This work was partially supported by NIH grant K99EB009096-01A1 to J.M.-M.

1. Preparation
<ol>
	<li>Make EGM-2 medium (500 mL)
	<ul>
		<li>Add 100 mL of fetal bovine serum (FBS) to 395 mL of Endothelial Basal Medium (EBM-2).</li>
		<li>Add 5 mL of 100x Glutamine-penicillin-streptomycin solution (GPS).</li>
		<li>Add all the EGM-2 SingleQuots supplements except for hydrocortisone (i.e., VEGF, hFGF-B, R-IGF-1, hEGF, Heparin, ascorbic acid, and GA-1000).</li>
		<li>Filter sterilized with a 0.2-&mu;m pore size vacuum filter.</li>
	</ul>
	</li>
	<li>Make MSCGM medium (500 mL)
	<ul>
		<li>Add 5 mL of 100x GPS to 440 mL of Mesenchymal Stem Cell Basal Medium (MSCBM).</li>
		<li>Add all the content of the MSCGM SingleQuots kit.</li>
		<li>Add hFGF-B aliquot from EGM-2 SingleQuots supplements.</li>
		<li>Filter sterilized with a 0.2-&mu;m pore size vacuum filter.</li>
	</ul>
	</li>
	<li>Make DMEM medium (500 mL)
	<ul>
		<li>Add 50 mL of fetal bovine serum (FBS) to 440 mL of 1x high glucose Dulbecco's Modified Eagle Medium (DMEM).</li>
		<li>Add 5 mL of 100x GPS.</li>
		<li>Add 5 mL of nonessential amino acid solution.</li>
		<li>Filter sterilized with a 0.2-&mu;m pore size vacuum filter.</li>
	</ul>
	</li>
	<li>Make collagen/fibronectin solution (3.6 mL; made same day of injection)
	<ul>
		<li>Add 0.4 mL of 10x DMEM to 150 &mu;L of distilled H2O.</li>
		<li>Add 100 &mu;L of 1M HEPES (25 mM final).</li>
		<li>Carefully, add 2.4 mL of bovine collagen (5 mg/mL stock; 3 mg/mL final) and mix gently on ice.</li>
		<li>Adjust the pH to neutral by adding 1N NaOH solution (approximately 30 &mu;L).</li>
		<li>Use phenol red indicator to assess neutral pH; alternative, use pH test paper to monitor pH.</li>
		<li>Add 120 &mu;L of human fibronectin (1 mg/mL stock; 30 &mu;g/mL final)</li>
		<li>Add 0.4 mL of FBS (10% final)</li>
		<li>keep solution on ice until use.</li>
	</ul>
	</li>
	<li>Make fibrinogen solution (1 mL; made same day of injection)
	<ul>
		<li>Add 30 mg of powdered fibrinogen to 1 mL of 0.9N NaCl (pH 7.4) solution (30 mg/mL final).</li>
		<li>Prior to use, incubate the fibrinogen solution at 37&deg; C for 30 minutes.</li>
		<li>Mix gently, without vortexing, prior to its addition to the collagen/fibronectin gel.</li>
	</ul>
	</li>
	<li>Make thrombin solution (200 mL)
	<ul>
		<li>Add 1 KU of powdered thrombin to 200 mL of 0.9% NaCl normal saline (50 U/mL).</li>
		<li>Filter sterilized with a 0.2-&mu;m pore size syringe filter and store at -20&deg; C until use.</li>
		<li>Prior to use, dilute with 0.9% NaCl normal saline to a final concentration of 10 U/mL.</li>
	</ul>
	</li>
	<li>Make 1% gelatin solution (500 mL)
	<ul>
		<li>Add 5 g of powdered gelatin to 500 mL of Dulbecco's phosphate buffered saline (PBS).</li>
		<li>Autoclaved at 121 &deg;C for 30 min.</li>
		<li>Filter sterilized with a 0.2-&mu;m pore size vacuum filter.</li>
	</ul>
	</li>
	<li>Coat 100-mm tissue culture plates with 1% gelatin coating solution
	<ul>
		<li>Add 10 mL of 1% gelatin solution to each 100-mm tissue culture plate.</li>
		<li>Incubate plates at 37&deg;C for 60 min.</li>
		<li>Prior to use, remove the gelatin solution and wash the plates once with PBS.</li>
	</ul>
	</li>
</ol>
2. Culture of Human Cord Blood-Derived Endothelial Colony-Forming Cells (ECFCs)
This protocol assumes frozen vials of ECFC are available in the laboratory before this experiment. ECFC can be isolated from the mononuclear cell fraction of either umbilical cord blood or adult peripheral blood as previously described 7.
<ol>
	<li>Thaw one vial of ECFCs (typically 0.5-1x106 cells in 1 mL of freezing media) taken from the liquid nitrogen storage tank and immediately dilute its content in a 15-mL conical tube containing 10 mL of DMEM medium. Spin at 1200 rpm for 5 min. Remove supernatant and resuspend the cell pellet in 10 mL of warm EGM-2 medium.</li>
	<li>Add the 10 mL of ECFC suspension into one 1% gelatin-coated 100-mm tissue culture plate. Place the plate in a humidified incubator at 37&deg;C and 5%CO2. Next day, gently aspirate out unbound cells and medium, and feed bound cells with 10 mL of fresh EGM-2 medium.</li>
	<li>Feed the plate every 2-3 days with EGM-2 medium. Allow cells to expand such that the plate is covered by a confluent cellular monolayer. At confluence, subculture the cells as follows:</li>
	<li>Aspirate out the culture medium and wash the cells with 10 mL of PBS.</li>
	<li>Remove PBS and add 2 mL of trypsin-EDTA solution to each 100-mm plate. Gently rock the plates to evenly distribute the trypsin-EDTA solution. Incubate at 37&deg;C and 5%CO2 for 3-5 minutes. Gently tap the plate to see the detached cells in suspension under an inverted microscope.</li>
	<li>When cells completely detach, add 8 mL of EGM-2 medium and collect the cell solution into a 15-mL conical tube. Take 10 &mu;L to count the cells in a haemocytometer and work out the total number of cells harvested.</li>
