Tissue-engineered vascular conduits for single ventricle repair

Abstract

The present invention provides a tissue-engineered vascular conduit comprising a graft and a plurality of stem-cell derived cells, wherein the plurality of stem-cell derived cells are seeded on the graft, wherein a shear stress is applied to the plurality of stem-cell derived cells.

Description

[0001] Attorney Docket No.: 047162-5388-00WO

[0002] Tissue-Engineered Vascular Conduits for Single Ventricle Repair

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 720,344 filed on November 14, 2024 incorporated by reference herein in its entirety.

[0005] STATEMENT OF GOVERNMENT SUPPORT

[0006] This invention was made with government support under R01HL171984 awarded by the National Institutes of Health, W81XWH-19-1-0557 awarded by the Department of Defense. The government has certain rights in the invention.

[0007] REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS AN XML FILE The present application hereby incorporates by reference the entire contents of the sequence listing xml document named “047162-5388-OOWO_Sequence_Listing. xml”. The xml file containing the Sequence Listing of the present application was created on November 14, 2025 and is 15,221 bytes in size.

[0008] BACKGROUND OF THE INVENTION

[0009] Single ventricle congenital heart defects (SVCHDs) are caused by an array of structural anomalies that result in the formation of one functional ventricle, affecting about 1 in 1,000 live births, (de Leval, M.R., et al., 2010, Nat Rev Cardiol 7:520-527; Hoffman, J.I., et al., 2002, J Am Coll Cardiol 39:1890-1900; Samanek, M., 1992, Pediatr Cardiol 13:152-158). The resulting abnormalities cause the single ventricle to pump blood to both pulmonary and systemic circulations, leading to mixing of oxygenated and deoxygenated blood and compromised systemic and / or pulmonary output. Without surgical repair, children bom with these defects have a 70% mortality rate. (Samanek, M., 1992, Pediatr Cardiol 13:152-158). The current standard of care is the Fontan operation, which reconfigures the great vessels so that the single functional ventricle pumps blood to the body while blood returning from the body passively flows to the lung via a synthetic or tissue-engineered vascular conduit (TEVC). (de Leval, M.R., et al., 2010, Nat Rev Cardiol 7:520-527; Hibino, N., et al., 2010, J Thorac Cardiovasc Surg 139:431-436). Synthetic conduits are prone to infection, thrombosis, and lack the capacity for growth, thus Attorney Docket No.: 047162-5388-00WO

[0010] limiting therapeutic efficacy and requiring potential repeat invasive surgical procedures. (Wilson, W.R., et al., 2016, Circulation 134:e412-e460; Attard, C., et al., 2018, Thromb Res 172:204-213).

[0011] As such, there is a pressing need for improved vascular conduits. The present invention meets this long felt, but unmet, need.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following detailed description of exemplary embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0014] Figure 1, comprising Figure 1A through Figure IF, depicts representative experiments demonstrating the development of tissue engineered vascular conduits (TEVCs) using human induced pluripotent stem cells (hiPSCs) for implantation as inferior vena cava interposition grafts in nude rats. Figure 1A depicts a schematic illustration of lumen endothelialization using perfusion bioreactors and decellularized human umbilical cord arteries (dHUAs; ~2.0 mm luminal diameter) with hiPSC-derived endothelial cells (hiPSC-ECs). HUAs were chosen because they are readily available and the luminal diameters match well with that of the rat inferior vena cava (IVC) for implantation. HUAs (~2.5 cm in length) were decellularized, and the luminal surface were coated with human fibronectin at 100 pg / mL overnight at 37°C, followed by two consecutive 4-hour seeding incubations of hiPSC-ECs (~2.0xl06cells / cm2). A three-way connector with an injection port was used to introduce the fibronectin and both rounds of cell seeding to maintain sterility. An EC medium containing 10% FBS, 5 ng / mL VEGF-A, and 30 mg / mL dextran was next applied and shear stress was initiated at around 1 dyne / cm2for 12 hours. The shear training was then gradually increased towards 15 dynes / cm2over 24 hours, which was then maintained for an additional 3.5 days. Prior to implantation, shear stress was ramped down to 10 dynes / cm2for 12 hours and then to around 5 dynes / cm2for 12 hours in order to match the shear stress in the IVC of nude rats, where the TEVC is implanted as an interposition graft. Figure IB depicts representative images of immunostaining of endothelial markers (CD31 and eNOS) and human leukocyte antigen type A (HLA-A). Cross-sectional Attorney Docket No.: 047162-5388-00WO

