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1.
Methods Mol Biol ; 2206: 193-203, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754819

RESUMO

The capability of forming functional blood vessel networks is critical for the characterization of endothelial cells. In this chapter, we will review a modified in vivo vascular network forming assay by replacing traditional mouse tumor-derived Matrigel with a well-defined collagen-fibrin hydrogel. The assay is reliable and does not require special equipment, surgical procedure, or a skilled person to perform. Moreover, investigators can modify this method on-demand for testing different cell sources, perturbation of gene functions, growth factors, and pharmaceutical molecules, and for the development and investigation of strategies to enhance neovascularization of engineered human tissues and organs.

2.
Sci Adv ; 6(30): eaba7606, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32832668

RESUMO

Human induced pluripotent stem cell (h-iPSC)-derived endothelial cells (h-iECs) have become a valuable tool in regenerative medicine. However, current differentiation protocols remain inefficient and lack reliability. Here, we describe a method for rapid, consistent, and highly efficient generation of h-iECs. The protocol entails the delivery of modified mRNA encoding the transcription factor ETV2 at the intermediate mesodermal stage of differentiation. This approach reproducibly differentiated 13 diverse h-iPSC lines into h-iECs with exceedingly high efficiency. In contrast, standard differentiation methods that relied on endogenous ETV2 were inefficient and notably inconsistent. Our h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. Timely activation of ETV2 was critical, and bypassing the mesodermal stage produced putative h-iECs with reduced expansion potential and inability to form functional vessels. Our protocol has broad applications and could reliably provide an unlimited number of h-iECs for vascular therapies.

3.
Blood Adv ; 3(24): 4166-4176, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31851760

RESUMO

Hemophilia A (HA) is a bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). Current treatments are based on regular infusions of FVIII concentrates throughout a patient's life. Alternatively, viral gene therapies that directly deliver F8 in vivo have shown preliminary successes. However, hurdles remain, including lack of infection specificity and the inability to deliver the full-length version of F8 due to restricted viral cargo sizes. Here, we developed an alternative nonviral ex vivo gene-therapy approach that enables the overexpression of full-length F8 in patients' endothelial cells (ECs). We first generated HA patient-specific induced pluripotent stem cells (HA-iPSCs) from urine epithelial cells and genetically modified them using a piggyBac DNA transposon system to insert multiple copies of full-length F8. We subsequently differentiated the modified HA-iPSCs into competent ECs with high efficiency, and demonstrated that the cells (termed HA-FLF8-iECs) were capable of producing high levels of FVIII. Importantly, following subcutaneous implantation into immunodeficient hemophilic (SCID-f8ko) mice, we demonstrated that HA-FLF8-iECs were able to self-assemble into vascular networks, and that the newly formed microvessels had the capacity to deliver functional FVIII directly into the bloodstream of the mice, effectively correcting the clotting deficiency. Moreover, our implant maintains cellular confinement, which reduces potential safety concerns and allows effective monitoring and reversibility. We envision that this proof-of-concept study could become the basis for a novel autologous ex vivo gene-therapy approach to treat HA.


Assuntos
Bioengenharia , Fator VIII/genética , Hemofilia A/genética , Hemofilia A/terapia , Microvasos/transplante , Animais , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Modelos Animais de Doenças , Células-Tronco Embrionárias , Fator VIII/metabolismo , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , Camundongos SCID , Mutação , Fenótipo , Transplante de Células-Tronco , Resultado do Tratamento
5.
Cell Mol Life Sci ; 76(3): 421-439, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30315324

