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

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Medical College of Wisconsin

We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome c and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding - (1) the promiscuous role of cytochrome b(5) in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.

Redox active molecules cytochrome c and vitamin C enhance heme-enzyme peroxidations by serving as non-specific agents for redox relay.

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.

A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells.

Mass spectrometry (MS) based proteomic technologies enable the identification and quantification of membrane proteins as well as their post-translational modifications. A prerequisite for their quantitative and reliable MS-based bottom-up analysis is the efficient digestion into peptides by proteases, though digestion of membrane proteins is typically challenging due to their inherent properties such as hydrophobicity. Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach. Multiple parameters, including the number of peptides and proteins identified, total protein sequence coverage, and digestion specificity were used to evaluate transmembrane protein digestion performance. A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage. Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.

Combine and conquer: surfactants, solvents, and chaotropes for robust mass spectrometry based analyses of membrane proteins.

Detailed knowledge of cell surface proteins present during early embryonic development remains limited for most cell lineages. Due to the relevance of cell surface proteins in their functional roles controlling cell signaling and their utility as accessible, nongenetic markers for cell identification and sorting, the goal of this study was to provide new information regarding the cell surface proteins present during early mouse embryonic development.

N-glycoprotein surfaceomes of four developmentally distinct mouse cell types.

Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.

Subarna Bhattacharya

Department of Biochemistry

Medical College of Wisconsin

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