Dr. Yoder is consultant to EndGenitor Technologies, Inc. and a member of the board of Rimedion Technologies, Inc.

Reagents and Solutions
EMG-2 media (Lonza, Cat. No. cc-3162 containing EBM-2 basal medium and EGM-2 SingleQuot kit Supplements, &amp; growth factors)
EBM-2 (Lonza, Cat. No. cc-3156) supplemented with the entire SingleQuot kit supplements &amp; growth factors (Lonza, Cat. No. cc-4176), 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin (10,000 U/ml)/streptomycin (10,000 &mu;g/ml)/amphotericin (25 &mu;g/ml). Store up to 1 month at 4 &deg;C. Recommended EGM-2 volumes to use for ECFC culture are 500 &mu;l/well for 24-well plates, 2 ml/well for 6-well plates, 5ml/25-cm2 flasks, and 10 ml/75-cm2 flasks, unless otherwise specified in the protocol.
Collagen I solution
Dilute 0.575 ml of glacial acetic acid (17.4N) in 495 ml of sterile distilled water (0.02 N final concentration). Sterile filter the dilute acetic acid with a 0.22-&mu;m vacuum filtration system. Add 25 mg rat tail collagen I to the dilute acetic acid to a final concentration of 50 &mu;g/ml. The amount of collagen added will vary depending on the collagen stock concentration. Store up to a month at 4 &deg;C.
Preparation of collagen I-coated tissue culture surfaces
Place 1 ml of collagen I solution in each well of a 6-well tissue culture plate (use 300 &mu;l/well for 24-well plates, 3ml/25-cm2 flasks, and 8 ml/75-cm2 flasks). Incubate 1 hour to overnight at 37 &deg;C. Remove the collagen I solution and wash surface two times, each time with PBS (use 500 &mu;l/well for 24-well plates, 2 ml/well for 6-well plates, 5ml/25-cm2 flasks, and 10 ml/75-cm2 flasks). Use plates immediately for cell cultures.
FACS staining buffer
Phosphate-buffered saline (PBS) supplemented with 2% (v/v) fetal bovine serum (FBS). Store up to 2 weeks at 4 &deg;C.
A. ECFC Outgrowth, Cloning and Expansion
<ol>
	<li>Collect CB with heparin (10 U heparin/ml blood) or EDTA as an anticoagulant and transport to the laboratory at room temperature and immediately (within 2 hours of infant's delivery) process the sample for mononuclear cell (MNC) isolation.</li>
	<li>Aliquot 15 ml of CB into each 50-ml conical centrifuge tube and add 20 ml of PBS to each tube of CB and pipette several times to mix. Draw up 15 ml Ficoll-Paque into a 20-ml syringe and attach a 20G needle or mixing cannula. Place the tip of the mixing cannula at the bottom of the tube of diluted blood and carefully underlay 15 ml Ficoll-Pague. Centrifuge the tubes 30 min at 740 x g, at room temperature, without the deceleration brake setting.</li>
	<li>Using a transfer pipette, remove the hazy layer of low-density MNCs located at the interface between the Ficoll-Paque and diluted plasma. Dispense the MNCs into a 50-ml conical tube containing 10 ml EGM-2 medium. Centrifuge the MNCs 10 min at 515 x g, at room temperature, with a high deceleration brake setting.</li>
	<li>Carefully aspirate and discard the supernatant. Wash the pelleted cells with EGM-2 twice (10 min at 515 x g) and re-suspend the MNCs in EGM-2 at 1.25 x 107 cells/ml. Prepare collagen I coated 6-well tissue culture plates by adding 1 ml rat-tail collagen type I (50 &mu;g/ml) per well and incubating the plate overnight. Pipette 4 ml of this MNC suspension into each well and place the cells in 37 &deg;C, 5% CO2 humidified incubator. A.1.5) After 24 hours (day 1), slowly remove the spent medium (do not disturb the loosely adherent cells) from the well with a pipette, slowly add 4 ml EGM-2 to the well, and return plate to the incubator. On day 2, refresh the medium by slowly removing the medium (do not disturb the loosely adherent cells) from the well with a pipette and adding 4 ml EGM-2 to each well. Repeat the medium change daily until day 7 and every other day thereafter until ECFC colonies appear for cloning. Typically colonies appear between day 5 and day 14 days7(Fig. 1).</li>
	<li>Visualize typical ECFC colonies with cobblestone morphology using inverted microscope (magnification 10x) and mark the colony boundaries with a marker on the underside of the well to indicate the position of each colony.</li>