	<li>Plate the cells in 1% gelatin-coated tissue culture plates at a seeding density of 5,000 cell/cm2 using EGM-2 medium. Place the plates in the incubator and feed them every 2-3 days with EGM-2 medium.</li>
</ol>
Repeat this procedure for subsequent passages. Keep track of the passage number as the cell population is expanded. ECFCs will be used between passages 4-8.
3. Culture of Human Bone Marrow-Derived Mesenchymal Stem Cells (MSCs)
This protocol assumes frozen vials of human MSC are available in the laboratory before this experiment. MSC can be isolated from bone marrow aspirates as previously described 11.
<ol>
	<li>Thaw one vial of MSCs (typically 0.50-1x106 cells in 1 mL freezing media) taken from the liquid nitrogen storage tank and immediately dilute its content in a 15-mL conical tube containing 10 mL of DMEM medium. Spin at 1200 rpm for 5 min. Remove supernatant and resuspend the cell pellet in 10 mL of warm MSCGM medium.</li>
	<li>Add the 10 mL of MSC suspension into one uncoated 100-mm tissue culture plate. Place the plate in a humidified incubator at 37&deg;C and 5%CO2. Next day, gently aspirate out unbound cells and medium, and feed bound cells with 10 mL of fresh MSCGM medium.</li>
	<li>Feed the plate every 2-3 days with MSCGM medium. Allow cells to expand such that the plate reaches 80% of confluent cellular monolayer. At 80% confluence, subculture the cells as follows:</li>
	<li>Aspirate out the culture medium and wash the cells with 10 mL of PBS.</li>
	<li>Remove PBS and add 2 mL of trypsin-EDTA solution to each 100-mm plate. Gently rock the plates to evenly distribute the trypsin-EDTA solution. Incubate at 37&deg;C and 5%CO2 for 3-5 minutes. Gently tap the plate to see the detached cells in suspension under an inverted microscope.</li>
	<li>When cells completely detach, add 8 mL of MSCGM medium and collect the cell solution into a 15-mL conical tube. Take 10 &mu;L to count the cells in a haemocytometer and work out the total number of cells harvested.</li>
	<li>Plate the cells in uncoated tissue culture plates at a seeding density of 10,000 cell/cm2 using MSCGM medium. Place the plates in the incubator and feed them every 2-3 days with MSCGM medium.</li>
</ol>
Repeat this procedure for subsequent passages. Keep track of the passage number as the cell population is expanded. MSCs will be used between passages 4-8.
4. Resuspension of Cells in Collagen/Fibronectin/Fibrinogen Solution (Day 0)
Prior to the experiment, make sure there are enough ECFCs and MSCs in culture; 0.8x106 ECFCs and 1.2x106 MSCs will be required for each implant and mouse.
<ol>
	<li>Aspirate out the medium of each culture plate and wash the cells with 10 mL of PBS. Remove PBS and add 2 mL of trypsin-EDTA solution to each 100-mm plate. Gently rock the plates to evenly distribute the trypsin-EDTA solution. Incubate for 3-5 minutes. Gently tap the plate to see the detached cells in suspension under an inverted microscope.</li>
	<li>When cells completely detach, add 8 mL of DMEM medium and collect the cell solution into a 15-mL conical tube. Take 10 &mu;L to count the cells in a haemocytometer and work out the total number of ECFCs and MSCs harvested.</li>
	<li>Transfer 4x106 ECFCs (5x 0.8x106 cells) and 6x106 MPCs (5x 1.2x106 cells) together into a single 50-mL conical tube. This is the total amount of cells required for five individual implants and mice. Centrifuge at 1200 rpm and remove the supernatant.</li>
	<li>Gently, add 100 uL of Fibrinogen solution to 0.9 mL of collagen/fibronectin solution (3 mg/mL final fibrinogen concentration); keep the mixture on ice.</li>
	<li>Resuspend the cell pellet on 1 mL of ice cold collagen/fibronectin/fibrinogen solution; mix the cells very gently to avoid bubbles. Load the mixture into a 1-mL sterile syringe, and place a 26-gauge needle with its cap on the tip of the syringe. Keep the loaded syringe on ice until injection.</li>
</ol>
5. Injection into Immunodeficient Nude Mouse (Day 0)
All animal experiments will be carried out with 6-week old athymic nude (nu/nu) mice.
<ol>
	<li>Prior to the injection, anesthetize the immunodeficient mice by placing them into a gas chamber delivering isoflurane. Allow the mice to inhale the isoflurane for approximately 2 minutes until they are anesthetized and unresponsive to toe pinch (monitor their heart beats by inspection).</li>
	<li>For each mouse, inject 50 &mu;L of thrombin solution (10 U/mL) subcutaneously into the upper dorsal region using a 26-gauge needle.</li>
	<li>In the same place where thrombin was injected, inject 200 &mu;L of the cell mixture using a 26-gauge needle. Collagen will gel at 37&deg;C and fibrinogen will form fibrin gel in the presence of thrombin. As a result, the implant should form a small, but appreciable, bump under the skin.</li>
	<li>After the injection, place the mice on a layer of gauze for comfort and warmth and observe them until they become ambulatory. Then after, observe the mice daily for the first three days.</li>