RESUMO

Tissue engineering holds great promise in regenerative medicine. However, the field of tissue engineering faces a myriad of difficulties. A major challenge is the necessity to integrate vascular networks into bioengineered constructs to enable physiological functions including adequate oxygenation, nutrient delivery, and removal of waste products. The last two decades have seen remarkable progress in our collective effort to bioengineer human-specific vascular networks. Studies have included both in vitro and in vivo investigations, and multiple methodologies have found varying degrees of success. What most approaches to bioengineer human vascular networks have in common, however, is the synergistic use of both (1) endothelial cells (ECs)-the cells used to line the lumen of the vascular structures and (2) perivascular cells-usually used to support EC function and provide perivascular stability to the networks. Here, we have highlighted trends in the use of various cellular sources over the last two decades of vascular network bioengineering research. To this end, we comprehensively reviewed all life science and biomedical publications available at the MEDLINE database up to 2018. Emphasis was put on selective studies that definitively used human ECs and were specifically related to bioengineering vascular networks. To facilitate this analysis, all papers were stratified by publication year and then analyzed according to their use of EC and perivascular cell types. This study provides an illustrating discussion on how each alternative source of cells has come to be used in the field. Our intention was to reveal trends and to provide new insights into the trajectory of vascular network bioengineering with regard to cellular sources.


Assuntos
Células Endoteliais/citologia , Microvasos/citologia , Pericitos/citologia , Engenharia Tecidual , Humanos , Células-Tronco Pluripotentes/citologia , Engenharia Tecidual/tendências
6.
Artif Cells Nanomed Biotechnol ; 46(sup3): S434-S447, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30146913

RESUMO

Timely tissue vascularization and integration of engineered tissues into a patient plays an important role in the successful translation of engineered tissues into clinically relevant therapies. To decrease the time needed to vascularize an engineered adipose tissue, suitable local microenvironments provided by hydrogels to support cell-based functional vascular network formation have been investigated. Using the same biomolecule in solution, two types of hydrogels can be obtained: a "physical hydrogel" which is thermal-induced self-assemble fibril initiation and growth, due to amino and carboxyl telopeptides on collagen chains, and a "chemical hydrogel" which results from the covalently cross-linking of the side chains induced by one step enzyme mediation in aqueous solution. In this paper, we compare the capability of engineering vascular network and large-sized vascularized adipose tissue in vivo in different types of collagen hydrogels, physical and chemical crosslinking. The relationships between vascular network formation and hydrogel properties for the two types of hydrogels are discussed. Finally, we successfully engineered a vascularized adipose tissue construct (∼877.6 adipocytes/mm2; 94% area of a construct) in the absence of exogenous cytokines in chemical covalently crosslinking cell-laden hydrogel. These results show manipulating the polymerized methods of a hydrogel could not only modulate vascular network formation, but also regenerate adipose tissue in vivo.


Assuntos
Tecido Adiposo , Colágeno/química , Hidrogéis/química , Neovascularização Fisiológica , Engenharia Tecidual , Tecidos Suporte/química , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Matriz Extracelular/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus
7.
Artigo em Inglês | MEDLINE | ID: mdl-28868207

RESUMO

Notwithstanding remarkable progress in vascular network engineering, implanted bioengineered microvessels largely fail to form anastomoses with the host vasculature. Here, we demonstrate that implants containing assembled human vascular networks (A-Grafts) fail to engraft due to their inability to engage non-inflammatory host neutrophils upon implantation into mice. In contrast, unassembled vascular cells (U-Grafts) readily engage alternatively polarized neutrophils, which in turn serve as indispensable mediators of vascular assembly and anastomosis. The depletion of host neutrophils abrogated vascularization in U-Grafts, whereas an adoptive transfer of neutrophils fully restored vascularization in myeloid-depleted mice. Neutrophil engagement was regulated by secreted factors and was progressively silenced as the vasculature matured. Exogenous addition of factors from U-Grafts reengaged neutrophils and enhanced revascularization in A-Grafts, a process that was recapitulated by blocking Notch signaling. Our data suggest that the pro-vascularization potential of neutrophils can be harnessed to improve the engraftment of bioengineered tissues.