[0015] segments of grafts after luminal endothelialization with hiPSC-ECs were stained for CD31, eNOS, and HLA-A. DNA was counterstained by 4,6-diamidino-2-phenylindole (DAPI). Graft lumen is indicated by the asterisk *. Scale bar = 200 gm. Figure 1C depicts a representative percentage quantification of the luminal surface of TEVCs covered by hiPSC-ECs based on the immunostaining results in Figure IB. Quantification was performed based on representative sections of pre-implant of dHUA grafts endothelialized using hiPSC-ECs for IVC implantation. Mean values and standard error of the mean indicated by the error bars are shown. Note that n=5 independent hiPSC-TEVCs were investigated. Figure ID depicts a representative image of the luminal surface of TEVCs after endothelialization using hiPSC-ECs via scanning electron microscopy. Note that hiPSC-ECs adhered to the surface of decellularized TEVCs were aligned in the direction of medium flow and presented a typical elongated morphology under pulsatile, unidirectional flow. The arrow indicates the direction of luminal flow during in vitro culture. Scale bar = 50 pm. Note that n=3 independent TEVCs were investigated. Figure IE depicts representative images of statically EC -coated (static ECs) or shear flow-trained, EC-coated (15-5 dynes / cm2ECs) TEVCs implanted as IVC interposition grafts before and after explantation. Grafts endothelialized with hiPSC-ECs (15-5 dynes / cm2ECs) were trained in bioreactors towards an arterial-like shear stress of 15 dynes / cm2over 36 hours and then maintained for an additional 3.5 days, which was followed by a ramp-down to 10 dynes / cm2for 12 hours and then to around 5 dynes / cm2for 12 hours prior to implantation. Control grafts were coated with hiPSC-ECs statically in bioreactors for overnight pre-implantation. The static coating period was kept under 24 hours as the volume of culture medium filling the luminal space is limited and having no circulation or medium change may impact EC viability negatively in long term. Additionally, coating grafts with hiPSC-ECs statically in bioreactors under 24 hours pre-implantation mimics the prior clinical trial in which patient bone marrow mononuclear cells were seeded onto the polymeric scaffolds statically on the same day the vascular conduits were assembled and implanted. (Drews, J.D., et al., 2020, Sci Transl Med 12). Statically EC-coated grafts were explanted 2 weeks after implantation. Shear flow-trained, EC-coated grafts were explanted 1 month after implantation. Dashed white rectangles indicate grafts implanted into the IVC. Black arrows indicate the formation of a thrombus. Scale bar = 1 mm (a graduation on the ruler). IVC, Inferior vena cava; AO, aorta. Note that n>3 independent grafts for each group were utilized and implanted into respective nude rats for experiments. Figure IF depicts representative images of Attorney Docket No.: 047162-5388-00WO

[0016] immunostaining of luminal endothelialized followed by 15-5 dynes / cm2flow trained grafts explanted 1 month after implantation. Cross-sectional segments of grafts were stained for eNOS and HLA-A. DNA was counterstained by DAPI. The graft lumen is indicated by the asterisk *. Scale bar = 200 pm. Note that n=4 independent TEVCs were investigated.

