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Kshitiz,University of Washington | Afzal J.,Institute of Cell Engineering | Kim D.-H.,University of Washington | Levchenko A.,Yale University
Stem Cells | Year: 2014

Mechanical cues can have pleiotropic influence on stem cell shape, proliferation, differentiation, and morphogenesis, and are increasingly realized to play an instructive role in regeneration and maintenance of tissue structure and functions. To explore the putative effects of mechanical cues in regeneration of the cardiac tissue, we investigated therapeutically important cardiosphere-derived cells (CDCs), a heterogeneous patient- or animal-specific cell population containing c-Kit+ multipotent stem cells. We showed that mechanical cues can instruct c-Kit+ cell differentiation along two lineages with corresponding morphogenic changes, while also serving to amplify the initial c-Kit+ subpopulation. In particular, mechanical cues mimicking the structure of myocardial extracellular matrix specify cardiomyogenic fate, while cues mimicking myocardium rigidity specify endothelial fates. Furthermore, we found that these cues dynamically regulate the same molecular species, p190RhoGAP, which then acts through both RhoA-dependent and independent mechanisms. Thus, differential regulation of p190RhoGAP molecule by either mechanical inputs or genetic manipulation can determine lineage type specification. Since human CDCs are already in phase II clinical trials, the potential therapeutic use of mechanical or genetic manipulation of the cell fate could enhance effectiveness of these progenitor cells in cardiac repair, and shed new light on differentiation mechanisms in cardiac and other tissues. © 2014 AlphaMed Press.

Agus D.B.,University of Southern California | Alexander J.F.,Methodist Hospital Research Institute | Arap W.,University of Texas M. D. Anderson Cancer Center | Ashili S.,Arizona State University | And 99 more authors.
Scientific Reports | Year: 2013

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

Newman R.H.,North Carolina A&T State University | Rho H.-S.,Center for High throughput Biology | Woodard C.,Center for High throughput Biology | Neiswinger J.,Center for High throughput Biology | And 24 more authors.
Molecular Systems Biology | Year: 2013

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans. © 2013 EMBO and Macmillan Publishers Limited.

Kshitiz,The Johns Hopkins Medical Institutions | Kshitiz,Institute of Cell Engineering | Kim D.-H.,The Johns Hopkins Medical Institutions | Kim D.-H.,Institute of Cell Engineering | And 4 more authors.
Trends in Biotechnology | Year: 2011

Current techniques used in stem cell research only crudely mimic the physiological complexity of the stem cell niches. Recent advances in the field of micro- and nanoengineering have brought an array of in vitro cell culture models that have enabled development of novel, highly precise and standardized tools that capture physiological details in a single platform, with greater control, consistency, and throughput. In this review, we describe the micro- and nanotechnology-driven modern toolkit for stem cell biologists to design novel experiments in more physiological microenvironments with increased precision and standardization, and caution them against potential challenges that the modern technologies might present. © 2011 Elsevier Ltd.

Perica K.,Institute of Cell Engineering | Kosmides A.K.,Institute of Cell Engineering | Schneck J.P.,Institute of Cell Engineering
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2015

Artificial antigen presenting cells (aAPCs) are engineered platforms for T cell activation and expansion, synthesized by coupling T cell activating proteins to the surface of cell lines or biocompatible particles. They can serve both as model systems to study the basic aspects of T cell signaling and translationally as novel approaches for either active or adoptive immunotherapy. Historically, these reductionist systems have not been designed to mimic the temporally and spatially complex interactions observed during endogenous T cell-APC contact, which include receptor organization at both micro- and nanoscales and dynamic changes in cell and membrane morphologies. Here, we review how particle size and shape, as well as heterogenous distribution of T cell activating proteins on the particle surface, are critical aspects of aAPC design. In doing so, we demonstrate how insights derived from endogenous T cell activation can be applied to optimize aAPC, and in turn how aAPC platforms can be used to better understand endogenous T cell stimulation. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling. © 2014 Elsevier B.V.

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