8.
Nat Commun ; 8(1): 383, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28851877

RESUMO

Release of promoter-proximally paused RNA polymerase II (RNAPII) is a recently recognized transcriptional regulatory checkpoint. The biological roles of RNAPII pause release and the mechanisms by which extracellular signals control it are incompletely understood. Here we show that VEGF stimulates RNAPII pause release by stimulating acetylation of ETS1, a master endothelial cell transcriptional regulator. In endothelial cells, ETS1 binds transcribed gene promoters and stimulates their expression by broadly increasing RNAPII pause release. 34 VEGF enhances ETS1 chromatin occupancy and increases ETS1 acetylation, enhancing its binding to BRD4, which recruits the pause release machinery and increases RNAPII pause release. Endothelial cell angiogenic responses in vitro and in vivo require ETS1-mediated transduction of VEGF signaling to release paused RNAPII. Our results define an angiogenic pathway in which VEGF enhances ETS1-BRD4 interaction to broadly promote RNAPII pause release and drive angiogenesis.Promoter proximal RNAPII pausing is a rate-limiting transcriptional mechanism. Chen et al. show that this process is essential in angiogenesis by demonstrating that the endothelial master transcription factor ETS1 promotes global RNAPII pause release, and that this process is governed by VEGF.


Assuntos
Neovascularização Patológica , Proteína Proto-Oncogênica c-ets-1/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Acetilação , Animais , Proteínas de Ciclo Celular , Movimento Celular , Cromatina , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
9.
Sci Rep ; 7(1): 770, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-28396600

RESUMO

Here we investigated whether endothelial colony forming cells (ECFC) and mesenchymal progenitor cells (MPC) form vascular networks and restore blood flow in ischemic skeletal muscle, and whether host myeloid cells play a role. ECFC + MPC, ECFC alone, MPC alone, or vehicle alone were injected into the hind limb ischemic muscle one day after ligation of femoral artery and vein. At day 5, hind limbs injected with ECFC + MPC showed greater blood flow recovery compared with ECFC, MPC, or vehicle. Tail vein injection of human endothelial specific Ulex europaeus agglutinin-I demonstrated an increased number of perfused human vessels in ECFC + MPC compared with ECFC. In vivo bioluminescence imaging showed ECFC persisted for 14 days in ECFC + MPC-injected hind limbs. Flow cytometric analysis of ischemic muscles at day 2 revealed increased myeloid lineage cells in ECFC + MPC-injected muscles compared to vehicle-injected muscles. Neutrophils declined by day 7, while the number of myeloid cells, macrophages, and monocytes did not. Systemic myeloid cell depletion with anti-Gr-1 antibody blocked the improved blood flow observed with ECFC + MPC and reduced ECFC and MPC retention. Our data suggest that ECFC + MPC delivery could be used to reestablish blood flow in ischemic tissues, and this may be enhanced by coordinated recruitment of host myeloid cells.


Assuntos
Células Progenitoras Endoteliais/citologia , Isquemia/fisiopatologia , Células-Tronco Mesenquimais/citologia , Músculos/irrigação sanguínea , Neovascularização Fisiológica , Fluxo Sanguíneo Regional , Animais , Células Progenitoras Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Músculo Esquelético/irrigação sanguínea , Células Mieloides/citologia , Células Mieloides/metabolismo
10.
Stem Cells Transl Med ; 6(5): 1316-1320, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28296182

RESUMO

Endothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term "EPC." It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping, potency assays, and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review, we seek to discourage the indiscriminate use of "EPCs," and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well-defined cell types that have been considered EPCs because they both promote vascular repair, albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing "EPC" nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and, in some cases, progress toward use in cell therapy. Stem Cells Translational Medicine 2017;6:1316-1320.