[0017] Figure 2, comprising Figure 2A through Figure 2G, depicts representative results demonstrating gradual shear flow training (15-5 dynes / cm2) enhances hiPSC-EC-derived vascular endothelium functions in IVC implanted grafts. Figure 2A depicts representative en face images of human fibrinogen adsorbed onto the luminal surface of statically endothelialized grafts exposed to an abrupt flow or of grafts coated with hiPSC-ECs under a gradual shear flow training regimen (15-5 dynes / cm2). Statically endothelialized grafts were prepared by seeding hiPSC-ECs onto dHUA luminal surface followed by an abrupt shear flow of around 5 dynes / cm2for 6 hours in bioreactors to mimic rat IVC shear flow for investigating the necessity of shear flow training for preventing thrombus formation. Scale bar = 25 pm. Figure 2B and Figure 2C depicts representative quantification of endothelial cell coverage and fibrinogen adsorption on graft luminal surface in Figure 2A. Coverage of hiPSC-ECs (marked by tdTomatao) on graft luminal surface was measured by tdTomatao positive areas (red) over total areas (Figure 2B). Fibrinogen adsorption (yellow) on graft luminal surface was assessed by mean fluorescence intensity (MFI) of adsorbed fibrinogen (Figure 2C). Segments (~5 mm in length) around midgraft regions were immersed in a solution containing 50 pg / mL human plasma fibrinogen conjugated with 647 Alexa Fluorophore for 1 hour at room temperature, followed by PBS washing, formalin (10%) fixation, and cutting into imageable flat sheets, prior to imaging for quantifying EC coverage and fibrinogen adsorption. Mean values and standard error of the mean indicated by the error bars are shown. Multiple independent grafts (n=5) were quantified for EC coverage and fibrinogen adsorption. A nonparametric Mann-Whitney test was used to compare two groups of grafts (**p<0.01). Figure 2D depicts representative en face scanning electron microscopy images from statically EC-coated grafts exposed to an abrupt flow and shear flow-trained (15-5 dynes / cm2), endothelialized grafts incubated with human whole blood. Segments (~5 mm in length) around mid-regions of grafts were immersed in human whole blood and incubated for 30 minutes at 37°C, followed by PBS washing, formalin (10%) fixation, and cutting into imageable flat sheets, which were further treated with 2.5% glutaraldehyde, ethanol, and carbon for scanning electron microscopy imaging. An appreciable amount of polymerized Attorney Docket No.: 047162-5388-00WO

[0018] fibrin and aggregated red blood cells was observed on the luminal surface of statically EC-coated grafts exposed to an abrupt flow compared to the gradually shear flow trained (15-5 dynes / cm2), endothelialized grafts. Scale bar = 25 pm. Multiple independent grafts (n=5) were investigated for whole blood assay. Figure 2E depicts representative immunofluorescent images characterizing endothelialized grafts explanted at 1 day, 7 days, 2 weeks, or 2 months postimplantation. Cross-sectional segments of grafts were stained for aSMA, CD31, CD68, HLA-A, and MYH11. DNA was counterstained by DAPI. The graft lumen is indicated by the asterisk *. Scale bar = 200 pm. Figure 2F depicts a representative quantification of the percentage of endothelial cell coverage on the luminal surface of hiPSC-TEVCs by CD31 and HLA-A immunofluorescence analysis in Figure 2E. Quantification was performed based on representative sections near mid-grafts, and three independent grafts were implanted into three respective nude rats for experiments (n=3). Mean values and standard error of the mean indicated by the error bars are shown. Figure 2G depicts a representative schematic illustrating generation of hiPSC-TEVCs for future therapy for single ventricle defects using allogeneic universal hiPSC-TEVCs. This novel strategy for generating endothelialized, functional venous conduits will help prevent thrombus formation and stenosis via hiPSC-EC-produced glycocalyx components, anticoagulant proteins, and basement membrane components enhanced under high shear stress (15 dynes / cm2) training in bioreactors. Prior to implantation of vascular conduits as IVC interposition grafts in nude rats, shear stress was gradually decreased to 5 dyne / cm2to mimic that of the IVC. As a potential future therapy for treating single ventricle defects, off-the-shelf, decellularized donor vascular grafts can be endothelialized with allogeneic universal hiPSC-ECs that are immunocompatible with any patient. These endothelialized grafts could provide immediate endothelial functions, including preventing blood coagulation on biomaterial surface, expediting host EC recruitment, and enabling the remodeling and maturation of grafts into functional host venous conduits for single ventricle treatment.