	<li>On day 14, wash these primary colonies 2 times with PBS and harvest individual colonies using cloning cylinders and just enough TrypLE express to cover the cells inside the cylinders. After aspirating the final wash of PBS, place a sterile gel coated cloning cylinder around each colony and press firmly against the plate using sterile forceps. Add 2-3 drops of warm TrypLE express into each cloning cylinder and incubate for 3-5 min until cells within the cylinder begin to detach. Add approximately 250 &mu;l EGM-2 medium into the center of the cylinder and pipette up and down to generate single cell suspension. Transfer the single cell suspension from each cloning cylinder separately into an individual micro-centrifuge tube.</li>
	<li>Wash the area within the cylinder 2-3 times with approximately 250 &mu;l EGM-2 medium to collect all remaining cells and transfer the wash from each cylinder into the respective micro-centrifuge tube. Centrifuge the cells at high speed (&le;300 x g) on tabletop centrifuge for 5 min.</li>
	<li>Remove the supernatant and re-suspend the cell pellet in 1.5 ml fresh EGM-2 medium. Transfer the cell suspension (containing all the cells from individual colonies) from each tube into one well of a 24-well tissue culture plate pre-coated with 500 &mu;L rat-tail collagen I (50 &mu;g/ml).</li>
	<li>Place inside the incubator for expansion with media change performed every other day.</li>
	<li>When cells approach 80-90% confluency, remove the spent medium then wash the cells 2 times with PBS and add 500 &mu;l TrypLE express medium to each well of 24-well tissue culture plate. Place the plate inside the incubator for 3-5 min until cells begin to round up and detach. Add 1 ml of EGM-2 (with 10% defined FBS) to each well to collect the cells by washing and transfer them to a 15 ml tube. Wash the well with 1 ml EGM-2 medium to collect all remaining cells and transfer the wash from each well into respective 15 ml tube. Centrifuge the cells at 300 x g for 5 min.</li>
	<li>Remove the medium and re-suspend the cell pellet in 1 ml fresh EGM-2 medium (with 10% defined FBS). Obtain the viable cell count of an aliquot using a hemacytometer and trypan blue exclusion.</li>
	<li>Expand the cells by seeding about 5000 cells/cm2 onto a collagen I pre-coated tissue culture surfaces in EGM-2 media with media change every other day until cells approach 80-90% confluency before sub-culturing again.</li>
</ol>
B. In vitro Phenotypic Characterization of ECFCs: Endothelial Cell Surface Antigen Expression and Single Cell Assay for Clonal Proliferative Potential
<ol>
	<li>For endothelial cell surface antigen expression, detach cells with TrypLE express to collect cells by using methods described for ECFC cloning and expansion.</li>
	<li>Re-suspend cells in FACS staining buffer (10 X 106 cells/ 1ml FACS staining buffer) then add FcR blocking reagent (20 &mu;l/ 10 X 106 cells), mix gently and place on ice for 10 min.</li>
	<li>Aliquot 100 &mu;l of this cell suspension in micro-centrifuge tube for each endothelial surface antigen and isotype controls. Add appropriate amount of fluorochrome labeled human monoclonal antibodies that recognize endothelial antigens (CD31, CD144, CD146, and CD105) or hematopoietic antigens (CD45, and CD14) and leave on ice for 30 min protected from light. Caution: 1 X 106 cells are not required for each test. The authors typically prepare 0.5 to 1 X 105 cells/tube to obtain 2 X 104 analyzed events. Also, follow the manufacturer's recommendation for the amount of antibody to be used for each test. Some antibodies may require titrating to determine the optimal antibody concentration. All staining that the authors have performed are single color staining.</li>
	<li>After 30 min of incubation on ice, centrifuge each tube at high speed on a tabletop centrifuge for 5 min then remove the supernatant and re-suspend the cell pellet in FACS buffer.</li>
	<li>Analyze the samples on a flow cytometer to determine the percentage of cells that stain positively or negatively for each antigen. ECFCs stain uniformly positive for CD31, CD144, and CD146, but negative for CD45 and CD14 compared to the corresponding isotype controls.</li>
	<li>For single cell assay to examine the clonal proliferative potential, detach early passage (2-3) cells with TrypLE express to collect cells using methods similar to that was described for ECFC cloning and expansion.</li>
	<li>Infect these cells with enhanced green fluorescent protein (EGFP) expressing lentiviruses 16 and collect cells expressing EGFP by fluorescence cytometry. Caution: Working with lentivirus is considered a biohazard and all bio-safety precautions should be obeyed. </li>
	<li>Culture them as described for culturing ECFC in the section for ECFC expansion.</li>
