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1.
Sci Rep ; 6: 26960, 2016 06 06.
Article in English | MEDLINE | ID: mdl-27264636

ABSTRACT

Therapeutic delivery of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) represents a novel clinical approach to regenerate the injured myocardium. However, methods for robust and accurate in vivo monitoring of the iCMs are still lacking. Although superparamagnetic iron oxide nanoparticles (SPIOs) are recognized as a promising tool for in vivo tracking of stem cells using magnetic resonance imaging (MRI), their signal persists in the heart even weeks after the disappearance of the injected cells. This limitation highlights the inability of SPIOs to distinguish stem cell viability. In order to overcome this shortcoming, we demonstrate the use of a living contrast agent, magneto-endosymbionts (MEs) derived from magnetotactic bacteria for the labeling of iCMs. The ME-labeled iCMs were injected into the infarcted area of murine heart and probed by MRI and bioluminescence imaging (BLI). Our findings demonstrate that the MEs are robust and effective biological contrast agents to track iCMs in an in vivo murine model. We show that the MEs clear within one week of cell death whereas the SPIOs remain over 2 weeks after cell death. These findings will accelerate the clinical translation of in vivo MRI monitoring of transplanted stem cell at high spatial resolution and sensitivity.


Subject(s)
Contrast Media/administration & dosage , Heart/diagnostic imaging , Magnetite Nanoparticles/administration & dosage , Myocytes, Cardiac/physiology , Animals , Bacteria , Cell Tracking , Humans , Induced Pluripotent Stem Cells/physiology , Male , Mice, SCID , Myocardium/pathology , Myocytes, Cardiac/transplantation , Rats , Symbiosis
2.
Nat Med ; 19(10): 1331-1337, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24037094

ABSTRACT

Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Insulin Receptor Substrate Proteins/physiology , Insulin/metabolism , Liver/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Diabetes Mellitus, Type 2/therapy , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction
3.
PLoS One ; 7(2): e32191, 2012.
Article in English | MEDLINE | ID: mdl-22363814

ABSTRACT

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1α and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.


Subject(s)
Embryonic Stem Cells/drug effects , Embryonic Stem Cells/physiology , Insulin-Like Growth Factor II/pharmacology , Insulin-Like Growth Factor I/pharmacology , Neovascularization, Physiologic/drug effects , Animals , Carrier Proteins/metabolism , Cell Differentiation/drug effects , Embryoid Bodies/cytology , Embryoid Bodies/drug effects , Embryoid Bodies/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/enzymology , Endothelium/cytology , Endothelium/drug effects , Endothelium/embryology , Extracellular Signal-Regulated MAP Kinases/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mesoderm/cytology , Mesoderm/drug effects , Mesoderm/embryology , Mice , Models, Biological , Podophyllotoxin/analogs & derivatives , Podophyllotoxin/pharmacology , Protein Stability/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptor, IGF Type 1/antagonists & inhibitors , Receptor, IGF Type 1/metabolism , Receptor, IGF Type 2/antagonists & inhibitors , Receptor, IGF Type 2/metabolism , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/metabolism
4.
Stem Cells Dev ; 20(5): 749-58, 2011 May.
Article in English | MEDLINE | ID: mdl-21091341

ABSTRACT

The ability to control stem cell differentiation is the holy grail of regenerative medicine. Although significant progress toward this goal has been achieved, few efficient and straightforward methods have been developed, necessitating a better understanding of the mechanisms that influence differentiation. The extracellular microenvironment is emerging as a major player in controlling stem cell fate. Cell surface and secreted heparan sulfate glycosaminoglycans (HSGAGs) are one element of the extracellular matrix that regulates complex cell signaling networks. HSGAGs facilitate binding and availability of cytokines to cells as they progress through development. For example, growth factors such as fibroblast growth factor and vascular endothelial growth factor bind to specific HSGAG sequences during vasculogenesis. HSGAGs have been shown to be critical for stem cell vasculogenesis as well as other differentiation lineages. Understanding the role that the extracellular microenvironment plays in controlling cell fate can lead us closer to directing differentiation for developmental models and regenerative therapies. This review will focus on the role of extracellular microenvironment in regulating cell differentiation, with particular attention to the role of HSGAGs in vasculogenesis.


Subject(s)
Extracellular Matrix/metabolism , Fibroblast Growth Factors/metabolism , Heparitin Sulfate , Neovascularization, Physiologic , Stem Cells/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Cell Differentiation/physiology , Cell Membrane/metabolism , Cytokines/metabolism , Extracellular Matrix/genetics , Fibroblast Growth Factors/genetics , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Glycomics , Heparitin Sulfate/biosynthesis , Heparitin Sulfate/metabolism , Humans , Mice , Mice, Knockout , Protein Binding , Regenerative Medicine , Signal Transduction , Stem Cells/cytology , Vascular Endothelial Growth Factor A/genetics
5.
Circulation ; 120(19): 1883-1892, 2009 Nov 10.
Article in English | MEDLINE | ID: mdl-19858418

ABSTRACT

BACKGROUND: Therapeutic vasculogenesis is an emerging concept that can potentially be harnessed for the management of ischemic pathologies. The present study elucidates the potential coregulation of vasculogenesis by the heparan sulfate glycosaminoglycan-rich cell-surface glycome and the transcriptome. METHODS AND RESULTS: Differentiation of embryonic stem cells into endothelial cells in an in vitro embryoid body is paralleled by an amplification of heparan sulfate glycosaminoglycan sulfation, which correlates with the levels of the enzyme N-deacetylase/N-sulfotransferase 1 (NDST1). Small hairpin RNA-mediated knockdown of NDST1 or modification of heparan sulfate glycosaminoglycans in embryonic stem cells with heparinases or sodium chlorate inhibited differentiation of embryonic stem cells into endothelial cells. This was translated to an in vivo zebrafish embryo model, in which the genetic knockdown of NDST1 resulted in impaired vascularization characterized by a concentration-dependent decrease in intersegmental vessel lumen and a large tail-vessel configuration, which could be rescued by use of exogenous sulfated heparan sulfate glycosaminoglycans. To explore the cross talk between the glycome and the transcriptome during vasculogenesis, we identified by microarray and then validated wild-type and NDST1 knockdown-associated gene-expression patterns in zebrafish embryos. Temporal analysis at 3 developmental stages critical for vasculogenesis revealed a cascade of pathways that may mediate glycocalyx regulation of vasculogenesis. These pathways were intimately connected to cell signaling, cell survival, and cell fate determination. Specifically, we demonstrated that forkhead box O3A/5 proteins and insulin-like growth factor were key downstream signals in this process. CONCLUSIONS: The present study for the first time implicates interplay between the glycome and the transcriptome during vasculogenesis, revealing the possibility of harnessing specific cellular glyco-microenvironments for therapeutic vascularization.


Subject(s)
Embryonic Stem Cells/physiology , Endothelial Cells/physiology , Gene Expression Profiling , Glycomics , Neovascularization, Physiologic/physiology , Animals , Cell Differentiation/physiology , Cells, Cultured , Embryonic Stem Cells/cytology , Endothelial Cells/cytology , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Glycocalyx/physiology , Heparan Sulfate Proteoglycans/genetics , Heparan Sulfate Proteoglycans/metabolism , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor II/metabolism , Mice , Models, Animal , Phenotype , Signal Transduction/physiology , Sulfotransferases/genetics , Sulfotransferases/metabolism , Zebrafish
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