[0019] Figure 3, comprising Figure 3A through Figure 3E, depicts representative experiments demonstrating endothelialization function and anti coagulation of the hiPSC-EC-coated grafts. Related to Figure 1 and Figure 2. Figure 3A depicts a representative micro-computed tomography (micro-CT) image of rats implanted with shear flow-trained (15-5 dynes / cm2) hiPSC-TEVCs as interposition IVC grafts one month after implantation. The intact flow of the contrast agent across the entire rat IVC indicates that the implanted graft is functionally patent in Attorney Docket No.: 047162-5388-00WO

[0020] vivo one-month post-implantation. The white rectangle indicates graft location after TVC implantation. The liver, right and left kidneys, aorta, (inferior) vena cava, and a common iliac vein in the rat are labeled. Note that four independent grafts were implanted into four respective nude rats in the studies (n=4). Figure 3B and Figure 3C depict representative images of preexplanted and explanted non-endothelialized dHUA grafts without (Figure 3B) and with 15-5 dynes / cm2shear flow (Figure 3C) training exhibited apparent thrombus formation after 2 weeks of implantation (n=3). Dashed white rectangles indicate grafts implanted into IVC. Dashed black rectangles indicate explanted grafts. Black arrows indicate the formation of a thrombus within the implanted grafts. Scale bar = 1 mm. IVC, Inferior vena cava; AO, aorta. Figure 3D depicts a cartoon illustrating the structure of the endothelial glycocalyx (EG) within blood vessels. The EG is composed of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium. Glycosaminoglycans (GAGs) linked to core proteins in proteoglycans are able to prevent thrombus formation through interactions with anticoagulant proteins, including tissue factor pathway inhibitor (TFPI) and tissue plasminogen activator (tPA). Pulsatile, laminar shear stress has been known to enhance endothelial functions, including increased production of anticoagulant proteins. Figure 3E depicts representative immunofluorescent images characterizing grafts in the presence or absence of luminal hiPSC-EC endothelialization and with or without shear flow training prior to implantation through immunostaining with lectin, heparan sulfate (HS), TFPI, tPA, and laminin to investigate their endothelial phenotype. DNA was counterstained by DAPI. Graft lumen is indicated by the asterisk *. Scale bar = 200 pm. Note that three independent non-endothelialized grafts with or without shear flow training as well as three independent hiPSC-EC-endothelialized grafts with or without shear flow training were utilized for staining on representative sections.

[0021] Figure 4, comprising Figure 4A through Figure 41, depicts representative results from experiments investigating antithrombotic marker expression of hiPSC-EC-coated grafts and characterizing decellularized HUA scaffolds. Related to Figure 1 and Figure 2. Figure 4A depicts representative images of en face immunostaining of anticoagulation markers including lectin-binding carbohydrates, HS, TFPI, and tPA in grafts coated with hiPSC-ECs statically followed by a 6-hr abrupt flow or in grafts under a gradual shear flow training (15-5 dynes / cm2) in bioreactors. Scale bar = 200 pm. Figure 4B depicts representative quantification of graft areas positive for expression of anticoagulant markers in Figure 4A. Segments (~5 mm in length) Attorney Docket No.: 047162-5388-00WO

[0022] across grafts were stained for lectin-binding carbohydrates, HS, TFPI, and tPA. Mean values and standard error of the mean (S.E.M.) indicated by the error bars are shown. Multiple independent grafts (n=4) were quantified for expression of anticoagulant markers. A nonparametric Mann-Whitney test was used to compare two groups of grafts (*p<0.05). Figure 4C depicts a representative mRNA expression analysis of antithrombotic (eNOS, KLF2, and KLF4) and thrombogenic (E-Selectin, P-Selectin, ICAM-1, and VCAM-1) markers in grafts coated with hiPSC-ECs statically followed by a 6-hr abrupt flow and in grafts coated with hiPSC-ECs under a gradual shear flow training (15-5 dynes / cm2). Mean values and standard error of the mean (S.E.M.) indicated by the error bars are shown. Multiple independent grafts (n=7) were quantified for expression of antithrombotic and thrombogenic markers. A nonparametric Mann-Whitney test was used to compare two groups of grafts (*p<0.05; **p<0.01; ***p<0.001).