Assuntos
Células Progenitoras Endoteliais/citologia , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Humanos , Neovascularização Fisiológica/fisiologia , Terminologia como Assunto
11.
Adv Biosyst ; 1(3)2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30221188

RESUMO

The objective of this work was to investigate the effect of devitalized human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) seeded on mineralized nanofiber microsheets on protein release, osteogenesis, vasculogenesis, and macrophage polarization. Calcium phosphate nanocrystals were grown on the surface of aligned, functionalized nanofiber microsheets. The microsheets were seeded with hMSCs, ECFCs, or a mixture of hMSCs+ECFCs, cultured for cell attachment, differentiated to the osteogenic or vasculogenic lineage, and devitalized by lyophilization. The release kinetic of total protein, bone morphogenetic protein-2 (BMP2), and vascular endothelial growth factor (VEGF) from the devitalized microsheets was measured. Next, hMSCs and/or ECFCs were seeded on the devitalized cell microsheets and cultured in the absence of osteo-/vasculo-inductive factors to determine the effect of devitalized cell microsheets on hMSC/ECFC differentiation. Human macrophages were seeded on the microsheets to determine the effect of devitalized cells on macrophage polarization. Based on the results, devitalized undifferentiated hMSC and vasculogenic-differentiated ECFC microsheets had highest sustained release of BMP2 and VEGF, respectively. The devitalized hMSC microsheets did not affect M2 macrophage polarization while vascular-differentiated, devitalized ECFC microsheets did not affect M1 polarization. Both groups stimulated higher M2 macrophage polarization compared to M1.

12.
J Control Release ; 223: 126-136, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26721447

RESUMO

Reconstruction of large bone defects is limited by insufficient vascularization and slow bone regeneration. The objective of this work was to investigate the effect of spatial and temporal release of recombinant human bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) on the extent of osteogenic and vasculogenic differentiation of human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) encapsulated in a patterned hydrogel. Nanogels (NGs) based on polyethylene glycol (PEG) macromers chain-extended with short lactide (L) and glycolide (G) segments were used for grafting and timed-release of BMP2 and VEGF. NGs with 12kDa PEG molecular weight (MW), 24 LG segment length, and 60/40L/G ratio (P12-II, NG(10)) released the grafted VEGF in 10days. NGs with 8kDa PEG MW, 26 LG segment length, and 60/40L/G ratio (P8-I, NG(21)) released the grafted BMP2 in 21days. hMSCs and NG-BMP2 were encapsulated in a patterned matrix based on acrylate-functionalized lactide-chain-extended star polyethylene glycol (SPELA) hydrogel and microchannel patterns filled with a suspension of hMSCs+ECFCs and NG-VEGF in a crosslinked gelatin methacryloyl (GelMA) hydrogel. Groups included patterned constructs without BMP2/VEGF (None), with directly added BMP2/VEGF, and NG-BMP2/NG-VEGF. Based on the results, timed-release of VEGF in the microchannels in 10days from NG(10) and BMP2 in the matrix in 21days from NG(21) resulted in highest extent of osteogenic and vasculogenic differentiation of the encapsulated hMSCs and ECFCs compared to direct addition of VEGF and BMP2. Further, timed-release of VEGF from NG(10) in hMSC+ECFC encapsulating microchannels and BMP2 from NG(21) in hMSC encapsulating matrix sharply increased bFGF expression in the patterned constructs. The results suggest that mineralization and vascularization are coupled by localized secretion of paracrine signaling factors by the differentiating hMSCs and ECFCs.


Assuntos
Proteína Morfogenética Óssea 2/administração & dosagem , Células-Tronco/efeitos dos fármacos , Fator de Crescimento Transformador beta/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Antígenos CD/genética , Proteína Morfogenética Óssea 2/farmacologia , Caderinas/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , DNA/metabolismo , Endotélio Vascular/citologia , Humanos , Hidrogéis , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Poliésteres/química , Polietilenoglicóis/química , Ácido Poliglicólico/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Células-Tronco/citologia , Fator de Crescimento Transformador beta/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia
13.
Acta Biomater ; 27: 151-166, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26348142

