This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.
Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.
Até recentemente, acreditava a geração pós-natal de novos vasos sanguíneos a ocorrer exclusivamente através de um processo conhecido como angiogênese, definida como o surgimento de novos vasos a partir das células endoteliais dos vasos pré-existentes. 1 Este processo contrasta da vasculogênese, ou a formação de novo de vasos sanguíneos a partir de células progenitoras endoteliais, o que foi pensado para ocorrer exclusivamente durante a embriogénese. 2 No entanto, estudos mais recentes foram identificados e isolados circulantes células progenitoras endoteliais (CPE) no sangue periférico de adultos. Estas células possuem a capacidade de se diferenciarem em células endoteliais maduras em cultura e são acreditados para participar na vasculogénese pós-natal. 3,4
Os protocolos para o isolamento e expansão destas EPCs envolvem tipicamente a cultura de células mononucleares de sangue periférico (PBMNCs) em meio contendo factores de crescimento endoteliais, incluindo Vasculfactor de crescimento endotelial AR (VEGF) e factor de crescimento de fibroblastos-2. 5-8 culturas EPC produzir uma variedade de tipos de células diferentes dramaticamente. Culturas iniciais (<7 dias) são dominadas por um tipo de célula monocítica, conhecido na literatura como "precoces" EPCs. Apesar do seu nome, estas células expressam o CD14 marcador de monócitos, são negativas para CD34 marcador de células progenitoras e expressar apenas níveis mínimos dos marcadores CD31 endotelial clássica e VEGF receptor 2 (VEGFR2). 5 cultura contínua dá origem a uma população secundária de células, conhecido como EPCs final excrescência ou células endoteliais conseqüência sangue (BOECs), que aparecem como colónias discretas de células endoteliais-like. Ao contrário das primeiras EPCs monocíticas, BOECs, que também tem sido chamado de formação de colónias de células endoteliais (ECFCs), células endoteliais ou células endoteliais excrescência de fim de crescimento, exibem a morfologia paralelepípedos que é típico de monocamadas de células endoteliais e são altamente semelhantes em marcador de superfície5 e 9, a expressão do gene para amadurecer as células endoteliais.
A geração de células endoteliais semelhantes a partir de sangue periférico oferece várias vantagens, particularmente para o estudo da disfunção das células endoteliais associadas com desordens vasculares, tais como hipertensão arterial pulmonar (HAP) 10 ou de von Willebrand doença. 11 Antes da disponibilidade de BOECs, endotelial células só pode ser derivada a partir de órgãos explantados no momento da morte ou transplante de órgãos, ou isolado a partir da veia umbilical ao nascimento. Esta disponibilidade reduzida representada uma limitação séria para a compreensão da biologia das células endoteliais a partir de pacientes com doenças cardiovasculares, assim como as interacções entre as células endoteliais e as células do sangue ou quer células murais. Além disso, o isolamento e a cultura de uma população pura de células endoteliais a partir destas fontes é tecnicamente exigente e as células obtidas por estes métodos apresentam apenas uma LIMITEd capacidade proliferativa. BOECs portanto oferecer um substituto valioso para o isolamento e cultura de células endoteliais primárias derivadas do paciente.
Para além das suas aplicações in vitro, BOECs também são potencialmente úteis em terapias de transplante de células autólogas. Estas aplicações incluem tanto o transplante de células endoteliais para promover neovascularização (ver 12 e referências ai), bem como a geração de células estaminais pluripotentes induzidas (iPSCs). 13 iPSCs BOEC-derivados pode ser usada para modelar doenças e oferecer imenso potencial como o ponto de partida material para terapias com células autólogas. BOECs reprogramar mais rápido e com uma maior eficiência do que os fibroblastos da pele. Além disso, também permitem que BOECs para a geração de iPSCs que estão livres de anomalias karyotypic, o que é uma característica essencial de qualquer tecnologia que será adequado para aplicações de translação. A capacidade para gerar iPSCs de uma amostra de sangue de um pacientelso elimina a necessidade de uma biópsia da pele e a geração de fibroblastos da pele, facilitando assim a geração de células de pacientes com distúrbios cicatrização de feridas, ou os muito jovens.
