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1.
Proc Natl Acad Sci U S A ; 110(31): 12601-6, 2013 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-23858432

RESUMEN

The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature's inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.


Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Matriz Extracelular/química , Neovascularización Fisiológica , Células Madre Pluripotentes/metabolismo , Ingeniería de Tejidos/métodos , Línea Celular , Células Endoteliales/citología , Endotelio Vascular/citología , Humanos , Células Madre Pluripotentes/citología , Medicina Regenerativa/métodos
2.
Arterioscler Thromb Vasc Biol ; 34(4): 913-20, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24526696

RESUMEN

OBJECTIVE: A critical regulator of the developing or regenerating vasculature is low oxygen tension. Precise elucidation of the role of low oxygen environments on endothelial commitment from human pluripotent stem cells necessitates controlled in vitro differentiation environments. APPROACH AND RESULTS: We used a feeder-free, 2-dimensional differentiation system in which we could monitor accurately dissolved oxygen levels during human pluripotent stem cell differentiation toward early vascular cells (EVCs). We found that oxygen uptake rate of differentiating human pluripotent stem cells is lower in 5% O2 compared with atmospheric conditions. EVCs differentiated in 5% O2 had an increased vascular endothelial cadherin expression with clusters of vascular endothelial cadherin+ cells surrounded by platelet-derived growth factor ß+ cells. When we assessed the temporal effects of low oxygen differentiation environments, we determined that low oxygen environments during the early stages of EVC differentiation enhance endothelial lineage commitment. EVCs differentiated in 5% O2 exhibited an increased expression of vascular endothelial cadherin and CD31 along with their localization to the membrane, enhanced lectin binding and acetylated low-density lipoprotein uptake, rapid cord-like structure formation, and increased expression of arterial endothelial cell markers. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogated the endothelial inductive effects of the low oxygen environments. CONCLUSIONS: Low oxygen tension during early stages of EVC derivation induces endothelial commitment and maturation through the accumulation of reactive oxygen species, highlighting the importance of regulating oxygen tensions during human pluripotent stem cell-vascular differentiation.


Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Endoteliales/metabolismo , Oxígeno/metabolismo , Células Madre Pluripotentes/metabolismo , Nicho de Células Madre , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Cadherinas/metabolismo , Hipoxia de la Célula , Línea Celular , Membrana Celular/metabolismo , Técnicas de Cocultivo , Células Nutrientes , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Lectinas/metabolismo , Lipoproteínas LDL/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo
3.
FASEB J ; 26(12): 4925-36, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22919069

RESUMEN

Extracellular matrix (ECM) production is critical to preserve the function and integrity of mature blood vessels. Toward the engineering of blood vessels, studies have centered on ECM production by supporting cells, whereas few studies implicate endothelial cells (ECs) with ECM synthesis. Here, we elucidate variations between cultured human arterial, venous, and progenitor ECs with respect to ECM deposition assembly, composition, and response to biomolecular and physiological factors. Our studies reveal that progenitor ECs, endothelial colony-forming cells (ECFCs), deposit collagen IV, fibronectin, and laminin that assemble to an organized weblike structure, as confirmed by decellularized cultures. Mature ECs only express these ECM proteins intracellularly. ECFC-derived ECM is abrogated in response to TGFß signaling inhibition and actin cytoskeleton disruption. Hypoxic (1%) and physiological (5%) O(2) tension stimulate ECM deposition from mature ECs. Interestingly, deposition of collagen I is observed only under 5% O(2) tension. ECM production from all ECs is found to be regulated by hypoxia-inducible factors 1α and 2α but differentially in the different cell lines. Collectively, we suggest that ECM deposition and assembly by ECs is dependent on maturation stage and oxygen supply and that these findings can be harnessed to advance engineered vascular therapeutics.