RESUMO

UNLABELLED: Tissue engineering promises to restore or replace diseased or damaged tissue by creating functional and transplantable artificial tissues. The development of artificial tissues with large dimensions that exceed the diffusion limitation will require nutrients and oxygen to be delivered via perfusion instead of diffusion alone over a short time period. One approach to perfusion is to vascularize engineered tissues, creating a de novo three-dimensional (3D) microvascular network within the tissue construct. This significantly shortens the time of in vivo anastomosis, perfusion and graft integration with the host. In this study, we aimed to develop injectable allogeneic collagen-phenolic hydroxyl (collagen-Ph) hydrogels that are capable of controlling a wide range of physicochemical properties, including stiffness, water absorption and degradability. We tested whether collagen-Ph hydrogels could support the formation of vascularized engineered tissue graft by human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSC) in vivo. First, we studied the growth of adherent ECFCs and MSCs on or in the hydrogels. To examine the potential formation of functional vascular networks in vivo, a liquid pre-polymer solution of collagen-Ph containing human ECFCs and MSCs, horseradish peroxidase and hydrogen peroxide was injected into the subcutaneous space or abdominal muscle defect of an immunodeficient mouse before gelation, to form a 3D cell-laden polymerized construct. These results showed that extensive human ECFC-lined vascular networks can be generated within 7 days, the engineered vascular density inside collagen-Ph hydrogel constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with the existing vasculature to further support the survival of host muscle tissues. Finally, optimized conditions of the cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse after 1 month of implantation. STATEMENT OF SIGNIFICANCE: We reported a method for preparing autologous extracellular matrix scaffolds, murine collagen-Ph hydrogels, and demonstrated its suitability for use in supporting human progenitor cell-based formation of 3D vascular networks in vitro and in vivo. Results showed extensive human vascular networks can be generated within 7 days, engineered vascular density inside collagen-Ph constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with existing vasculature to further support the survival of host muscle tissues. Moreover, optimized conditions of cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse.


Assuntos
Derme Acelular , Vasos Sanguíneos/crescimento & desenvolvimento , Colágeno/química , Hidrogéis/química , Transplante de Células-Tronco Mesenquimais/instrumentação , Engenharia Tecidual/instrumentação , Animais , Órgãos Bioartificiais , Vasos Sanguíneos/citologia , Reagentes para Ligações Cruzadas/química , Células Endoteliais/citologia , Células Endoteliais/transplante , Análise de Falha de Equipamento , Matriz Extracelular/química , Peroxidase do Rábano Silvestre/química , Injeções , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Desenho de Prótese , Pele/química , Enxerto Vascular/instrumentação
14.
Acta Biomater ; 19: 85-99, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25749296

RESUMO

To manufacture tissue engineering-based functional tissues, scaffold materials that can be sufficiently vascularized to mimic the functionality and complexity of native tissues are needed. Currently, vascular network bioengineering is largely carried out using natural hydrogels as embedding scaffolds, but most natural hydrogels have poor mechanical stability and durability, factors that critically limit their widespread use. In this study, we examined the suitability of gelatin-phenolic hydroxyl (gelatin-Ph) hydrogels that can be enzymatically crosslinked, allowing tuning of the storage modulus and the proteolytic degradation rate, for use as injectable hydrogels to support the human progenitor cell-based formation of a stable and mature vascular network. Porcine gelatin-Ph hydrogels were found to be cytocompatible with human blood-derived endothelial colony-forming cells and white adipose tissue-derived mesenchymal stem cells, resulting in >87% viability, and cell proliferation and spreading could be modulated by using hydrogels with different proteolytic degradability and stiffness. In addition, gelatin was extracted from mouse dermis and murine gelatin-Ph hydrogels were prepared. Importantly, implantation of human cell-laden porcine or murine gelatin-Ph hydrogels into immunodeficient mice resulted in the rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, the degree of enzymatic crosslinking of the gelatin-Ph hydrogels could be used to modulate cell behavior and the extent of vascular network formation in vivo. Our report details a technique for the synthesis of gelatin-Ph hydrogels from allogeneic or xenogeneic dermal skin and suggests that these hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues.