	<li>Prepare Collagen I coated 96-well plates by adding 50 &mu;l rat-tail collagen type I (50 &mu;g/ml) per well and incubate the plate for overnight. Collect the EGFP+ ECFCs by using TrypLE express and resuspend them in EGM-2 medium with final concentration ~106 cells /ml.</li>
	<li>Use FACS Vantage (Becton Dickson) or other comparable sorter with low flow rate of 20 cells/second to sort one single EGFP+ ECFC per well of a 96-well plate and adjust the final volume to 200 &mu;l per well with EGM-2. Use an inverted fluorescence microscope to ensure that each well received only one cell. Alternatively, single cells can be sorted based on FSC and SSC and Sytox nuclear dye staining the colonies can be used for cell scoring and quantification.</li>
	<li>Incubate the plate over a period of two weeks with two media changes (200 &mu;l EGM-2 using multichannel pipetter) performed on day 5 and day 10. On day 14 of culture, wash each well with 100 &mu;l PBS before fixing the cells with 100 &mu;l of 4% paraformaldehyde for 30 min. For ECFCs that are not expressing EGFP, add Sytox reagent, a green fluorescent nuclear dye, following paraformaldehyde fixation and incubate at 4 &deg;C overnight.</li>
	<li>Use a fluorescence microscope to examine each well to quantitate the number of endothelial cells that expanded from a single cell cultured for 14 days. For quantitative analysis, score wells with 2 or more endothelial cells as positive (for proliferation) and examine them further for total endothelial cell count by visual counting with the fluorescence microscope.</li>
	<li>To display the obtained data, wells with an endothelial cell number of: 2 to 50, 51-2000, and 2001 or more, are considered as: endothelial cell clusters, low proliferative potential (LPP)-ECFCs, and high proliferative potential (HPP)-ECFCs respectively. ECFCs exhibit complete hierarchy of clonal proliferative potential by giving rise to endothelial clusters, LPPs, and HPPs when cultured at a single cell level for 14 days.7</li>
</ol>
C. In vivo Functional Characterization of ECFCs: In vivo Vessel Formation Assay to Examine the ECFC's Potential for Vasculogenesis
<ol>
	<li>Prepare cellularized gel implants by calculating the total volume (ml) of each of the following gel materials (with the final concentration in the parenthesis) needed to cast 1-ml gel (which will be later bisected to generate 2 implants): FBS (10%), EBM-2 10:1 (adjust the final volume to 1 ml), sodium bicarbonate (1.5 mg/ml), NaOH (adjust pH to 7.4), HEPES (25 mM), fibronectin (100 &mu;g/ml), and collagen I (1.5 mg/ml).</li>
	<li>After gel material calculation, detach cells with TrypLE express to collect cells using methods similar to that was described for ECFC cloning and expansion. Obtain a viable cell count of an aliquot using a hemacytometer and trypan blue. Transfer 2.4 million viable cells into a 50 ml-conical tube and pellet the cells by centrifugation at 515 x g, room temperature. Simultaneously, prepare gel matrix solution by adding calculated volumes of HEPES, sodium bicarbonate, EBM-2 10:1, FBS, fibronectin, and collagen to an ice-cold 50-ml conical tube (in the same order as they are listed). Mix thoroughly and adjust the pH to 7.4 with NaOH while keeping solution on ice.</li>
	<li>Discard the supernatant from the cells after centrifugation and re-suspend the pellet in 360 &mu;l warm EGM-2 and to it add pH adjusted 840 &mu;l of gel matrix solution, making the final volume of gel implant 1 ml. Mix the cells into gel matrix solution slowly until cells are thoroughly suspended in the gel solution.</li>
	<li>Transfer this 1 ml cellularized gel suspension to one well of a 12-well tissue culture plate and incubate for 20-30 min until the gel polymerizes. Gently cover the gel with 2 ml warm EGM-2 and incubate overnight.</li>
	<li>Immediately before the implantation, bisect the overnight incubated gel into two equal pieces using iris scissors and return the gel pieces to the same culture well containing EGM-2 medium.</li>
	<li>Use the animal surgical facility to sedate the animal (5-6 week old NOD/SCID mice) by administrating isoflurane anesthesia. Shave the lower part of abdomen and thoroughly clean the surgical site with alcohol pads and betadine or other antiseptic. Then isolate the area with drapes as per IACUC and institutional guidelines.</li>
