RESUMO
OBJECTIVE: Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. METHODS AND RESULTS: To enhance endothelial cell differentiation above a baseline of approximately 2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31(+) cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31(+) cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFalpha, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. CONCLUSIONS: VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. This enrichment method increases endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células Endoteliais/citologia , Traumatismo por Reperfusão Miocárdica/terapia , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Colágeno , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Laminina , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Proteoglicanas , Ratos , Ratos Nus , Células U937 , Veias Umbilicais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.
RESUMO
The ability to control proliferation of grafted cells in the heart and consequent graft size could dramatically improve the efficacy of cell therapies for cardiac repair. To achieve targeted graft cell proliferation, we created a chimeric receptor (F36Vfgfr-1) composed of a modified FK506-binding protein (F36V) fused with the cytoplasmic domain of the fibroblast growth factor receptor-1 (FGFR-1). We retrovirally transduced mouse C2C12 and MM14 skeletal myoblasts with this construct and treated them with AP20187, a dimeric F36V ligand ("dimerizer"), in vitro and in vivo to induce receptor dimerization. Dimerizer treatment in vitro activated the mitogen-activated protein kinase pathway and induced proliferation in myoblasts expressing F36Vfgfr-1 comparable with the effects of basic FGF. Wild-type myoblasts did not respond to dimerizer. Subcutaneous grafts composed of myoblasts expressing F36Vfgfr-1 showed a dose-dependent increase in DNA synthesis with dimerizer treatment. When myoblasts expressing F36Vfgfr-1 were injected into infarcted hearts of nude mice, dimerizer treatment resulted in a dose-dependent increase in graft size, from 20 +/- 3 to 42.9 +/- 4.3% of the left ventricle. Blinded echocardiographic analysis demonstrated that larger graft size was associated with a dose-dependent reduction in ventricular dilation after myocardial infarction, although animals with the largest grafts showed an increased incidence of ventricular tachycardia. Thus, selective proliferation of genetically modified graft cells can be induced with a systemically administered synthetic molecule in vitro or in vivo. Control of intramyocardial graft size by this approach may allow optimization of cell-based therapy to obtain desired cardiac function postinfarction.
Assuntos
Infarto do Miocárdio/terapia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Animais , Linhagem Celular , Proliferação de Células , Dimerização , Terapia Genética/métodos , Técnicas In Vitro , Camundongos , Camundongos Nus , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Infarto do Miocárdio/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/uso terapêutico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução GenéticaRESUMO
Basic fibroblast growth factor (bFGF) is a potent angiogenic molecule, but its therapeutic use is limited by mitogenic effects on multiple cell types. To specifically activate FGF signaling in endothelial cells, a chimeric FGF receptor was generated that contained a modified FK506 drug-binding domain (F36V) fused to the FGF receptor-1 (FGFR1) cytoplasmic domain. Human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells were retrovirally transduced with this chimeric receptor, and the effects of administering synthetic receptor-dimerizing ligands were studied. As expected, both control and transduced cells proliferated in response to bFGF treatment; however, only transduced endothelial cells exhibited dose-dependent proliferative responses to dimerizer treatment. Dimerizer-induced proliferation was MEK-dependent and was accompanied by MAP kinase phosphorylation, indicating that the chimeric receptor utilizes signaling pathways similar to endogenous FGFR1. Although bFGF stimulated wound re-epithelialization in HUVECs (which natively express FGFR1 and FGFR4), chemical dimerization of FGFR1 did not; this suggests FGFR4 may control migration in these cells. The ability to selectively activate receptor subtypes should facilitate the study of signaling pathways in vitro and in vivo beyond what can be accomplished with nonselective natural ligands, and it may eventually permit stimulation of graft cell angiogenesis without driving overgrowth of host cells.