This work was supported by The British Heart Foundation and The Oak Foundation.

Perform all animal experiments according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals.
1. Preparation of Culture Media
<ol>
	<li>Make culture media for human fibroblast cell line CCL-153: F-12K Medium, 10% fetal bovine serum (FBS) and 100 U/ml penicillin and streptomycin.</li>
	<li>Make Reprogramming Media for PiPSC generation: Knockout Dulbecco&#8217;s modified eagle&#8217;s medium (DMEM) containing 20% Knockout Serum Replacement, 0.1 mM &#946;-mercaptoethanol, 0.1 mM Minimum Essential Medium (MEM) Non-Essential Amino Acids, 10 ng/ml basic Fibroblast Growth Factor 2 (bFGF-2) and 100 U/ml penicillin and streptomycin. Add bFGF-2 freshly each time before using the media.</li>
	<li>Make Differentiation Media (DM) to induce smooth muscle cell (SMC) differentiation: &#945;-MEM media containing 10% FBS, 0.1 mM &#946;-mercaptoethanol, 100 U/ml penicillin and streptomycin and 25 ng/ml Platelet Derived Growth Factor (PDGF-&#946;&#946;).</li>
	<li>Make Endothelial Differentiation Media (EC-DM) to induce endothelial cell (EC) differentiation: Endothelial growth media-2 (EGM-2) media containing 50 ng/ml Vascular Endothelial Growth Factor (VEGF) and 100 U/ml penicillin and streptomycin.</li>
</ol>
2. Reprogramming Human Fibroblasts into Partially-induced Pluripotent Stem Cells (PiPSC)
<ol>
	<li>Culture human fibroblast cell line CCL-153 on 0.04% gelatin solution coated flasks in culture medium.</li>
	<li>Passage cells every 3 days at a ratio of 1:6. Use cells before passage 9 for optimal efficiency of PiPSC generation. Cells are ready for transfection upon reaching 80 - 90% confluency.</li>
	<li>Linearize polycistronic plasmid pCAG2LMKOSimO containing 4 reprogramming factors octamer-binding transcription factor 4 (OCT4), SOX2, Kruppel-like factor 4 (KLF4) and C-MYC (pCAG-OSKM). Digest 5 &#181;g of plasmid with 5 units of PvuI restriction enzyme for 3 hr at 37 &#176;C. Purify the plasmid with a commercial kit according to manufacturer&#8217;s protocol.</li>
	<li>Transfect 2 x 106 human fibroblasts with 4 &#181;g of linearized pCAG-OSKM plasmid by electroporation with human dermal fibroblast (NHDF) nucleofection kit according to manufacturer&#8217;s protocol. Gently seed transfected cells onto pre- 0.04% gelatin coated T25 flask containing 5 ml of pre-warmed Reprogramming Media.</li>
	<li>After 24 hr, change medium with supplements of 25 &#181;g/ml neomycin to select a pure population of transfected cells.</li>
	<li>Change medium every other day until day 4. 
	Note: Human fibroblasts become PiPSC after 4 days of reprogramming.</li>
</ol>
3. Differentiation of PiPSC into Endothelial and Smooth Muscle Cells
<ol>
	<li>Seed PiPSC on collagen IV pre-coated dishes containing DM for 4 days to induce differentiation into SMCs. Change medium every other day until day 4.</li>
	<li>Seed PiPSC on collagen IV pre-coated dishes containing endothelial-DM for 6 days to induce differentiation into endothelial cells. Change medium every other day until day 6.</li>
</ol>
4. Decellularized Aorta Graft Preparation
Note: All solutions and equipment should be sterile.
<ol>
	<li>Sacrifice the mouse by cervical dislocation and fix the mouse in a supine position under dissection microscope.</li>
	<li>Cut through the sternum and around the rib cage to open the thoracic cavity. Remove the heart, lungs and esophagus to expose the aorta. Gently remove the peri-aorta fat with forceps.</li>
	<li>Cut the aorta from the anterior end with scissors and gently hold the end with blunt forceps. Detach the aorta from the spine column behind by blunt dissection. Carefully close all the branching arteries from the aorta by ligation with a bipolar electrocoagulator and cutting from the far end of the ligation with scissors.</li>
	<li>Use a 5 ml syringe to flush the aorta lumen with 3 ml of saline solution containing 100 U of heparin to prevent the formation of blood clots.</li>
	<li>Cut the posterior end of the aorta before it branches into the renal. Keeping the integrity of the aorta is very important during the whole procedure.</li>
	<li>Flush the aorta lumen with 5 ml of 0.075% sodium dodecyl sulphate (SDS) solution diluted in phosphate buffered saline (PBS).</li>
	<li>Soak the thoracic aorta in 0.075% SDS solution in a 10 cm Petri dish. Place the Petri dish on an orbital shaker for 2 hr at 150 rpm at RT.</li>
	<li>Flush the aorta lumen with 5 ml of PBS.</li>
	<li>Soak the aorta in PBS in a 10 cm Petri dish. Place the Petri dish on the orbital shaker for 2 hr at 150 rpm at RT. Refresh PBS every 20 min.</li>
	<li>Flush the aorta lumen with 5 ml of PBS.</li>
	<li>Keep decellularized aorta graft in PBS at 4 &#176;C for up to one week.</li>
</ol>
5. Dual Seeding of PiPSC and Bioreactor Conditioning
Note: All solutions and equipment should be sterile. Perform all operations in a tissue culture hood.
<ol>
	<li>Assemble the bioreactor flow circuit as illustrated in Figure 1. Connect incubation chamber, media reservoir, peristaltic pump and compliance chamber with tubing. Minimal volume of medium needed for circulation is 40 ml.</li>
	<li>Pre-condition the decellularized vessel with culture medium by soaking the aorta in DM medium in a 10 cm Petri dish. Place the Petri dish on the orbital shaker for 1 hr at 50 rpm at RT.</li>