[0023] Figure 4D depicts representative immunostaining images of anticoagulant markers expressed by hiP SC -EC-coated grafts (15-5 dynes / cm2) explanted after 1 or 7 days of implantation. Graft lumen is indicated by the asterisk *. Scale bar = 200 pm. Three independent grafts were utilized and implanted into three respective nude rats for experiments. Figure 4E depicts representative images characterizing non-decellularized and decellularized HU As. (i) Representative images of histological characterizations (H&E, Masson’s Trichrome, EVG, Collagen IV, and Alizarin Red) of the non-decellularized and decellularized HUAs revealed efficient removal of nuclear materials, well preserved extracellular collageneous matrix, effective luminal expression of basement membrane protein collagen IV, appreciable amounts of elastin, and absence of calcification after vessel decellularization (n=4 grafts in each group). Scale bar = 75 pm. (ii) Highly efficient removal of nuclear materials in decellularized HUAs (n=7 grafts in each group). DNA contents were measured using the Invitrogen™ Quant-iT™ PicoGreen™ assay per the vendor’s instruction, (iii) Mechanical characterizations of the non-decellularized and decellularized HUAs revealed comparable mechanical properties including suture retention (iii), rupture pressure (iv), failure strain (v), maximum tensile stress (vi), wall thickness (vii), and inner diameter (viii). Mean values and standard error of the mean indicated by the error bars are shown (ii-viii). Multiple grafts (n=5 for each group) were quantified for mechanical characterizations of the non-decellularized and decellularized HUAs (iii-viii). A nonparametric Mann-Whitney test was used to compare two groups of grafts (ii-viii; ***p<0.001; ns, not significant). Figure 4F depicts representative images of immunostaining of endothelial markers Attorney Docket No.: 047162-5388-00WO

[0024] (CD31 and eNOS) and human leukocyte antigen type A (HLA-A) in TEVCs coated with ECs derived from an additional hiPSC line reported in previous studies (Luo, J., et al., 2021, Acta Biomater 119:184-196) under a gradual shear flow training (15-5 dynes / cm2). Cross-sectional segments of grafts after luminal endothelialization with hiPSC-ECs were stained for CD31, eNOS, and HLA-A. DNA was counterstained by DAPI. Graft lumen is indicated by the asterisk *. Scale bar = 200 pm. Figure 4G depicts representative percentage quantification of the luminal surfaces of TEVCs covered by hiPSC-ECs based on the immunostaining results in Figure 4F. Quantification was performed based on three grafts that were independently endothelialized by vascular hiPSC-ECs derived from an independent hiPSC line (Luo, J., et al., 2021, Acta Biomater 119:184-196) using an alternative vascular endothelial cell differentiation protocol. (Patsch, C., et al., 2015, Nat Cell Biol 17:994-1003). Mean values and standard error of the mean (S.E.M.) indicated by the error bars are shown. Figure 4H depicts representative images of pre-explant and explanted hiPSC-EC-endothelialized grafts. Grafts with luminal endothelialization followed by 15-5 dynes / cm2shear training were implanted as IVC interposition grafts and explanted 1 -month post-implantation. Dashed white rectangles indicate grafts implanted into IVC. Scale bar = 1 mm. IVC, Inferior vena cava; AO, aorta. Note that five independent hiPSC-EC-endothelialized (15-5 dynes / cm2) grafts were implanted into five respective nude rats for investigation. Figure 41 depicts representative immunostaining images of luminal endothelialized grafts explanted 1- month post-implantation. Cross-sectional segments of grafts were stained for eNOS and HLA-A. DNA was counterstained by DAPI. Graft lumen is indicated by the asterisk *. Scale bar = 200 pm. Note that five independent hiPSC-EC-endothelialized (15-5 dynes / cm2) grafts were utilized for experiments.

[0025] Figure 5, comprising Figure 5A and Figure 5B, depicts representative images of explanted grafts coated with hiPSC-ECs under venous-like, 5 dynes / cm2or arterial-like, 15 dynes / cm2flow shear stress training. Figure 5A depicts a representative image of a graft with hiPSC-ECs coated to the luminal surface of decellularized human umbilical arteries (dHUA) in a medium containing 10% FBS, 5 ng / mL VEGF-A, and 30 mg / mL dextran. The shear stress training was initiated at around 1 dyne / cm2and gradually increased towards 5 dynes / cm2over 24 hours, which was continued for an additional 4 or 5 days. Three grafts were implanted as inferior vena cava (IVC) interposition grafts in nude rats for 2 or 4 weeks, and two grafts showed evidence of thrombosis. Figure 5B depicts a representative image of a graft with hiPSC-ECs Attorney Docket No.: 047162-5388-00WO