Asunto(s)
Células Endoteliales/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Células Madre/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Hipoxia de la Célula , Línea Celular , Células Cultivadas , Colágeno Tipo IV/metabolismo , Células Endoteliales/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Laminina/metabolismo , Microscopía Fluorescente , Oxígeno/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Madre/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo
4.
Biophys J ; 99(11): 3563-70, 2010 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-21112280

RESUMEN

Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells. To this end, we use the method of particle tracking microrheology to compare the viscoelastic properties of the cytoplasm of hES cells, hiPS cells, and the terminally differentiated parental human fibroblasts from which our hiPS cells are derived. Our results indicate that although the cytoplasm of parental fibroblasts is both viscous and elastic, the cytoplasm of hiPS cells does not exhibit any measurable elasticity and is purely viscous over a wide range of timescales. The viscous phenotype of hiPS cells is recapitulated in parental cells with disassembled actin filament network. The cytoplasm of hES cells is predominantly viscous but contains subcellular regions that are also elastic. This study supports the hypothesis that intracellular elasticity correlates with the degree of cellular differentiation and reveals significant differences in the mechanical properties of hiPS cells and hES cells. Because mechanical stimuli have been shown to mediate the precise fate of differentiating stem cells, our results support the concept that stem cell "softness" is a key feature of force-mediated differentiation of stem cells and suggest there may be subtle functional differences between force-mediated differentiation of hiPS cells and hES cells.


Asunto(s)
Células Madre Embrionarias/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Reología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Línea Celular , Difusión , Células Madre Embrionarias/citología , Fibroblastos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Simulación de Dinámica Molecular , Nanopartículas/química , Viscosidad
6.
J Tissue Eng Regen Med ; 11(3): 855-861, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-25641688

RESUMEN

The multifaceted extracellular milieu presents biochemical and biophysical stimuli that influence stem cell differentiation. Two-dimensional (2D) micropatterned substrates allow the presentation of these cues in spatially defined geometries that have been demonstrated to guide stem cell fate decisions. Leveraging stem cells to reconstruct microvasculature, made up of an inner lining of endothelial cells (ECs) supported by pericytes, is critical to tissue-engineering advances; thus, methods to improve endothelial differentiation efficiency are vital to these efforts. In this study, we examine the hypothesis that the diameter of micropatterned islands influences endothelial differentiation from human induced pluripotent stem cells (hiPSCs). Comparing island diameters of 80, 140, 225 and 500 µm, we found that co-cultures of control ECs and pericytes did not yield variable ratios of cell types; however, when hiPSCs were differentiated toward a bicellular population of ECs and pericytes on these varying micropattern feature sizes, we found that smaller islands promoted EC differentiation efficiency, yielding a derived population composed of 70% ECs, which exhibited a greater sprouting propensity. Differentiation on the largest feature size exhibited a smaller EC yield, similar to that on non-patterned substrates. Taken together, these data demonstrate that micropatterned islands of varying diameters can be used to modulate EC differentiation efficiency. Copyright © 2015 John Wiley & Sons, Ltd.


Asunto(s)
Diferenciación Celular , Células Endoteliales de la Vena Umbilical Humana/citología , Células Madre Pluripotentes Inducidas/citología , Técnicas de Cocultivo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neovascularización Fisiológica , Pericitos/citología , Pericitos/metabolismo
7.
Methods Mol Biol ; 1307: 213-22, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-25515528

RESUMEN

Blood vessels serve as the lifeline of nearly all living tissue. Vascular cells derived from human pluripotent stem cells hold great potential for clinical use in the regeneration of diseased vasculature and construction of blood vessels in engineered tissue. By deriving these cells in a controllable and clinically relevant manner harnessing physiological cues, we can obtain populations of cells amenable for transplantation. In this chapter, we describe methods to differentiate human pluripotent stem cells toward a bicellular population of early vascular cells using low oxygen cues, guide these subpopulations into mature endothelial cells and pericytes, and expand the vascular derivatives.