Assuntos
Células Endoteliais/fisiologia , Gelatina/química , Hidrogéis/química , Microvasos/fisiologia , Neovascularização Fisiológica/fisiologia , Tecidos Suporte , Materiais Biocompatíveis/síntese química , Proliferação de Células/fisiologia , Células Cultivadas , Reagentes para Ligações Cruzadas/química , Células Endoteliais/citologia , Desenho de Equipamento , Análise de Falha de Equipamento , Peroxidase do Rábano Silvestre/química , Humanos , Teste de Materiais , Fenóis/química
15.
Tissue Eng Part C Methods ; 21(3): 274-83, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25091645

RESUMO

INTRODUCTION: Endothelial colony-forming cells (ECFCs) are endothelial progenitors that circulate in peripheral blood and are currently the subject of intensive investigation due to their therapeutic potential. However, in adults, ECFCs comprise a very small subset among circulating cells, which makes their isolation a challenge. MATERIALS AND METHODS: Currently, the standard method for ECFC isolation relies on the separation of mononuclear cells and erythrocyte lysis, steps that are time consuming and known to increase cell loss. Alternatively, we previously developed a novel disposable microfluidic platform containing antibody-functionalized degradable hydrogel coatings that is ideally suited for capturing low-abundance circulating cells from unprocessed blood. In this study, we reasoned that this microfluidic approach could effectively isolate rare ECFCs by virtue of their CD34 expression. RESULTS: We conducted preclinical experiments with peripheral blood from four adult volunteers and demonstrated that the actual microfluidic capture of circulating CD34(+) cells from unprocessed blood was compatible with the subsequent differentiation of these cells into ECFCs. Moreover, the ECFC yield obtained with the microfluidic system was comparable to that of the standard method. Importantly, we unequivocally validated the phenotypical and functional properties of the captured ECFCs, including the ability to form microvascular networks following transplantation into immunodeficient mice. DISCUSSION: We showed that the simplicity and versatility of our microfluidic system could be very instrumental for ECFC isolation while preserving their therapeutic potential. We anticipate our results will facilitate additional development of clinically suitable microfluidic devices by the vascular therapeutic and diagnostic industry.


Assuntos
Células Sanguíneas/citologia , Ensaio de Unidades Formadoras de Colônias/métodos , Células Endoteliais/citologia , Microfluídica/métodos , Adulto , Animais , Humanos , Camundongos Nus , Neovascularização Fisiológica , Fenótipo , Reprodutibilidade dos Testes
16.
Nat Commun ; 5: 5200, 2014 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-25335460

RESUMO

Targeting the vasculature remains a promising approach for treating solid tumours; however, the mechanisms of tumour neovascularization are diverse and complex. Here we uncover a new subpopulation of melanoma cells that express the vascular cell adhesion molecule PECAM1, but not VEGFR-2, and participate in a PECAM1-dependent form of vasculogenic mimicry (VM). Clonally derived PECAM1(+) tumour cells coalesce to form PECAM1-dependent networks in vitro and they generate well-perfused, vascular endothelial growth factor (VEGF)-independent channels in mice. The neural crest specifier AP-2α is diminished in PECAM1(+) melanoma cells and is a transcriptional repressor of PECAM1. Re-introduction of AP-2α into PECAM1(+) tumour cells represses PECAM1 and abolishes tube-forming ability, whereas AP-2α knockdown in PECAM1(-) tumour cells upregulates PECAM1 expression and promotes tube formation. Thus, VM-competent subpopulations, rather than all cells within a tumour, may instigate VM, supplant host-derived endothelium, and form PECAM1-dependent conduits that are not diminished by neutralizing VEGF.