[0026] coated to the luminal surface of dHUA, and shear stress was started at around 1 dyne / cm2for 12 hours. The flow shear stress training was next gradually increased towards 15 dynes / cm2over 24 hours, which was maintained for an additional 3.5 days. This endothelialized graft was then implanted as IVC interposition graft in a nude rat model for 4 weeks. Explant histological analysis showed evident blood clotting in this graft. Dashed rectangles indicate grafts implanted into the IVC. Arrows indicate the formation of a thrombus. Scale bar = 1 mm (a graduation on the ruler).

[0027] Figure 6, comprising Figures 6A through 6L, depicts the generation and characterization of immunocompatible universal human induced pluripotent stem cells (iPSCs) and their endothelial derivatives. Figure 6A depicts a schematic illustration of the genome engineering strategy used to generate universal hiPSCs. CRISPR / Cas9-mediated knockout of B2M and OITA eliminated expression of MHC class I and II molecules, respectively, while TALEN-mediated knock-in of CD47 at the AAVS1 safe-harbor locus conferred “don’t eat me” signaling to evade immune clearance by NK cells. Figure 6B depicts representative images of the differentiation of wild-type and universal hiPSCs into VE-cadherin-positive endothelial cells (hiPSC-ECs). Scale bar, 25 pm. Figure 6C depicts a flow cytometry analysis of HLA class I and HLA-DR expression in wild-type and universal hiPSC-ECs, confirming the loss of MHC expression in the engineered lines. Figure 6D depicts a flow cytometric analysis of CD47 surface expression in wild-type, B2M- / CIITA- double-knockout, and CD47 knock-in universal hiPSC-ECs. Figure 6E depicts representative images of a capillary network formation assay of wild-type and universal hiPSC-ECs. Figure 6F depicts a quantification of the number of branch points of a capillary network formation assay of wild-type and universal hiPSC-ECs demonstrating comparable angiogenic capacity between the two groups. Figure 6F depicts a quantification of the number of branch points of a capillary network formation assay of wild-type and universal hiPSC-ECs demonstrating comparable angiogenic capacity between the two groups. Figure 6G depicts a quantification of total tube length of a capillary network formation assay of wild-type and universal hiPSC-ECs demonstrating comparable angiogenic capacity between the two groups. Figure 6H depicts representative images showing uptake of acetylated low-density lipoprotein (Ac-LDL) by wild-type and universal hiPSC-ECs, indicating preserved endothelial functionality. Figure 61 depicts a quantification of Ac-LDL-positive cells performed using Imaged. Figure 6J depicts representative images showing detection of nitric oxide (NO) Attorney Docket No.: 047162-5388-00WO

[0028] production in wild-type and universal hiPSC-ECs cultured under static and swirling conditions using the fluorescent probe DAF-FM diacetate. Figure 6K depicts a graph of mean fluorescence intensity corresponding to intracellular NO levels quantified using ImageJ. Figure 6L depicts a graph of nitrite concentration in the culture medium, reflecting NO release, was measured in wild-type and universal hiPSC-ECs.

[0029] Figure 7, comprising Figures 7A through 7E, depicts in vitro immune responses of universal hiPSC-ECs. Figure 7A depicts a flow cytometric analysis of CD4+T cell activation following co-culture with wild-type or universal hiPSC-ECs. T cell activation was assessed by the expression of activation markers after antigen-independent co-culture. Figure 7B depicts a quantification of HLA-DR. CD4 T cells demonstrating reduced activation in response to universal hiPSC-ECs compared with wild-type controls. Figure 7C depicts a measurement of tumor necrosis factor-a (TNF-a) secretion in the supernatant from CD4+T cell and hiPSC-EC co-cultures, showing attenuated cytokine release in response to universal hiPSC-ECs. Figure 7D depicts TNF-a release from CD8+T cells co-cultured with wild-type or universal hiPSC-ECs, indicating reduced cytotoxic T cell activation against universal cells. Figure 7E depicts an assessment of natural killer (NK) cell-mediated cytotoxicity using the NK-92 cell line cocultured with hiPSC-ECs. Universal hiPSC-ECs exhibit decreased NK cell-induced lysis compared with wild-type hiPSC-ECs, confirming enhanced immune evasion capacity.