	<li>Using sterile sharp iris scissors to make an approximately 5 mm incision in the lower quadrant of the abdomen, exposing the subcutaneous space between the skin and abdominal muscle. Perform blunt-dissection through the dermal layer from the abdominal muscle to create a 15-20 mm wide pocket leading superior into the upper abdominal quadrant. Repeat similar procedure to create similar open pocket in another side of the same mouse abdomen.</li>
	<li>Insert one half piece of gel into one side of abdominal pocket and another half piece of gel into another side of the abdominal pocket by lifting the dermal layer just caudal to the incision.</li>
	<li>After insertion of gels, close each incision with 2-3 stitches using 5-0 polypropylene suture on a cutting needle. Appropriately label the cage cards and perform post-surgical monitoring and analgesia as per the institutional and protocol requirements and allow 14 days for cells in these implanted gels to generate de novo vasculature.</li>
	<li>Finally, harvesting of gel implants is typically performed 14 days after implantation after euthanizing the mouse.</li>
	<li>Swab the abdominal area with alcohol pads and cut the abdominal skin caudal to the original incision line. Carefully, dissect out the implant by excising a flap of skin caudal to the probable location of the gel. Excise the implant by cutting circumferentially around the gel then place in zinc fixative (BD Biosciences, follow manufacturer's recommendation for optimal results) and allow them to fix for 1-2 hours at room temperature.</li>
	<li>Prepare the paraffin-embedded gels using standard histochemical protocols and prepare 5 &mu;m sections on glass slides to perform staining with hematoxylin and eosin, anti-human CD31 or anti-mouse CD31 to visualize the vasculature within the gel. ECFCs undergo de novo vasculogenesis to generate humanized vessels in cellularized gel implants inserted into immunodeficient mice for 14 days 4,8,11.</li>
</ol>
D. Representative Results
Using this ECFC derivation technique we have observed out-growth of primary colony formation as early as day 5 (Fig. 1). The out-growth ECFC colonies exhibited typical cobblestone appearance and gave rise to &gt;40 population doublings upon long term expansion after colony pickup by cloning. Expanded colonies expressed endothelial antigens, but did not express hematopoietic antigens (Fig. 2). Importantly, they displayed a complete hierarchy of clonal proliferative potential at a single cell level (Fig. 3). Moreover, ECFCs formed humanized blood vessels that are perfused with host RBCs when implanted into immunodeficient mice4, 6, 8, 11 (Fig. 4).
<img alt="Figure 1." src="/files/ftp_upload/3872/3872fig1.jpg" /> 
Figure 1. Isolation of mononuclear cells (MNCs) from cord blood and outgrowth of endothelial colony forming cells (ECFCs) from cultured MNCs. MNCs form buffy coat layer during Ficoll-Pague density gradient separation of cord blood cells. Isolation of buffy coat layer to culture MNCs on rat-tail collagen I coated plates results in outgrowth of ECFC colony in 5 to 14 days. The outgrowth ECFC colony (indicated by arrow heads) displayed cobblestone morphology7.
<img alt="Figure 2." src="/files/ftp_upload/3872/3872fig2.jpg" /> 
Figure 2. Representative in vitro phenotypic assessment of endothelial and hematopoietic cell surface antigen expression. Immunophenotyping of cord blood-derived ECFCs revealed that ECFCs expressed endothelial antigens CD31, CD34, CD144, CD146, Flt-1, Flk-1, Flt-4, and Nrp2 but did not express hematopoietic antigens CD45, CD14, CD11b, cKit, CXCR4 or AC1337, 8.
<img alt="Figure 3." src="/files/ftp_upload/3872/3872fig3.jpg" /> 
Figure 3. Representative in vitro quantitation of the clonogenic and proliferative potential of CB derived ECFCs. (A) cord blood-derived ECFCs display clonal proliferative potential with a hierarchy of colonies ranging from clusters of 2-50 cells up to colonies of &gt;2001. (B) Micrographs of hierarchy of colonies (colonies stained with, Sytox, a green fluorescent nuclear dye to take better quality pictures) obtained after cord blood-derived ECFCs were cultured at a single cell level for 14 days. Scale bar represents 100&mu;m7, 8.
<img alt="Figure 4." src="/files/ftp_upload/3872/3872fig4.jpg" /> 
Figure 4. Representative in vivo functional characterization of cord blood-derived ECFCs. A) H&amp;E staining of cord blood-derived ECFC containing cellularized gel implants indicated microvessel (filled with host RBCs) formation in collagen-fibronectin gel after 14 days of implantation. B) Anti-human CD31 staining (brown staining) further confirms the human origin of these vessels.