O protocolo detalhado abaixo, aprovado pelo e conduzida de acordo com as orientações do Comitê de Serviço de Ética em Pesquisa Nacional (leste da Inglaterra), fornece um método simples e confiável para a geração de BOECs com rendimento superior a 90% de um volume relativamente pequeno (60 ml) de sangue periférico. Estas células são altamente proliferativas e podem ser passadas várias vezes, permitindo a geração de centenas de milhões de células a partir de uma única amostra de sangue.
We present a detailed protocol that allows for the robust and efficient derivation of BOECs from adult peripheral blood mononuclear cells (PBMNCs). Our protocol includes two important refinements that represent advances on previous methods of BOEC isolation.14-16 These include the absence of heparin in the initial PBMNC culture medium and the use of defined, embryonic stem cell-qualified serum. This latter refinement is of particular importance. Embryonic stem cell (ESC)-qualified serum is a more consistent grade of serum and, although it is not known yet what component(s) are enriched in the serum that benefit BOEC isolation, the impact of this defined serum on the efficiency of BOEC generation is clear in our hands. In addition, we have also had success in isolating BOECs using human serum, thereby allowing for the generation of BOECs for clinical translation. In our hands, this refined protocol results in the successful isolation of stable BOEC cultures from greater than 90% of donors, making it one of the most reliable BOEC generation methods reported thus far. Although the use of particular sera is critical to BOEC generation, it also represents a primary limitation of the current protocol. Future improvements to the technique could include the generation of these cells in serum-free, defined culture conditions.
Critical Steps in the protocol include processing blood samples as soon as possible after collection, complete harvesting of the buffy coat cells after density gradient centrifugation and the timely passaging of initial colonies from P0 to P1. This passaging step is critical to establishment of a stable isolation. Like other endothelial cells, BOECs appear to be very sensitive to plating density. If the plating density after passaging is too low, the BOECs will not proliferate. Conversely, if the colonies are allowed to become overconfluent before passaging, the cells will also cease to proliferate and have the tendency to convert into an elongated, mesenchymal cell phenotype. If few colonies appear from days 7 to 14, or if the colonies are small in size, troubleshooting can include increasing cell density by passaging P0 colonies into a T-25 flask instead of a T-75.
Once the technique is mastered, the resultant BOECs can be used in several applications, including in vitro studies of endothelial cell biology, disease modeling and drug screening, as well as in vivo cell transplantation therapies. An important consideration for the development of any cell therapy process is to use cells that are free from pathogenic mutations. We have previously shown that BOECs isolated using our protocol possess genomes that are free from copy number variations and are thus representative of the individual from which they were collected. In addition, we have also demonstrated that the majority of BOEC-derived iPSC lines are free from copy number variations.13 This contrasts with previous reports of copy number variation in fibroblast-derived iPSCs. To date, these cells remain the only iPSCs for which this degree of genomic fidelity has been reported. This feature is important for the field of iPSC biology and the use of iPSCs in disease modeling, drug screening and future cell transplantation therapies.
The authors have nothing to disclose.
This work was supported by grants funded by the British Heart Foundation (BHF), Dinosaur Trust, McAlpine Foundation, Fondation Leducq, Fight for Sight, the Cambridge Biomedical Research Centre, National Institute of Health Research including (i) the BHF Oxbridge Centre of Regenerative Medicine [RM/13/3/30159], (ii) the BHF Cambridge Centre of Research Excellence, (iii) Addenbrooke’s Hospital, Cambridge University Hospitals NHS Foundation Trust and (iv) Papworth Hospital NHS Foundation Trust, and supported the Cambridge NIHR BRC Cell Phenotyping Hub. MLO is funded by a BHF Intermediate Fellowship. FNK is funded by a BHF PhD Studentship.
