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.
Fino a poco tempo, la generazione post-natale di nuovi vasi sanguigni è creduto avvenire esclusivamente attraverso un processo noto come angiogenesi, definito come il germogliare di nuovi vasi dalle cellule endoteliali dei vasi preesistenti. 1 Questo processo contrasta dalla vasculogenesi, o la formazione de novo di vasi sanguigni da progenitori endoteliali, che è stato pensato per manifesta esclusivamente durante l'embriogenesi. 2 Tuttavia, studi più recenti hanno identificato e isolato cellule progenitrici endoteliali circolanti (EPC) nel sangue periferico di adulti. Queste cellule possiedono la capacità di differenziarsi in cellule endoteliali mature di cultura e si ritiene che partecipare a vasculogenesi postnatale. 3,4
Protocolli per l'isolamento e l'espansione di queste EPC tipicamente coinvolgono la coltura di cellule mononucleate del sangue periferico (PBMNCs) in supporti contenenti fattori di crescita endoteliali, tra cui vascular fattore di crescita endoteliale (VEGF) e le culture di crescita dei fibroblasti fattore-2. 5-8 EPC produrre una varietà di tipi di cellule drammaticamente diversi. Culture iniziali (<7 giorni) sono dominati da un tipo di cellula monocitica, noto in letteratura come "early" EPC. Nonostante il loro nome, queste cellule esprimono il CD14 marcatore monociti, sono negativi per il CD34 marcatore progenitore ed esprimono livelli solo minimi della classica endoteliale marcatori CD31 e VEGF recettore 2 (VEGFR2). 5 coltura continua dà luogo ad una popolazione secondaria di cellule, noto come EPC fine escrescenza o cellule endoteliali escrescenza del sangue (BOECs), che appaiono come colonie discreti di cellule endoteliali-like. A differenza dei primi monocitiche EPC, BOECs, che sono stati anche chiamati formanti colonie cellule endoteliali (ECFCs), cellule endoteliali escrescenza o cellule endoteliali tardo-escrescenza, presentano la morfologia ciottoli che è tipica di monostrati cellule endoteliali e sono molto simili a marcatore di superficie5 e l'espressione genica 9 a maturare cellule endoteliali.
La generazione di cellule endoteliali-simili da sangue periferico offre numerosi vantaggi, in particolare per lo studio della disfunzione delle cellule endoteliali associata a disturbi vascolari quali ipertensione arteriosa polmonare (PAH) 10 o malattia di von Willebrand. 11 Prima della disponibilità di BOECs, endoteliale cellule potrebbero essere derivate solo da organi espiantati al momento della morte o trapianto di organi, o isolate dalla vena ombelicale al momento della nascita. Questa ridotta disponibilità rappresentato una seria limitazione alla comprensione della biologia delle cellule endoteliali di pazienti con disturbi cardiovascolari, nonché le interazioni tra cellule endoteliali e sia cellule del sangue o cellule murali. Inoltre, isolando e coltivando una popolazione pura di cellule endoteliali da queste fonti è tecnicamente difficile e le cellule derivate da questi metodi presentano solo un limited capacità proliferativa. BOECs quindi offrire un surrogato valido per l'isolamento e la coltura di cellule endoteliali primarie derivate paziente.
Oltre alle loro applicazioni in vitro, BOECs sono anche potenzialmente utili in autologhe terapie di trapianto cellulare. Queste applicazioni comprendono sia il trapianto di cellule endoteliali di promuovere la neovascolarizzazione (vedi 12 e riferimenti ivi), nonché la generazione di cellule staminali pluripotenti indotte (iPSCs). 13 iPSCs BOEC derivati possono essere utilizzate per modellare la malattia e offrono un potenziale immenso come partenza materiale per terapie cellulari autologhe. BOECs riprogrammare più velocemente e con un rendimento superiore fibroblasti cutanei. Inoltre, BOECs permettono anche per la generazione di iPSCs che siano privi di anomalie del cariotipo, che è una caratteristica essenziale di qualsiasi tecnologia che sarà adatto per applicazioni traslazionali. La capacità di generare iPSCs da un campione di sangue del paziente unaLSO elimina la necessità di una biopsia cutanea e la generazione di fibroblasti cutanei, facilitando in tal modo la generazione di cellule da pazienti con disturbi guarigione delle ferite, o molto giovane.
Il protocollo di seguito dettagliato, approvato e condotto in conformità con le linee guida del Comitato Research Service Etico Nazionale (Est Inghilterra), fornisce un metodo semplice e affidabile per la generazione di BOECs con efficienza superiore al 90% da un volume relativamente piccolo (60 ml) di sangue periferico. Queste cellule sono altamente proliferative e possono essere diversi passaggi più volte, permettendo la generazione di centinaia di milioni di cellule da un singolo campione di 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 |