</ol>
6. Harvesting (Day 7)
<ol>
	<li>One week after the injections, euthanize the mice by placing them into a gas chamber delivering compressed CO2 gas.</li>
	<li>Once euthanized, cut open the skin near the area of the injection and surgically remove the gel plug. Digital photographs of the retrieved gel plugs with a scale are advised.</li>
	<li>Place the harvested gel plugs into histological cassette and deep them into 10% neutral buffered formalin overnight at room temperature.</li>
	<li>After fixation, wash the 10% neutral buffered formalin away with distilled H2O and place the histological cassettes at 4&deg;C in PBS until histological evaluation.</li>
</ol>
7. Evaluation: Histology (H&amp;E) and Immunohistochemistry (hCD31)
<ol>
	<li>For histological evaluation, the implants are embedded in paraffin and sectioned (7 &mu;m-thick sections) using standard histological procedures.</li>
	<li>Quantify microvessel density by evaluation of Hematoxilin and Eosin (H&amp;E) stained sections taken from the middle part of the implants. Standard protocols for H&amp;E staining can be found elsewhere. Microvessels can be identified as lumenal structures containing red blood cells. Report microvessels density as the average number of red blood cell-filled microvessels from the fields analyzed and expressed as vessels/mm2.</li>
	<li>To demonstrate the human nature of the microvascular vessels, sections of the retrieved implant should be immunohistochemically stained with a human-specific CD31 (hCD31) antibody using standard staining protocols. We recommend using the monoclonal mouse anti-human CD31 antibody from DakoCytomation (Clone JC70A; cat. # M0823) at a 1:100 dilution. The human specificity of this antibody has been confirmed by the negative reaction obtained with a diversity of mouse tissue sections that were stained in parallel 7,11. Of note, mouse blood vessels are often seen inside the implants (specially around the border), but mouse vessels will not stain positive with this antibody. In addition, the presence of human MSC can be detected by immunohistochemical staining with human-specific antibodies against CD90 or &alpha;-Smooth muscle actin (&alpha;-SMA). Human MSC are found both in the perivascular region of newly-formed blood vessels and interstitially located throughout the implant 11.</li>
</ol>
8. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/3065/3065fig1.jpg" /> 
Figure 1. Typical appearance of ECFC and MSC cultures. Phase contrast micrographs displaying the typical appearance of ECFCs and MSCs in culture. (A) Confluent monolayer of cord blood-derived ECFCs displaying the characteristic cobble-stone morphology of endothelial cells. (B) Human bone marrow-derived MSCs displaying a spindle shape morphology. Scaler bars, 200 um.
<img alt="Figure 2" src="/files/ftp_upload/3065/3065fig2.jpg" /> 
Figure 2. Appearance of explanted plugs at day 7. Human cord blood-derived ECFCs and bone marrow-derived MSCs were embedded in collagen/fibronectin/fibrin gel and implanted subcutaneously into nude mice as described in the text. (A) After 7 days, once the mouse has been euthanized, cut open the skin near the area of the injection and expose the cell/gel plug by flipping the skin. (B) Appearance of the plug surgically removed from the mouse and prior to formalin fixation. The red color of the implant is an indication of vascularization.
<img alt="Figure 3" src="/files/ftp_upload/3065/3065fig3.jpg" /> 
Figure 3. Histological identification of vascular network in explanted plugs. Hematoxilin and Eosin (H&amp;E) stained sections taken from the middle part of the explanted plugs. (A) Low magnification (10x) micrograph displaying the implant (marked by a yellow dashed line) in the context of surrounding host tissues (i.e., adipose tissue and skeletal muscle). (B) High magnification (40x) micrograph displaying multiple microvessels (yellow arrowhead pointing at some of them) inside the plug; microvessels can be identified as lumenal structures containing red blood cells.
<img alt="Figure 4" src="/files/ftp_upload/3065/3065fig4.jpg" /> 
Figure 4. Immunohistochemical identification of human lumens. Immunohistochemically stained sections taken from the middle part of the explanted plugs. Staining was carried out using a monoclonal mouse anti-human CD31 (hCD31) antibody from DakoCytomation (Clone JC70A; cat. # M0823) at a 1:100 dilution; cell nuclei were counterstained with hematoxilin. (A) Low magnification (10x) micrograph displaying the implant (delineated by a black dashed line) in the context of surrounding host tissues. Human specific, CD31-positive microvessels are stained in brown (peroxidase staining). (B) High magnification (40x) micrograph displaying multiple human microvessels (black arrowhead pointing at some hCD31-positive lumens) inside the plug.