Asunto(s)
Células Endoteliales/citología , Pericitos/citología , Células Madre Pluripotentes/citología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Citometría de Flujo , Humanos , Oxígeno/farmacología , Pericitos/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos
8.
Stem Cells Dev ; 24(4): 451-8, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25233291

RESUMEN

Tissue-engineered constructs are rendered useless without a functional vasculature owing to a lack of nutrients and oxygen. Cell-based approaches to reconstruct blood vessels can yield structures that mimic native vasculature and aid transplantation. Vascular derivatives of human induced pluripotent stem cells (hiPSCs) offer opportunities to generate patient-specific therapies and potentially provide unlimited amounts of vascular cells. To be used in engineered vascular constructs and confer therapeutic benefit, vascular derivatives must exhibit additional key properties, including extracellular matrix (ECM) production to confer structural integrity and growth factor production to facilitate integration. In this study, we examine the hypothesis that vascular cells derived from hiPSCs exhibit these critical properties to facilitate their use in engineered tissues. hiPSCs were codifferentiated toward early vascular cells (EVCs), a bicellular population of endothelial cells (ECs) and pericytes, under varying low-oxygen differentiation conditions; subsequently, ECs were isolated and passaged. We found that EVCs differentiated under low-oxygen conditions produced copious amounts of collagen IV and fibronectin as well as vascular endothelial growth factor and angiopoietin 2. EVCs differentiated under atmospheric conditions did not demonstrate such abundant ECM expression, but exhibited greater expression of angiopoietin 1. Isolated ECs could proliferate up to three passages while maintaining the EC marker vascular endothelial cadherin. Isolated ECs demonstrated an increased propensity to produce ECM compared with their EVC correlates and took on an arterial-like fate. These findings illustrate that hiPSC vascular derivates hold great potential for therapeutic use and should continue to be a preferred cell source for vascular construction.


Asunto(s)
Diferenciación Celular , Células Endoteliales/citología , Células Madre Pluripotentes Inducidas/citología , Ingeniería de Tejidos , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Animales , Línea Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Neovascularización Fisiológica , Oxígeno/metabolismo
9.
Sci Rep ; 5: 12617, 2015 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-26227093

RESUMEN

Stem cell differentiation underlies many fundamental processes such as development, tissue growth and regeneration, as well as disease progression. Understanding how stem cell differentiation is controlled in mixed cell populations is an important step in developing quantitative models of cell population dynamics. Here we focus on quantifying the role of cell-cell interactions in determining stem cell fate. Toward this, we monitor stem cell differentiation in adherent cultures on micropatterns and collect statistical cell fate data. Results show high cell fate variability and a bimodal probability distribution of stem cell fraction on small (80-140 µm diameter) micropatterns. On larger (225-500 µm diameter) micropatterns, the variability is also high but the distribution of the stem cell fraction becomes unimodal. Using a stochastic model, we analyze the differentiation dynamics and quantitatively determine the differentiation probability as a function of stem cell fraction. Results indicate that stem cells can interact and sense cellular composition in their immediate neighborhood and adjust their differentiation probability accordingly. Blocking epithelial cadherin (E-cadherin) can diminish this cell-cell contact mediated sensing. For larger micropatterns, cell motility adds a spatial dimension to the picture. Taken together, we find stochasticity and cell-cell interactions are important factors in determining cell fate in mixed cell populations.


Asunto(s)
Diferenciación Celular/fisiología , Modelos Biológicos , Células Madre/citología , Cadherinas/metabolismo , Comunicación Celular , Movimiento Celular , Células Cultivadas , Humanos , Células Madre/fisiología , Procesos Estocásticos
10.
Methods Mol Biol ; 1202: 1-9, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24155232

RESUMEN

As the lifeline of almost all living tissues, blood vessels are a major focus of tissue-regenerative therapies. Rebuilding blood vessels has vast implications for the study of vascular growth and treatment of diseases in which vascular function is compromised. Toward this end, human pluripotent stem cells have been widely studied for their differentiation capacity toward vascular lineages. We demonstrate methods to derive a bicellular population of early specialized vascular cells from human pluripotent stem cells, to differentiate these toward mature endothelial cells and pericytes, and to utilize a collagen scaffold to facilitate organization into vascular networks.