[0030] Figure 8, comprising Figures 8A through 8J, depicts in vivo immune responses of universal hiPSC-EC coated grafts. Figure 8A depicts a schematic illustration of the preparation of universal hiPSC-EC-coated vascular grafts and the shear-conditioning protocol prior to implantation into the rat inferior vena cava (IVC). Grafts were endothelialized with either wildtype or universal hiPSC-ECs and subjected to arterial-like shear stress (15-5 dyne / cm2) to promote endothelial maturation and stability before transplantation. Figure 8B depicts a flow cytometric analysis confirming successful reconstitution of human immune cells in rats prior to graft implantation. Two to four weeks prior to surgery, human peripheral blood mononuclear cells (PBMCs; 100 million cells / kg body weight) were administered via tail vein injection. The presence of human CD45-positive circulating cells confirmed human immune reconstitution. Representative plots from a reconstituted and a non-reconstituted rat are shown. Figure 8C depicts representative images of wild-type and universal hiPSC-EC-coated, shear-trained grafts one week post-transplantation and after explantation from the rat IVC interposition model. The Attorney Docket No.: 047162-5388-00WO

[0031] white rectangle highlights the region of graft implantation. The native rat TVC and aorta are indicated. Scale bar = 1 mm. Data derived from n = 4 grafts per group. Figure 8D depicts representative immunofluorescence images showing CD3+T cell infiltration within the graft wall 1 week after implantation. Scale bar, 100 pm. Figure 8E depicts a quantification of CD3+T cell infiltration in wild-type and universal hiP SC -EC-coated grafts (n = 4 per group). Five representative sections spanning the entire graft were analyzed per sample. Figure 8F depicts representative images of immunofluorescence staining for Granzyme B expression in explanted grafts demonstrating cytotoxic immune cell infiltration. Scale bar, 100 pm. Figure 8G depicts a quantification of granzyme B-positive puncta per field in wild-type and universal hiPSC-EC-coated grafts, indicating attenuated cytotoxic immune activity in universal grafts. Figure 8H depicts representative images of Immunostaining for CD31 showing endothelial coverage in preimplanted and explanted grafts 1 week post-transplantation. Scale bar, 200 pm. Figure 81 depicts a quantification of luminal endothelial coverage (CD31+area) in pre-implanted grafts. The CD31+surface area was measured using ImageJ and expressed as the percentage of the total CD31+EC luminal area. Figure 8J depicts a quantification of luminal endothelial coverage (CD31+area) in 1-week post-explanted grafts. The CD31+surface area was measured using ImageJ and expressed as the percentage of the total CD31+EC luminal area.

[0032] Figure 9, comprising Figures 9A through 9E, depicts a characterization of pluripotency and cardiovascular differentiation potential of universal hiPSCs. Figure 9A depicts an immunofluorescence analysis of wild-type and universal hiPSCs showing robust expression of pluripotency markers. Scale bar, 100 pm. Figure 9B depicts directed differentiation of universal hiPSCs into major cardiovascular lineages, including cardiac troponin T (cTnT)-positive cardiomyocytes. Figure 9C depicts directed differentiation of universal hiPSCs into major cardiovascular lineages, including smooth muscle a-actin (SMA)-positive vascular smooth muscle cells. Figure 9D depicts directed differentiation of universal hiPSCs into major cardiovascular lineages, including VE-cadherin-positive endothelial cells. The presence of the major lineages confirms preserved multilineage potential following genome editing. Figure 9E depicts an immunofluorescence characterization of universal hiPSC-derived endothelial cells (hiPSC-ECs) demonstrating expression of canonical endothelial markers CD31 and endothelial nitric oxide synthase (eNOS). Scale bar, 100 pm. Attorney Docket No.: 047162-5388-00WO