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	<li>Melero-Martin, J. M., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+13.+An+in+vivo+experimental+model+for+postnatal+vasculogenesis.">Chapter 13. An in vivo experimental model for postnatal vasculogenesis.</a> Methods Enzymol. 445, 303-329 (2008).</li><li>Koike, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering:+creation+of+long-lasting+blood+vessels.">Tissue engineering: creation of long-lasting blood vessels.</a> Nature. 428, 138-139 (2004).</li><li>Schechner, J. S. <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+formation+of+complex+microvessels+lined+by+human+endothelial+cells+in+an+immunodeficient+mouse.">In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse.</a> Proc Natl Acad Sci USA. 97, 9191-9196 (2000).</li><li>N&amp;ouml, r., E, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+characterization+of+functional+human+microvessels+in+immunodeficient+mice.">Engineering and characterization of functional human microvessels in immunodeficient mice.</a> Lab Invest. 81, 453-463 (2001).</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>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>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>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, 1302-1305 (2008).</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>Traktuev, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+Functional+Vascular+Network+Formation+In+Vivo+by+Cooperation+of+Adipose+Progenitor+and+Endothelial+Cells.">Robust Functional Vascular Network Formation In Vivo by Cooperation of Adipose Progenitor and Endothelial Cells.</a> Circ Res. 104, 1410-1420 (2009).</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=Host+Myeloid+Cells+Are+Necessary+for+Creating+Bioengineered+Human+Vascular+Networks+In+Vivo.">Host Myeloid Cells Are Necessary for Creating Bioengineered Human Vascular Networks In Vivo.</a> Tissue Eng Part A. 16, 2457-2466 (2010).</li></ol>