	<li>Insert 1 cm length Nylon tubes (OD 0.9 mm, ID 0.75 mm) into both ends of the decellularized vessel under microscope. Tie the vessel and the tubes with 8-0 silk sutures.</li>
	<li>Assemble the decellularized aorta graft in the incubation chamber by connecting the Nylon tubes with the inlet and outlet ports (1/32&#8221; tubing) from the chamber wall. Maintain incubation chamber with 5mls of medium each time.</li>
	<li>Trypsinize PiPSC by adding pre-warmed trypsin to cover the culture dish and gently rock the dish 10 times. Discard the trypsin and leave the dish in incubator for 2 - 3 min. Add pre-warmed culture medium in the dish and mix the medium with the cells.</li>
	<li>Count cell number using a hemocytometer. Centrifuge two aliquots of cell suspensions containing 5 x 105 cells each for 5 min at 300 x g. Aspirate the supernatant completely.</li>
	<li>After aspirating the supernatant completely, resuspend 1 pellet of the PiPS cell pellets in 50 &#181;l of DM. Inject this cell suspension into the decellularized vessel lumen.</li>
	<li>Then, resuspend the other PiPS cell pellet in 100 &#181;l of Matrigel. Carefully pipette the mixture onto the decellularized aorta graft.</li>
	<li>Wait for 10 - 15 min until the mixture turns into a gel-like state and evenly wraps around the vessel external surface. Fill the incubation chamber with DM.</li>
	<li>Place the whole bioreactor setting in a 5% CO2 incubator at 37 &#176;C. Manually rotate the decellularized graft 90&#176; around the longitudinal axis every half an hour in the first 2 hr.</li>
	<li>Keep static culture for 12 hr to enable cell adhesion.</li>
	<li>Deliver DM through the lumen by the peristaltic pump to induce smooth muscle cell differentiation. Start initial flow rate at 5 ml/min and stepwise increase to 20 ml/min over 24 hr.</li>
	<li>Keep the circulating medium flow rate at 20 ml/min for 24 hr.</li>
	<li>Stop the medium flow and move the bioreactor setting out of the incubator.</li>
	<li>Re-seed the lumen of the graft with PiPSC. Trypsinize, count and centrifuge 1 x 106 PiPSC as in steps 5.5 - 5.6. Resuspend the cells in 50 &#181;l of EGM-2 medium containing 50 ng/ml VEGF. Inject the cell mixture into the lumen of the graft.</li>
	<li>Change the medium in the incubation chamber and whole circulation to EGM-2 medium containing 50 ng/ml VEGF to induce endothelial differentiation.</li>
	<li>Move the bioreactor setting back to the incubator. Manually rotate the graft 90&#176; every 30 min in the first 2 hr and then keep a static culture for 12 hr.</li>
	<li>Start circulating flow from 5 ml/min and stepwise increase to 35 ml/min. Keep the flow rate at 35 ml/min for 5 days. Change circulating and chamber medium every other day.</li>
	<li>Harvest the engineered graft for further grafting to mouse.</li>
</ol>
6. Grafting of Double-seeded PiPSC Graft into Mice
<ol>
	<li>Use NOD.CB17-Prkdcscid/NcrCrl male mice as vessel graft recipients. Always ensure that the mice are about 10 weeks old, weigh approximately 25 - 35 g, and are in good health conditions.</li>
	<li>Anesthetize recipient mouse by administering a combination of Hypnorm (25 mg/kg) and Hypnovel (25 mg/kg) intraperitoneally. Apply petrolatum ophthalmic ointment on eyes to prevent dryness while under anesthesia. Confirm efficient anesthetization by ensuring that mice have relaxed muscles and are breathing steadily.
	<ol>
		<li>Fix the mouse in a supine position with its neck shaved and extended. Administer atropine sulfate (1.7 mg/kg) in combination with the anaesthetics to ensure that the mouse respiratory tract remains clear.</li>
	</ol>
	</li>
	<li>Prepare a midline incision from the mandible to sternum of the mouse. Under a dissecting microscope with 5- to 10-fold amplification, lift the right salivary glands laterally and remove the right cleidomastoid muscle to expose the right common carotid artery.</li>
	<li>Gently remove attached tissues to mobilize the right common carotid artery from the distal end toward the proximal end. Ligate the middle portion of the common carotid artery twice with an 8-0 silk suture and dissect between the two ties.</li>
	<li>Pass through a cuff made of autoclavable nylon tube in each vessel end and fix each end with microhemostat clamps.</li>
	<li>Remove suture tie&#160;on one end and apply a drop of heparin (100 U/ml). Immediately after, invert the distal end of the artery to cover the whole cuff body and fix the inverted vessel with an 8-0 silk suture to the cuff.
	<ol>
		<li>Repeat the same procedure to the other portion of the artery. Flush artery with saline solution to remove blood clots.</li>
	</ol>
	</li>
	<li>Implant a decellularized vessel graft between two ends of the carotid artery by sliding the decellularized vessel ends of the over the artery cuffs and fixing it with 8-0 silk sutures. Remove vascular clamps from either ends before evaluating the pulsation of the graft.</li>
	<li>Place the right salivary gland back to its original position. Close the skin on the surgical location with an interrupted suture using a 6-0 polyglactin suture.</li>
	<li>Consistently observe vital signs of mouse after the procedure is completed, until it resumes consciousness. Sacrifice recipient mice by cervical dislocation and harvest vessel grafts either 24 hr or 3 weeks later for further analysis.</li>
</ol>