Asunto(s)
Vasos Sanguíneos/citología , Técnicas de Cultivo de Célula/métodos , Neovascularización Fisiológica , Células Madre Pluripotentes/citología , Animales , Vasos Sanguíneos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Colágeno/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Geles/farmacología , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Pericitos/citología , Pericitos/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Ratas
11.
Stem Cell Reports ; 2(5): 561-75, 2014 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-24936446

RESUMEN

Distinguishing between perivascular cell types remains a hurdle in vascular biology due to overlapping marker expressions and similar functionalities. Clarifying and defining heterogeneities in vitro among perivascular cells could lead to improved cell-based tissue regeneration strategies and a better understanding of human developmental processes. We studied contractile vascular smooth muscle cells (vSMCs), synthetic vSMCs, and pericytes derived from a common human pluripotent stem cell source. Using in vitro cultures, we show unique cell morphology, subcellular organelle organization (namely endoplasmic reticulum, mitochondria, and stress fibers), and expression of smooth muscle myosin heavy chain and elastin for each cell type. While differences in extracellular matrix deposition and remodeling were less pronounced, the multipotency, in vivo, migratory, invasion, and contractile functionalities are distinctive for each cell type. Overall, we define a repertoire of functional phenotypes in vitro specific for each of the human perivascular cell types, enabling their study and use in basic and translational research.


Asunto(s)
Pericitos/citología , Células Madre Pluripotentes/citología , Diferenciación Celular , Elastina/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Metaloendopeptidasas/metabolismo , Contracción Muscular , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Cadenas Pesadas de Miosina/metabolismo , Pericitos/metabolismo
12.
Biotechnol J ; 8(4): 434-47, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23554249

RESUMEN

Vascular engineering seeks to design and construct functional blood vessels comprising endothelial cells (ECs) and perivascular cells (PCs), with the ultimate goal of clinical translation. While EC behavior has been extensively investigated, PCs play an equally significant role in the development of novel regenerative strategies, providing functionality and stability to vessels. The two major classes of PCs are vascular smooth muscle cells (vSMCs) and pericytes; vSMCs can be further sub-classified as either contractile or synthetic. The inclusion of these cell types is crucial for successful regeneration of blood vessels. Furthermore, understanding distinctions between vSMCs and pericytes will enable improved therapeutics in a tissue-specific manner. Here we focus on the approaches and challenges facing the use of PCs in vascular regeneration, including their characteristics, stem cell sources, and interactions with ECs. Finally, we discuss biochemical and microRNA (miR) regulators of PC behavior and engineering approaches that mimic various cues affecting PC function.


Asunto(s)
Músculo Liso Vascular/fisiología , Pericitos/fisiología , Regeneración/fisiología , Ingeniería de Tejidos/métodos , Animales , Humanos , Músculo Liso Vascular/citología , Pericitos/citología
13.
Macromol Biosci ; 12(1): 21-8, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22083795

RESUMEN

A biodegradable, temperature-sensitive dextran-allyl isocyanate-ethylamine (TSDAIE) as a nonenzymatic cell detachment polymeric substrate for human endothelial progenitor cells (EPCs) is developed and examined. The lower critical solution temperature of TSDAIE is determined; its phase transition occurrs at 18 to 22 °C. For EPC culture, cell culture flasks are coated with TSDAIE and type I collagen. The TSDAIE coating enables EPC detachment when the culture is cooled to 4 °C. The concentration of TSDAIE affects EPC attachment, which is thereby used to optimize the concentration of TSDAIE for coating. At the determined optimal concentration, TSDAIE is found to be compatible for use in EPC culture as revealed by cell attachment, spreading, proliferation, and phenotype. Overall, biodegradable TSDAIE shows promise for applications that culture and expand EPCs including vascular regenerative medicine and tissue engineering.