No conflicts of interest declared.

This is an experimental model of bioengineering human vascular networks. The main characteristics of this model are: 1) microvessels are formed from human cells isolated from post-natal tissues (i.e., blood and bone marrow); 2) microvessels are formed in an adult animal; and 3) microvessels do not arise from pre-existing (host) vessels but instead they are formed, de novo, from single cells suspended in a suitable gel.
Angiogenesis plays an important role in this assay because connections to the murine vasculature are needed to achieve red blood cell-filled vessels, one of the functional read-out in this assay. In addition, host myeloid cells are recruited to the implant in the early days post transplantation, and they are necessary for the formation of the new vascular bed 12.
This experimental model offers a versatile, quantifiable, and relatively simple model system to study post-natal formation of human vascular networks in vivo. Additionally, this assay is simple to perform and it does not require an incision or surgical procedure. The model could be used to study the vasculogenic potential of different sources of human endothelial and perivascular cells. The model could be used to screen for anti- and/or pro-vasculogenic compounds. Finally, the model can be used to study the role(s) of specific genes in the formation and function of a vascular network composed of human endothelium.

<table><tbody><tr><td>Endothelial Basal Medium, EBM-2</td><td>Lonza Inc.</td><td>CC-3156</td><td></td></tr><tr><td>EGM-2 SingleQuot supplements</td><td>Lonza Inc.</td><td>CC-3162</td><td></td></tr><tr><td>Glutamine-penicillin-streptomycin solution, 100x GPS</td><td>Mediatech, Inc.</td><td>30-009-CI</td><td></td></tr><tr><td>Mesenchymal Stem Cell Growth Medium BulletKit, MSCBM/MSCGM</td><td>Lonza Inc.</td><td>PT-3001</td><td></td></tr><tr><td>High glucose Dulbecco&amp;rsquo;s Modified Eagle Medium, 1x DMEM </td><td>Invitrogen</td><td>11995-073</td><td></td></tr><tr><td>MEM Non-essential amino acid solution, 100x NEAA</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>High glucose Dulbecco&amp;rsquo;s Modified Eagle, powdered DMEM </td><td>Invitrogen</td><td>12100-046</td><td></td></tr><tr><td>HEPES Buffer solution</td><td>Invitrogen</td><td>15630-080</td><td></td></tr><tr><td>Cultrex Bovine Collagen I solution</td><td>Trevigen Inc.</td><td>3442-050-01</td><td></td></tr><tr><td>Cultrex Human Fibronectin </td><td>Trevigen Inc.</td><td>3420-001-01</td><td></td></tr><tr><td>Fibrinogen from bovine plasma</td><td>Sigma-Aldrich</td><td>F8630-1G</td><td></td></tr><tr><td>Gelatin</td><td>Fisher Scientific</td><td>DF0143-17-9</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline, PBS</td><td>Invitrogen</td><td>14190-250</td><td></td></tr><tr><td>Trypsin-EDTA solution, 1x</td><td>Invitrogen</td><td>25300-054</td><td></td></tr><tr><td>Isoflurane, liquid for inhalation</td><td>Buxter Healthcare Corporation</td><td>NDC 10019-360-40</td><td></td></tr><tr><td>Thrombin from bovine plasma</td><td>Sigma-Aldrich</td><td>T4648-1KU</td><td></td></tr><tr><td>Formalin Solution, Neutral Buffered</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Monoclonal Mouse Anti-Human CD31 Antibody</td><td>Dako</td><td>M0823</td><td>Clone JC70A</td></tr></tbody></table>

bioengineering human microvascular networks in immunodeficient mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Watch this Scientific Journal Video about Bioengineering Human Microvascular Networks in Immunodeficient Mice at JoVE.com

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Video: Bioengineering Human Microvascular Networks in Immunodeficient Mice

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Cancer Research

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Developmental Biology

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Medicine

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Immunology and Infection

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Bioengineering

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Injecting Human HSPCs into the Femur of Sub-Lethally Irradiated Mice

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell populations containing HSCs into syngeneic or immunocompromised mice. The size and multilineage composition of the graft is then measured over time, usually by flow cytometry. Classically, a population containing HSCs is injected into the circulation of the animal, after which the HSCs home to the bone marrow, where they lodge and begin blood production. Alternatively, HSCs and/or progenitor cells (HSPCs) can be placed directly in the bone marrow cavity. 
This paper describes a protocol for intrafemoral injection of human HSPCs into immunodeficient mice. In short, preconditioned mice are anesthetized, and a small hole is drilled through the knee into the femur using a needle. Using a smaller insulin needle, cells are then injected directly into the same conduit created by the first needle. This method of transplantation can be applied in varied experimental designs, using either mouse or human cells as donor cells. It has been most widely used for xenotransplantation, because in this context, it is thought to provide improved engraftment over intravenous injections, therefore improving statistical power and reducing the number of mice to be used.

Author Spotlight: Exploring the Lifespan Dynamics of Healthy Human Hematopoiesis

Intrafemoral injections allow for the engraftment of a small number of hematopoietic stem and progenitor cells (HSPCs), by placing the cells directly in the bone marrow cavity. Here we describe an experimental protocol of intrafemoral injection of human HSPCs into immunodeficient mice.

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell ...