Cardioinductive Network Guiding Stem Cell Differentiation Revealed by Proteomic Cartography of Tumor Necrosis Factor α-Primed Endodermal Secretome

Abstract

In the developing embryo, instructive guidance from the ventral endoderm secures cardiac program induction within the anterolateral mesoderm. Endoderm-guided cardiogenesis, however, has yet to be resolved at the proteome level. Here, through cardiopoietic priming of the endoderm with the reprogramming cytokine tumor necrosis factor ␣ (TNF␣), candidate effectors of embryonic stem cell cardiac differentiation were delineated by comparative proteomics. Differential two-dimensional gel electrophoretic mapping revealed that more than 75% of protein species increased >1.5-fold in the TNF␣-primed versus unprimed endodermal secretome. Protein spot identification by linear ion trap quadrupole (LTQ) tandem mass spectrometry (MS/MS) and validation by shotgun LTQ-Fourier transform MS/MS following multidimensional chromatography mapped 99 unique proteins from 153 spot assignments. A definitive set of 48 secretome proteins was deduced by iterative bioinformatic screening using algorithms for detection of canonical and noncanonical indices of secretion. Protein-protein interaction analysis, in conjunction with respective expression level changes, revealed a nonstochastic TNF␣-centric secretome network with a scale-free hierarchical architecture. Cardiovascular development was the primary developmental function of the resolved TNF␣-anchored network. Functional cooperativity of the derived cardioinductive network was validated through direct application of the TNF␣-primed secretome on embryonic stem cells, potentiating cardiac commitment and sarcomerogenesis. Conversely, inhibition of primary network hubs negated the procardiogenic effects of TNF␣ priming. Thus, proteomic cartography establishes a systems biology framework for the endodermal secretome network guiding stem cell cardiopoiesis. STEM CELLS 2008;26:387-400 Disclosure of potential conflicts of interest is found at the end of this article.

Enhancing the Outcome of Cell Therapy for Cardiac Repair

“…[131][132][133] Because endoderm-derived signals are of crucial importance in cardiac development in vivo and drive cardiogenesis of ESCs in vitro, other investigators studied the endodermal secretome to identify factors guiding cardiac differentiation. 134 Based on the findings that secreted factors derived from tumor necrosis factor-␣-primed endoderm enhance cardiac commitment, an ongoing clinical study is addressing whether adult MSCs cultured with endoderm-derived factors improve cardiac function in patients with heart failure (C-Cure Trial, www.clinicaltrials.gov, NCT00810238).…”

Genetic Enhancement of Stem Cell Engraftment, Survival, and Efficacy

“…94 -96 These studies have suggested potential pathways that could be activated in adult stem cells in an attempt to have them take on a cardiac phenotype. 94,96,97 Perhaps the most studied strategy to date with adult stem cells is the effect of 5-azacytidine, a DNA demethylation reagent, on cardiac protein expression in mesenchymal stem cells. 98,99 Several studies have demonstrated an increase in cardiac protein expression after treatment of mesenchymal stem cells with 5-azacytidine, and some have observed spontaneously beating cells.…”

Substrate-Guided Proteomics Enhances Degradome Resolution

“…This will necessitate a cyclic return from molecular specificity to systematic physiological function, possibly with iterative incorporation of additional proteomic and other high-throughput data. As demonstrated by other extracellular proteomic 17 and combined metabolomic/proteomic 18 analyses, high-throughput identification and characterization of relationships between components of the extracellular domain can be critical in defining functional transitions between cellular states and cell fate decisions. Systems biology approaches offer an integrative approach by which to achieve data inclusivity and to maximize biological comprehension for disease diagnosis, determination of disease predilection, and assessment of therapeutic modalities, 3,5,19,20 and thus are ideally suited to addressing the biological complexity associated with future investigations into MMP isoform-dependent human ECM remodeling.…”