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The authors have no conflicting financial interests.

The current protocol indicates a sound, fast, simple, efficient and reproducible strategy in which functional tissue engineered vessels can be generated using PiPSC from human fibroblasts. This technique represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell therapy in the near future. Critical steps to ensure the efficacy of the protocol include the preparation of PiPSC, preparation of sterile and fully decellularized aortic grafts, successful seeding and different...

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. To date, graft materials used in surgery include biocompatible synthetic polymers (polytetrafluoroethylene [Teflon], expanded polytetrafluoroethylene [ePTFE; Gore-Tex] or polyethylene terephthalate [Dacron]), allografts, autologous tissue (pericardium or saphenous vein) and xenografts1. Whilst artificial grafts (e.g., Gore-Tex and Dacron) are most commonly used, these materials likely cause numerous short- and long-term complications that include stenosis, calcium deposition, thrombo-embolization and infections. Although patients with biological grafts present with decreased thrombo-embolic events, they still encounter limitations such as secondary graft failure and shortened durability owing to calcification degradation2. Therefore, despite significant improvements in surgical techniques over the years, researchers and clinicians are still burdened with the need for identifying the ideal conduit for vascular diseases. More recently, the research field of vascular tissue engineering has generated a concept in which cells are incorporated into biodegradable scaffolds, with the aim of creating a biomimetic environment that epitomizes a functional vessel for successful grafting1. Fundamentally, the success of the vascular constructs depend on three essential components; cells that comprise the scaffold, i.e., an endothelial cell inner layer and a smooth muscle cell layer, a scaffold containing the appropriate extracellular matrix to provide mechanical properties comparable to the native vasculature, and the molecular/cellular signaling that is required for initiating/regulating repair.
Long term graft patency and sustained development of the neo-tissues are highly dependent on effective cell seeding of scaffolds, thereby rendering the decision of cell type of critical importance. Several reports demonstrate the use of mature endothelial and smooth muscle cells from various sources to develop small diameter conduits3-6. Although promising, the lack of sufficient autologous vessels to obtain mature endothelial and smooth muscle cells remain a considerable burden. More recently, stem cells from various sources have been exploited for vascular tissue engineering applications. Indeed, a variety of stem cell types including embryonic stem cells7, induced pluripotent stem cells (iPSCs)8,9, PiPSC10,11, bone marrow-derived mononuclear cells12, mesenchymal stem cells13, endothelial progenitor cells and adult vessel wall-derived stem cell antigen-1 (Sca-1)+ stem/progenitor cells14,15 have all been demonstrated to be capable of differentiation into either functional endothelial or smooth muscle cells in response to defined media and culture conditions. Furthermore, the unlimited self renewal capacity of the stem cells make them better candidates unlike mature endothelial and smooth muscle cells which can only divide for a finite number of times before undergoing growth arrest and senescence.
The selection of scaffold material to generate successful tissue engineered vessel for grafting depends on several factors such as biocompatibility, biomechanical properties, and rate of biodegradation. Fundamentally, materials used to create scaffolds for the grafts should be biodegradable and will not mount unnecessary recipient immune responses. Additionally, it must encompass a suitable porosity and microstructure for cell attachment and subsequent survival. To date, the most common materials used for scaffolds in vascular tissue engineering include polymers of polyglycolic acid, polylactic acid, and poly &#949;-caprolactone16. More recently, decellularized biological materials have also been applied with some success. Several laboratories have shown that seeding decellularized human, canine or porcine vessels with autologous cells provided a biological graft that resisted clotting and intimal hyperplasia17-19. Other strategies in vascular tissue engineering include extracellular matrix proteins-based vascular grafts e.g., seeding cells in fibrin gel13 and generating cell sheets without scaffold support20, 21.