Assuntos
Melanoma/metabolismo , Neovascularização Patológica/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Inibidores da Angiogênese/química , Animais , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , RNA Interferente Pequeno/metabolismo , Neoplasias Cutâneas/metabolismo , Fator de Transcrição AP-2/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
18.
Angiogenesis ; 17(4): 897-907, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24986520

RESUMO

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane glycoprotein that regulates cell motility and proliferation. TM4SF1 is an attractive cancer target because of its high expression in both tumor cells and on the vascular endothelial cells lining tumor blood vessels. We generated mouse monoclonal antibodies against human TM4SF1 in order to evaluate their therapeutic potential; 13 of the antibodies we generated reacted with extracellular loop-2 (EL2), TM4SF1's larger extracellular, lumen-facing domain. However, none of these antibodies reacted with mouse TM4SF1, likely because the EL2 of mouse TM4SF1 differs significantly from that of its human counterpart. Therefore, to test our antibodies in vivo, we employed an established model of engineered human vessels in which human endothelial colony-forming cells (ECFC) and human mesenchymal stem cells (MSC) are incorporated into Matrigel plugs that are implanted subcutaneously in immunodeficient nude mice. We modified the original protocol by (1) preculturing human ECFC on laminin, fibronectin, and collagen-coated plates, and (2) increasing the ECFC/MSC ratio. These modifications significantly increased the human vascular network in Matrigel implants. Two injections of one of our anti-TM4SF1 EL2 monoclonal antibodies, 8G4, effectively eliminated the human vascular component present in these plugs; they also abrogated human PC3 prostate cancer cells that were incorporated into the ECFC/MSC Matrigel mix. Together, these studies provide a mouse model for assessing tumor xenografts that are supplied by a human vascular network and demonstrate that anti-TM4SF1 antibodies such as 8G4 hold promise for cancer therapy.


Assuntos
Antígenos de Superfície/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Mesenquimais , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , Neovascularização Patológica , Engenharia Tecidual/métodos , Transcrição Genética
19.
Proc Natl Acad Sci U S A ; 111(28): 10137-42, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24982174

RESUMO

Endothelial colony-forming cells (ECFCs) are endothelial precursors that circulate in peripheral blood. Studies have demonstrated that human ECFCs have robust vasculogenic properties. However, whether ECFCs can exert trophic functions in support of specific stem cells in vivo remains largely unknown. Here, we sought to determine whether human ECFCs can function as paracrine mediators before the establishment of blood perfusion. We used two xenograft models of human mesenchymal stem cell (MSC) transplantation and studied how the presence of ECFCs modulates MSC engraftment and regenerative capacity in vivo. Human MSCs were isolated from white adipose tissue and bone marrow aspirates and were s.c. implanted into immunodeficient mice in the presence or absence of cord blood-derived ECFCs. MSC engraftment was regulated by ECFC-derived paracrine factors via platelet-derived growth factor BB (PDGF-BB)/platelet-derived growth factor receptor (PDGFR)-ß signaling. Cotransplanting ECFCs significantly enhanced MSC engraftment by reducing early apoptosis and preserving stemness-related properties of PDGFR-ß(+) MSCs, including the ability to repopulate secondary grafts. MSC engraftment was negligible in the absence of ECFCs and completely impaired in the presence of Tyrphostin AG1296, an inhibitor of PDGFR kinase. Additionally, transplanted MSCs displayed fate-restricted potential in vivo, with adipose tissue-derived and bone marrow-derived MSCs contributing exclusive differentiation along adipogenic and osteogenic lineages, respectively. This work demonstrates that blood-derived ECFCs can serve as paracrine mediators and regulate the regenerative potential of MSCs via PDGF-BB/PDGFR-ß signaling. Our data suggest the systematic use of ECFCs as a means to improve MSC transplantation.


Assuntos
Células Endoteliais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Becaplermina , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Feminino , Xenoenxertos , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Tirfostinas/farmacologia
20.
Hypertension ; 64(1): 165-71, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24752434

RESUMO

Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation, and migration toward vascular endothelial growth factor-A and fibroblast growth factor-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P=0.04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events.


Assuntos
Células Endoteliais/patologia , Sangue Fetal/citologia , Pré-Eclâmpsia/patologia , Células-Tronco/patologia , Adulto , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Gravidez , Estudos Prospectivos , Fatores de Risco , Fator A de Crescimento do Endotélio Vascular/farmacologia
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