For blood collection | |||
60 mL syringe with luer-lok tip | BD | 309653 | |
19G Surflo Winged Infusion Set | Terumo | SV-19BL | |
50 mL conical centrifuge tube | StarLab | E1450 | 2 per donor |
Sodium Citrate | Martindale Pharmaceuticals | 270541 | |
Name | Company | Catalog Number | コメント |
For buffy coat isolation | |||
Ficoll-Paque Plus | GE Healthcare | 17-1440-03 | |
Dulbecco’s PBS (without Ca2+ and Mg2+) | Sigma-Aldrich | D8537 | |
Sterile wrapped plastic transfer pipettes | Appleton Woods | KC231 | |
Turk’s Solution | Millipore | 1.093E+09 | |
Name | Company | Catalog Number | コメント |
For cell culture, passaging and freezing cells | |||
Type 1 Collagen (derived from rat tail) | BD Biosciences | 35-4236 | |
Dulbecco’s PBS (without Ca2+ and Mg2+) | Sigma-Aldrich | D8537 | |
0.02M Acetic Acid | Sigma-Aldrich | A6283 | prepared in reagent grade water |
Endothelial Growth Medium-2MV (containing Bullet Kit, but not serum) |
Lonza | CC-3202 | Note: It is essential that the medium does not contain heparin. Do not use EGM-2. |
Fetal Bovine Serum (U.S.), Defined | Hyclone | SH30070 | |
10x Trypsin EDTA | Gibco | T4174 | Dilute to 1x in PBS prior to use |
Heat Inactivated FBS | Gibco | 10500-064 | |
DMEM | Gibco | 41965-039 | |
DMSO | Sigma-Aldrich | 276855 | |
Nalgene Mr. Frosty Freezing Container | Sigma-Aldrich | C1562 | |
Name | Company | Catalog Number | コメント |
For flow cytometric characterization | |||
FITC-conjugated mouse anti-human CD14 | BD Biosciences | 555397 | Mouse IgG1k, Clone: WM59 Dilution: 1:20 |
FITC-conjugated mouse anti-human CD31 | BD Biosciences | 555445 | Mouse IgG1k, Clone: WM59 Dilution: 1:20 |
APC-conjugated mouse anti-human CD34 | BD Biosciences | 555824 | Mouse IgG1k, Clone: 581/CD34 Dilution: 1:20 |
FITC-conjugated mouse anti-human CD45 | BD Biosciences | 560976 | Mouse IgG1k, Clone: HI30 Dilution: 1:20 |
APC-conjugated mouse anti-human VEGFR2 | R&D Systems | FAB357A | Mouse IgG1, Clone: 89106 Dilution: 1:10 |
FITC-conjugated mouse IgG1k isotype control | BD Biosciences | 555748 | Clone: MOPC-21 Dilution: 1:20 |
APC-conjugated mouse IgG1k isotype control | BD Biosciences | 555751 | Clone: MOPC-21 Dilution: 1:20 |
APC-conjugated mouse IgG1k isotype control | R&D Systems | IC002A Dilution: 1:10 |
Clone: 11711 |
EDTA, 0.5M solution | Sigma-Aldrich | E7889 | |
Name | Company | Catalog Number | コメント |
For immunofluorescent microscopy | |||
Corning Costar 24-well tissue culture plate | Sigma-Aldrich | CLS3527 | |
Paraformaldehyde | Sigma-Aldrich | 158127 | |
BSA | Sigma-Aldrich | A7906 | |
Polysorbate 20 | Sigma-Aldrich | P2287 | |
Monoclonal mouse anti-human CD34 antibody | R&D Systems | MAB72271 | Clone 756510, IgG1, use at 10 μg/ml |
Polyclonal goat anti-human VE-cadherin (CD144) | R&D Systems | AF938 | Antigen affinity- purified IgG, use at 1:300 |
Monoclonal rabbit anti-human Von Willebrand Factor (vWF) | Abcam | ab154193 | Clone EPSISR15, use at 1:250 |
Donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate | Life Technologies | A-21202 | Polyclonal, 2 mg/ml, use at 1:200 |
Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate | Life Technologies | A-11055 | Polyclonal, 2 mg/ml, 1:200 |
Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 568 conjugate | Life Technologies | A-10042 | Polyclonal, 2 mg/ml, 1:200 |
DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) | Sigma-Aldrich | D9542 | use at 1 μg/ml |