Asunto(s)
Materiales Biocompatibles/química , Adhesión Celular , Dextranos/química , Polímeros/química , Temperatura de Transición , Rastreo Diferencial de Calorimetría/métodos , Adhesión Celular/fisiología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fenotipo , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Células Madre/citología , Células Madre/metabolismo , Propiedades de Superficie , Temperatura
14.
PLoS One ; 7(5): e36689, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22574215

RESUMEN

The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape.


Asunto(s)
Diferenciación Celular , Núcleo Celular/metabolismo , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Fibras de Estrés/metabolismo , Animales , Línea Celular , Forma del Núcleo Celular , Fibroblastos/citología , Regulación de la Expresión Génica , Humanos , Lamina Tipo A/metabolismo , Pulmón/citología , Ratones , Transporte de Proteínas
15.
Macromol Biosci ; 11(1): 36-49, 2011 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-20967797

RESUMEN

The biochemical cues and topographical architecture of the extracellular environment extensively influence ES cell fate. The microenvironment surrounding the developing embryo presents these instructive cues in a complex and interactive manner in order to guide cell fate decisions. Current stem cell research aims to reconstruct this multifaceted embryonic niche to recapitulate development in vitro. This review focuses on 2D and 3D differentiation niches created from natural and synthetic biomaterials to guide the differentiation of ES cells toward specific lineages. Biomaterials engineered to present specific physical constraints are also reviewed for their role in differentiation.


Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Matriz Extracelular/química , Nicho de Células Madre , Animales , Técnicas de Cultivo de Célula , Colágeno/química , Colágeno/fisiología , Combinación de Medicamentos , Cuerpos Embrioides/citología , Células Madre Embrionarias/fisiología , Matriz Extracelular/fisiología , Fibronectinas/química , Fibronectinas/fisiología , Humanos , Laminina/química , Laminina/fisiología , Nanotecnología , Proteoglicanos/química , Andamios del Tejido
16.
Biomaterials ; 32(1): 95-106, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20870284

RESUMEN

Slow vascularization of functional blood limits the transplantation of tissue constructs and the recovery of ischemic and wounded tissues. Despite the widespread investigation of polysaccharide-based hydrogel scaffolds for their therapeutic applications, blood vessel ingrowth into these hydrogel scaffolds remains a challenge. We hypothesized that modifying the properties of biodegradable hydrogel scaffolds with immobilization of multiple angiogenic growth factors (GFs) would induce a rapid proliferation of functional vasculature into the scaffolds. To this end, we remodeled the hydrogel structure by decreasing crosslinking density via reduced degree of substitution of crosslinking groups, which resulted in improved hydrogel properties including reduced rigidity, increased swelling, increased vascular endothelial GF (VEGF) release capability, and facilitated rapid hydrogel disintegration and tissue ingrowth. Immobilizing VEGF in the scaffolds promoted tissue ingrowth and expedited biodegradation. Furthermore, a synergistic effect of multiple angiogenic GFs was established; the coimmobilization of VEGF+ angiopoietin-1, and VEGF+ insulin-like GF+ stromal cell-derived factor-1 induced more and larger blood vessels than any individual GF, while the combination of all GFs dramatically increased the size and number of newly formed functional vessels. Altogether, our data demonstrate that rapid, efficient, and functional neovascularization can be achieved by precisely manipulating hydrogel scaffold properties and immobilizing defined angiogenic GFs.


