A pinboard by
Matthew Waas

Graduate Student, Medical College of Wisconsin


Discovery and validation of a cell surface marker for human stem cell-derived cardiomyocytes

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) are invaluable for the study of human development, disease modeling, drug testing, and regenerative medicine. However, the use of hPSC-CM for these applications is currently limited by our inability to identify and select homogeneous populations of functionally defined hPSC-CM from heterogeneous cultures. Though immunophenotyping may overcome these current challenges, there remains a paucity of suitable cell surface markers that can be used as cell type specific markers for hPSC-CM. To address this need, Cell Surface Capture (CSC) Technology, a chemoproteomic approach for identifying extracellular domains of cell surface proteins, was applied to hPSC-CM. Applying the CSC to 19 time points across the first 100 days of hPSC-CM differentiation has identified >1300 cell surface proteins. From these data, we have selected one previously undocumented cell surface transmembrane protein that is unique to hPSC-CM, NCSP. Targeted quantitation by MS has confirmed the presence of this protein emerges during in vitro differentiation once the cells have committed to the CM fate and it is present in human heart tissue. Novel monoclonal antibodies have been developed to this protein and antibody specificity has been validated at the epitope and cell type level and efforts to validate at the protein-level are ongoing. The function of the protein is being studied through use of a CRISPR/Cas9 generated KO and functional analyses of cells sorted by this marker are ongoing. As phylogenetic analyses of this novel protein reveals high sequence conservation and that it emerged in species with functionally four-chambered hearts, we expect that NCSP will be relevant to hPSC-CM function and will be useful as a marker of cardiomyocyte identity.


Cardiomyocyte Differentiation Promotes Cell Survival During Nicotinamide Phosphoribosyltransferase Inhibition Through Increased Maintenance of Cellular Energy Stores.

Abstract: To address concerns regarding the tumorigenic potential of undifferentiated human pluripotent stem cells (hPSC) that may remain after in vitro differentiation and ultimately limit the broad use of hPSC-derivatives for therapeutics, we recently described a method to selectively eliminate tumorigenic hPSC from their progeny by inhibiting nicotinamide phosphoribosyltransferase (NAMPT). Limited exposure to NAMPT inhibitors selectively removes hPSC from hPSC-derived cardiomyocytes (hPSC-CM) and spares a wide range of differentiated cell types; yet, it remains unclear when and how cells acquire resistance to NAMPT inhibition during differentiation. In this study, we examined the effects of NAMPT inhibition among multiple time points of cardiomyocyte differentiation. Overall, these studies show that in vitro cardiomyogenic commitment and continued culturing provides resistance to NAMPT inhibition and cell survival is associated with the ability to maintain cellular ATP pools despite depletion of NAD levels. Unlike cells at earlier stages of differentiation, day 28 hPSC-CM can survive longer periods of NAMPT inhibition and maintain ATP generation by glycolysis and/or mitochondrial respiration. This is distinct from terminally differentiated fibroblasts, which maintain mitochondrial respiration during NAMPT inhibition. Overall, these results provide new mechanistic insight into how regulation of cellular NAD and energy pools change with hPSC-CM differentiation and further inform how NAMPT inhibition strategies could be implemented within the context of cardiomyocyte differentiation. © Stem Cells Translational Medicine 2017.

Pub.: 23 Feb '17, Pinned: 19 Jun '17

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

Abstract: 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.

Pub.: 08 Jan '14, Pinned: 19 Jun '17