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.
Inntil nylig var det post-natal generasjon av nye blodkar antas å oppstå utelukkende gjennom en prosess som kalles angiogenese, definert som spirende av nye skip fra endotelcellene av eksisterende fartøy. 1 Denne prosessen kontraster fra vaskulogenese, eller de novo dannelsen av blodkar fra endotel-forløpere, noe som ble antatt å skje utelukkende under embryogenese. 2. Imidlertid har nyere studier identifisert og isolert sirkulerende endoteliale progenitorceller (PP-) i perifert blod hos voksne. Disse cellene har evnen til å differensiere til modne endotelceller i kultur og antas å delta i postnatal vaskulogenese. 3,4
Protokoller for isolering og utvidelse av disse EPCs involverer typisk kultur av perifere mononukleære blodceller (PBMNCs) i media inneholdende endoteliale vekstfaktorer, inkludert vascular endotelial vekstfaktor (VEGF), og fibroblast vekstfaktor-2. 5-8 EPC kulturer produsere en rekke dramatisk forskjellige celletyper. Initial kulturer (<7 dager) er dominert av et monocyttisk celletype, som er kjent i litteraturen som "tidlig" EPCs. Til tross for sitt navn, er disse cellene uttrykker monocytt markør CD14, er negative for progenitor markør CD34 og uttrykker bare minimale nivåer av den klassiske endotel markørene CD31 og VEGF-reseptor 2 (VEGFR2). 5 Fortsatt kultur gir opphav til et sekundært populasjon av celler, kjent som sene utvekst EPCs eller blod utvekst endotelceller (BOECs), som vises som diskrete kolonier av endotelceller lignende celler. I motsetning til monocyttiske tidlige EPCs, BOECs, som også har blitt kalt endoteliale kolonidannende celler (ECFCs), utvekst endotelceller eller sen-utvekst endotelceller, oppviser den brostein morfologi som er typisk for endotel-cellemonolagene og er svært like i overflatemarkør5 og genuttrykk 9 til modne endotelceller.
Genereringen av endotel-lignende celler fra perifert blod byr på flere fordeler, særlig for studiet av endotelial celledysfunksjon i forbindelse med vaskulære forstyrrelser slik som pulmonal arteriell hypertensjon (PAH) 10 eller von Willebrands sykdom. 11 Før tilgjengeligheten av BOECs, endotelial Cellene kan bare bli avledet fra eksplanterte organer på tidspunktet for død eller organtransplantasjon, eller isolert fra navlevenen ved fødselen. Dette reduserte tilgjengelighet representerte en alvorlig begrensning for å forstå biologien til endotelceller fra pasienter med kardiovaskulære lidelser, så vel som interaksjonen mellom endotelceller og enten blodceller eller veggceller. Videre isolering og dyrking av en ren populasjon av endotelceller fra disse kildene er teknisk krevende og cellene avledet av disse metodene oppviser bare en Limited proliferativ kapasitet. BOECs gir derfor en verdifull surrogat for isolering og kultur av pasient-avledet primære endotelceller.
I tillegg til deres in vitro-applikasjoner, BOECs er også potensielt nyttige i autologe celle transplantasjonsterapi. Disse programmene inkluderer både endotelial celletransplantasjon for å fremme neovaskularisering (se 12 og referanser deri), samt generering av induserte pluripotente stamceller (iPSCs). 13 BOEC-avledet iPSCs kan brukes til sykdom modellering og har enormt potensial som utgangs materiale for autologe cellen terapi. BOECs omprogrammere raskere og med høyere virkningsgrad enn hudfibroblaster. Videre BOECs også gi rom for generering av iPSCs som er fri for karyotypic abnormiteter, som er en viktig funksjon i enhver teknologi som vil være egnet for translasjonsforskning applikasjoner. Evnen til å generere iPSCs fra en pasient blodprøve enlso eliminerer behovet for en hud biopsi og generering av hudfibroblaster, for derved å lette genereringen av celler fra pasienter med sårheling lidelser, eller de svært unge.
Protokollen beskrevet nedenfor, godkjent av og utføres i samsvar med retningslinjene fra Den nasjonale forskningsetiske Tjenesten Committee (East of England), gir en enkel og pålitelig metode for generering av BOECs med mer enn 90% effektivitet fra et relativt lite volum (60 ml) i perifert blod. Disse cellene er svært proliferativ og kan passaged gjentatte ganger, noe som åpner for generering av hundrevis av millioner av celler fra en enkelt blodprøve.
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 | Comments |
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 | Comments |
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 | Comments |
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 | Comments |
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 |