Asunto(s)
Inductores de la Angiogénesis/farmacología , Materiales Biocompatibles/farmacología , Dextranos/farmacología , Hidrogeles/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Compuestos Alílicos/farmacología , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/crecimiento & desarrollo , Sistemas de Liberación de Medicamentos , Humanos , Isocianatos/farmacología , Fenómenos Mecánicos/efectos de los fármacos , Polietilenglicoles/farmacología , Implantación de Prótesis , Ratas , Andamios del Tejido , Factor A de Crecimiento Endotelial Vascular/metabolismo
17.
Expert Rev Cardiovasc Ther ; 8(10): 1433-45, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20936930

RESUMEN

Vascular disease is the leading cause of mortality in the USA, providing the impetus for new treatments and technologies. Current therapies rely on the implantation of stents or grafts to treat injured blood vessels. However, these therapies may be immunogenic or may incompletely recover the functional integrity of the vasculature. In light of these shortcomings, cell-based therapies provide new treatment options to heal damaged areas with more suitable substitutes. Current clinical trials employing stem cell-based therapies involve the transfusion of harvested endothelial progenitor cells. While the results from these trials have been encouraging, utilizing tissue-engineered approaches could yield technologically advanced solutions. This article discusses engineered stem cell-based therapies from three angles: the differentiation of adult stem cells, such as mesenchymal stem cells and endothelial progenitor cells, into vascular lineages; investigation of human embryonic stem cells and induced pluripotent stem cells as inexhaustible sources of vascular cells; and tissue-engineering approaches, which incorporate these vascular progenitor cells into biomimetic scaffolds to guide regeneration. The optimal solution to vascular disease lies at the interface of these technologies--embedding differentiated cells into engineered scaffolds to impart precise control over vascular regeneration.


Asunto(s)
Trasplante de Células Madre/métodos , Ingeniería de Tejidos/métodos , Enfermedades Vasculares/terapia , Animales , Prótesis Vascular , Diferenciación Celular , Ensayos Clínicos como Asunto , Humanos , Regeneración , Andamios del Tejido
18.
J Biomed Mater Res A ; 93(3): 1080-90, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-19753626

RESUMEN

Modification of dextran backbone allows the development of a hydrogel with specific characteristics. To enhance their functionality for tissue-engineered scaffolds, a series of dextran-based macromers was synthesized by incorporating various functional groups, including allyl isocyanate (Dex-AI), ethylamine (Dex-AE), chloroacetic acid (Dex-AC), or maleic-anhydride (Dex-AM) into dextrans. The dextran-based biodegradable hybrid hydrogels are developed by integrating polyethylene glycol diacrylate (PEGDA). To explore the effect of different derivatives on hydrogel properties, three different ratios of Dex/PEGDA are examined: low (20/80), medium (40/60), and high (60/40). Differences in physical and biological properties of the hydrogels are found, including swelling, degradation rate, mechanics, crosslinking density, biocompatibility (in vitro and in vivo), and vascular endothelial growth factor release. The results also indicate that the incorporation of amine groups into dextran gives rise to hydrogels with better biocompatible and release properties. We, therefore, conclude that the incorporation of different functional groups affects the fundamental properties of a dextran-based hydrogel network, and that amine groups are preferred to generate hydrogels for biomedical use.


Asunto(s)
Dextranos/química , Dextranos/farmacología , Hidrogeles/química , Hidrogeles/farmacología , Ensayo de Materiales , Fenómenos Mecánicos/efectos de los fármacos , Animales , Reactivos de Enlaces Cruzados/farmacología , Femenino , Polietilenglicoles/farmacología , Ratas , Ratas Endogámicas Lew , Factor A de Crecimiento Endotelial Vascular/farmacología
19.
Stem Cell Rev Rep ; 6(2): 237-47, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20425149

RESUMEN

Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers--including alpha-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain--to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.


Asunto(s)
Vasos Sanguíneos/citología , Células Madre Embrionarias/citología , Miocitos del Músculo Liso/citología , Diferenciación Celular/fisiología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ingeniería de Tejidos/métodos
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