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473, 298-307 (2011).</li><li>Asahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+putative+progenitor+endothelial+cells+for+angiogenesis.">Isolation of putative progenitor endothelial cells for angiogenesis.</a> Science. 275, 964-967 (1997).</li><li>Urbich, C., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells:+characterization+and+role+in+vascular+biology.">Endothelial progenitor cells: characterization and role in vascular biology.</a> Circ. Res. 95, 343-353 (2004).</li><li>Critser, P. J., Voytik-Harbin, S. L., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+and+defining+cells+to+engineer+human+blood+vessels.">Isolating and defining cells to engineer human blood vessels.</a> Cell. Prolif. 44, 15-21 (2011).</li><li>Matthias, M., David, N., Josef, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+bench+to+bedside:+what+physicians+need+to+know+about+endothelial+progenitor+cells.">From bench to bedside: what physicians need to know about endothelial progenitor cells.</a> Am. J. Med. 124, 489-4897 (2011).</li><li>Ingram, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105, 2783-276 (2005).</li><li>Ingram, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104, 2752-2760 (2004).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Reinisch, A., Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Animal+Serum+Free+Expansion+of+Human+Umbilical+Cord+Derived+Mesenchymal+Stromal+Cells+(MSCs)+and+Endothelial+Colony+Forming+Progenitor+Cells+(ECFCs.">Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs.</a> J. Vis. Exp. (32), e1525 (2009).</li><li>Au, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+in+vivo+potential+of+endothelial+progenitor+cells+from+human+umbilical+cord+blood+and+adult+peripheral+blood+to+form+functional+long-lasting+vessels.">Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels.</a> Blood. 111, 1302-135 (2008).</li><li>Critser, P. J., Kreger, S. T., Voytik-Harbin, S. L., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+matrix+physical+properties+modulate+endothelial+colony+forming+cell-derived+vessels+in+vivo.">Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo.</a> Microvasc. Res. 80, 23-30 (2010).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+robust+and+functional+vascular+networks+in+vivo+with+human+adult+and+cord+blood-derived+progenitor+cells.">Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells.</a> Circ. Res. 103, 194-202 (2008).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109, 4761-4768 (2007).</li><li>Hofmann, N. A., Reinisch, A., Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Large+Scale+Expansion+of+Adult+Human+Endothelial+Colony+Forming+Progenitor+Cells.">Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells.</a> J. Vis. Exp. (32), e1524 (2009).</li><li>Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+circulating+endothelial+cells+and+endothelial+outgrowth+from+blood.">Origins of circulating endothelial cells and endothelial outgrowth from blood.</a> J. Clin. Invest. 105, 71-77 (2000).</li><li>Witting, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Large+Volume+Lentiviral+Vector+Production+Using+Flow+Electroporation.">Efficient Large Volume Lentiviral Vector Production Using Flow Electroporation.</a> Hum. Gene. Ther. , (2011).</li><li>Yoon, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+neovascularization+by+mixed+transplantation+of+early+endothelial+progenitor+cells+and+late+outgrowth+endothelial+cells:+the+role+of+angiogenic+cytokines+and+matrix+metalloproteinases.">Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases.</a> Circulation. , 112-1618 (2005).</li><li>Dubois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+progenitor+cell+populations+on+left+ventricular+remodeling+and+myocardial+neovascularization+after+myocardial+infarction.">Differential effects of progenitor cell populations on left ventricular remodeling and myocardial neovascularization after myocardial infarction.</a> J. Am. Coll. Cardiol. 55, 2232-2243 (2010).</li><li>Medina, R. J., O'Neill, C. L., Humphreys, M. W., Gardiner, T. A., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowth+endothelial+cells:+characterization+and+their+potential+for+reversing+ischemic+retinopathy.">Outgrowth endothelial cells: characterization and their potential for reversing ischemic retinopathy.</a> Invest. Ophthalmol. Vis. Sci. 51, 5906-5913 (2010).</li></ol>

