light-chain mRNA upon culturing at 32C in our model CHO cell line, this did not result in increased total product yield; however, changes in cell-specific yields were observed that reflected the metabolic rate of glucose utilization and changes in mRNA levels