The current protocol demonstrates the differentiation of human PiPSC into functional endothelial and smooth muscle cells, the generation of a bioreactor consisting of a decellularized vessel scaffold to harbor functional PiPSC-derived vascular cells, and grafting of the tissue engineered vessels into severe combined immunodeficiency (SCID) mice. PiPSC are an optimum cell type to use for tissue engineering of vessel grafts because these cells do not form tumors in mice or raise ethical and allo-immune responses. Furthermore, we have shown that the strategy for generating PiPS-endothelial cells and PiPS-smooth muscle cells is efficient and reproducible10,11. Thereafter, we designed a decellularized vessel for seeding of PiPSC-derived vascular cells to closely mimic the matrix proteins that exists within a native vessel, thus enhancing grafting and survival efficacy. Furthermore, the decellularization of the vessels prior to PiPSC seeding prevents the occurrence of inflammatory responses mounted by immune cell types such as macrophages. More importantly, this protocol does not only represent a methodology to generate promising vascular conduits for translation into humans, but also provides valuable means of studying and understanding the molecular mechanisms that govern vascular tissue regeneration through mouse models.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Human Fibroblasts CCL-153</td>
			<td >ATCC</td>
			<td >CCL-153</td>
			<td>Prenatal human embryonic fibroblasts</td>
		</tr>
		<tr >
			<td >ATCC F-12K Medium (Kaighn&#39;s Modification of Ham&#39;s F-12 Medium)</td>
			<td >ATCC</td>
			<td >30-2004</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ATCC</td>
			<td >30-2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout DMEM medium optimized for embryonic stem cells</td>
			<td >Life technologies (Gibco)</td>
			<td >12660-012</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout Serum Replacement</td>
			<td >Life technologies (Invitrogen)</td>
			<td >10828-028</td>
			<td></td>
		</tr>
		<tr >
			<td >Human Basic FGF-2&#160;</td>
			<td >Miltenyi Biotech</td>
			<td >130-093-837</td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Life technologies (Invitrogen)</td>
			<td >32571093</td>
			<td></td>
		</tr>
		<tr >
			<td >Human PDGF</td>
			<td >R&#38;D System</td>
			<td >120-HD-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Gelatin Solution 2%</td>
			<td >Sigma</td>
			<td >G1393</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td>
			<td >Addgene</td>
			<td >20866</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;PvuI Restriction Enzyme</td>
			<td >New England Biolabs</td>
			<td >RO150S</td>
			<td></td>
		</tr>
		<tr >
			<td >SureClean Plus</td>
			<td >Bioline</td>
			<td >BIO-37047</td>
			<td></td>
		</tr>
		<tr >
			<td >Nucelofection Kit (NHDF Kit)</td>
			<td >LONZA</td>
			<td >VPD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Neomycin</td>
			<td >SIGMA</td>
			<td >G418</td>
			<td>Selection of&#160;</td>
		</tr>
		<tr >
			<td >KL 1500 LCD, Illumination for Stereo Microscopy</td>
			<td >SCHOTT</td>
			<td >KL 1500 LCD</td>
			<td>Cold light illumination for stereo microscopy</td>
		</tr>
		<tr >
			<td >Nikon Zoom Steromicroscope SMZ800</td>
			<td >Nikon</td>
			<td >SMZ800</td>
			<td></td>
		</tr>
		<tr >
			<td >Heparin sodium salt</td>
			<td >Sigma</td>
			<td >H3393</td>
			<td></td>
		</tr>
		<tr >
			<td >10% SDS Stock Solution Molecular Biology Reagent</td>
			<td >Severn Biotech</td>