No conflicts of interest declared.

Phenotypic and functional characterization of putative endothelial progenitor cells is important to identify the bona fide ECFCs that are capable of clonally and serially re-plating in culture and give rise to durable and functional implantable blood vessels in vivo. Human umbilical cord blood is enriched with ECFCs and the concentration of these circulating cells declines with aging or disease10. Recent studies suggest that ECFC may play important roles in vascular repair or regeneration in ...

<table><tbody><tr><td>Heparin Sodium Injection, USP</td><td>APP Pharmaceuticals</td><td>504031</td><td></td></tr><tr><td>Ficoll-Pague</td><td>Amersham</td><td>17-1440-03</td><td></td></tr><tr><td>Mixing cannula</td><td>Maersk Medical</td><td>500.11.012</td><td></td></tr><tr><td>EGM-2</td><td>Lonza Inc.</td><td>CC-3162</td><td></td></tr><tr><td>Defined FBS</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>TrypLE express</td><td>GIBCO, by Life Technologies</td><td>12605</td><td></td></tr><tr><td>Rat type I collagen</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>356234</td><td></td></tr><tr><td>FcR Block</td><td>Miltenyi Biotec</td><td>130-059-901</td><td></td></tr><tr><td>hCD31, FITC conjugated</td><td>BD Biosciences</td><td>555445</td><td></td></tr><tr><td>hCD45, FITC conjugated</td><td>BD Biosciences</td><td>555482</td><td></td></tr><tr><td>hCD14, FITC conjugated</td><td>BD Biosciences</td><td>555397</td><td></td></tr><tr><td>hCD144, PE conjugated</td><td>eBioscience</td><td>12-1449-80</td><td></td></tr><tr><td>hCD146, PE conjugated</td><td>BD Biosciences</td><td>550315</td><td></td></tr><tr><td>hCD105, PE conjugated</td><td>Invitrogen</td><td>MHCD10504</td><td></td></tr><tr><td>Ms IgG&lt;sub&gt;1,k&lt;/sub&gt; antibody, FITC conjugated</td><td>BD Biosciences</td><td>555748</td><td></td></tr><tr><td>Ms IgG&lt;sub&gt;1,k&lt;/sub&gt; antibody, PE conjugated</td><td>BD Biosciences</td><td>559320</td><td></td></tr><tr><td>Ms IgG&lt;sub&gt;2a,k&lt;/sub&gt; antibody, FITC conjugated</td><td>BD Biosciences</td><td>555573</td><td></td></tr><tr><td>Anti-human CD31</td><td>Dako</td><td>clone JC70/A</td><td></td></tr><tr><td>Anti-mouse CD31</td><td>BD Biosciences</td><td>553370</td><td></td></tr><tr><td>0.22-&amp;mu;m vacuum filtration system</td><td>EMD Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>Glacial acetic acid, 17.4N</td><td>Fisher Scientific</td><td>A38-500</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Invitrogen</td><td>15240-062</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>IHC Zinc Fixative</td><td>BD Biosciences</td><td>550523</td><td></td></tr><tr><td>Sytox green reagent</td><td>Invitrogen</td><td>S33025</td><td></td></tr><tr><td>Cloning cylinders, sterile</td><td>Fisher Scientific</td><td>07-907-10</td><td></td></tr></tbody></table>

phenotypic and functional characterization of endothelial colony forming cells derived from human umbilical cord blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Watch this Scientific Journal Video about Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood at JoVE.com

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

phenotypic-functional-characterization-endothelial-colony-forming

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.2K Views.  Indiana University School of Medicine. Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Video: Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Immunology and Infection

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Developmental Biology

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.