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.

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

Bioengineering Human Microvascular Networks in Immunodeficient Mice

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

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JoVE Journal

Biology

12.2K Views.  Harvard Medical School. Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Video: Bioengineering Human Microvascular Networks in Immunodeficient Mice

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Title Cell Encapsulation by Droplets

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...