			<td >CAS 151-21-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td >Sigma</td>
			<td >D8537</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrigel (10mg/ml)</td>
			<td >BD</td>
			<td >A6661</td>
			<td></td>
		</tr>
		<tr >
			<td >Shaker IKA Vibrax with Shaking platform VX 7&#160;</td>
			<td >Jepson Bolton&#39;s, Janke&#38;Kunkel</td>
			<td >S32-102</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S Digital Pump Drive</td>
			<td >Cole-Parmer</td>
			<td >WZ-07523-80</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S 6-channel, 6-roller cartridge pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S large cartridges for pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-75</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td>
			<td >Cole-Parmer&#160;</td>
			<td >WZ-96410-14</td>
			<td >Tubing goes through the peristaltic pump</td>
		</tr>
		<tr >
			<td >0.5mm ID, 0.8 mm OD Silicone Tubing</td>
			<td >SILEX</td>
			<td >N/A</td>
			<td>Tubings connect incubation chamber, media reservoir and compliance chamber&#160;</td>
		</tr>
		<tr >
			<td >Fitting Reducer 0.5 to 1.6, natural Polypropyline</td>
			<td >Ibidi</td>
			<td >10829</td>
			<td>Adapter connect above two types of tubings</td>
		</tr>
		<tr >
			<td >1/32&#34; Tubing, ID 0.01&#34; (250&#181;m) Material: PEEK</td>
			<td >LabSmith</td>
			<td >T-132-010P</td>
			<td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td>
		</tr>
		<tr >
			<td >One-Piece Fittings&#160;</td>
			<td >LabSmith</td>
			<td >T-132-100</td>
			<td>Fix the above tubings through the incubation chamber wall</td>
		</tr>
		<tr >
			<td >Nylon tubes (OD 0.9mm, ID 0.75mm)&#160;</td>
			<td >Smiths Medical</td>
			<td >N/A</td>
			<td>Tubings insert into two ends of the aorta graft</td>
		</tr>
		<tr >
			<td >NOD.CB17-Prkdcscid/NcrCrl mouse</td>
			<td >Charles River</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 8-0&#160; silk</td>
			<td >ETHICON</td>
			<td >W819</td>
			<td ></td>
		</tr>
		<tr >
			<td >Hypnorm</td>
			<td >Vetapharm</td>
			<td >Vm21757/4000</td>
			<td >Neuroleptanalgesic for use in mice</td>
		</tr>
		<tr >
			<td >Hypnovel (Midazolam)</td>
			<td >Roche</td>
			<td >59467-70-8</td>
			<td >Induction of anaesthesia</td>
		</tr>
		<tr >
			<td >Dissecting microscope</td>
			<td >Carl Zeiss</td>
			<td >Stemi 2000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nylon Tubing</td>
			<td >Portex LTD</td>
			<td >800/200/100/200</td>
			<td >0.65 mm in diameter and 1 mm in length; to make artery cuff</td>
		</tr>
		<tr >
			<td >Electrocoagulator</td>
			<td >Martin&#160;</td>
			<td >SN 54.131</td>
			<td >Ligation of artery branches on aorta</td>
		</tr>
		<tr >
			<td >Bipolar micro hemostat forceps</td>
			<td >Martin</td>
			<td >80-91-12-04</td>
			<td >Fixation of vessel ends</td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFX-7</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFL-3dZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >D-5aZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini applier&#160;</td>
			<td >AESCULAP</td>
			<td >FE572K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micro hemostats clips</td>
			<td >AESCULAP</td>
			<td >FE720K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 6-0 VICRYL</td>
			<td >ETHICON</td>
			<td >V489</td>
			<td ></td>
		</tr>
	</tbody>
</table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

The successful generation of PiPSC was confirmed 4 days after nucleofecting human fibroblasts with a linearized pCAG2LMKOSimO plasmid carrying 4 transcription factors, OCT4, SOX2, KLF4 and c-MYC (OSKM). PiPSC displayed a markedly distinct phenotype when compared to fibroblasts (Figure 2A) and expressed the 4 reprogramming factors at mRNA (Figure 2B) and protein (Figure 2C) levels10. The efficacy of a PiPSC-based vascular graft is highly dependent on the abilit...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

generation-and-grafting-of-tissue-engineered-vessels-in-a-mouse-model

Research

JoVE Journal

Bioengineering

11.9K Views.  King's College London BHF Centre. Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.