[0033] Figure 10, comprising Figures 10A through 10D, depicts an in vitro evaluation of endothelial coverage and antithrombotic function of universal hiPSC-EC-coated vascular grafts. Figure 10A depicts an analysis of fibrinogen adsorption on the luminal surface of universal hiPSC-EC-coated, shear-trained grafts compared with non-EC-coated controls. Reduced fibrinogen deposition on EC-coated grafts indicates diminished thrombogenic surface activity. Scale bar, 20 pm. Figure 10B depicts a graph showing phalloidin staining shows cytoskeletal organization and continuous endothelial coverage on the luminal surface of universal hiPSC-EC-coated, flow-conditioned grafts. Figure 10C depicts a quantification of fibrinogen adsorption on the luminal surfaces of universal hiPSC-EC-coated shear-trained and non-EC-coated grafts. Decellularized human umbilical artery (dHUA) grafts were endothelialized with universal hiPSC-ECs and subjected to arterial-like shear stress (15-5 dyne / cm2) in a flow bioreactor.

[0034] Midsections (~5 mm) of grafts were incubated with 50 mg / mL Alexa Fluor 647647-conjugated human plasma fibrinogen, and mean fluorescence intensity was quantified using ImageJ. Figure 10D depicts an assessment of thrombus formation on universal hiPSC-EC-coated, flow-trained grafts following exposure to whole human blood. Graft segments were incubated with freshly drawn human blood for 30 minutes, rinsed three times with phosphate-buff ered saline (PBS), and analyzed by scanning electron microscopy (SEM). Representative SEM images show markedly reduced fibrin and platelet aggregates on EC-coated grafts. The white arrow indicates the direction of shear flow during the preconditioning process. Scale bar, 100 pm.

[0035] Figure 11 depicts an in vivo assessment of antithrombotic performance and patency of universal hiPSC-EC-coated vascular grafts in immunodeficient rats. Prior to transplantation, the luminal surfaces of decellularized human umbilical artery (dHUA) grafts were endothelialized with either wild-type (left) or universal (right) hiPSC-derived endothelial cells (hiPSC-ECs) and subjected to arterial-like shear conditioning (15-5 dyne / cm2) in a flow bioreactor to promote endothelial maturation and adhesion stability. The grafts were implanted into the rat inferior vena cava (IVC) via end-to-end anastomosis. Representative images of grafts one week posttransplantation (pre-explant) are shown. The implanted segments are indicated by white dashed rectangles. The native rat IVC and aorta are labeled for anatomical reference. In total, n = 5 wildtype hiPSC-EC-coated and n = 2 universal hiPSC-EC-coated grafts were evaluated. Scale bar, 1 mm. Attorney Docket No.: 047162-5388-00WO

[0036] Figure 12, comprising Figures 12A through 12D, depicts infiltration of human CD8 and CD4+T cells in wild-type and universal hiPSC-EC-coated vascular grafts. Figure 12A depicts representative immunofluorescence images of explanted wild-type hiPSC-EC-coated grafts stained for human CD8+and CD4+T cells infiltrating the graft wall. Asterisks (*) indicate the graft lumen. Scale bar, 100 pm. Figure 12B depicts a quantification of human CD8+and CD4+T cell infiltration in wild-type hiPSC-EC-coated grafts. Data were collected from n = 4 explanted grafts. Figure 12C depicts representative immunofluorescence images of explanted universal hiPSC-EC-coated grafts stained for human CD8+and CD4+T cells, showing markedly reduced immune cell infiltration compared with wild-type grafts. Asterisks (*) denote the graft lumen. Scale bar, 100 pm. Figure 12D depicts a quantification of human CD8+and CD4+T cell infiltration in universal hiPSC-EC-coated grafts (n = 4). Data represent mean values per graft from multiple representative fields across the graft wall.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention is based in part on the discovery that applying a shear stress training / schedule / regimen to a graft seeded with human induced pluripotent stem cell-derived endothelial cells provides immediate antithrombotic function. In various embodiments, the disclosure relates to tissue-engineered vascular conduits (TEVCs) used to treat heart defects such as single ventricle congenital heart defects (SVCHDs). In some embodiments, the TEVC comprises a graft and one or more population of cells, wherein the one or more population of cells is seeded on the graft, wherein a shear stress is applied to the population of cells seeded on the graft. In various embodiments, the disclosure relates to methods of generating the TEVC of the present invention.

[0039] Definitions

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described. Attorney Docket No.: 047162-5388-00WO

[0041] As used herein, each of the following terms has the meaning associated with it in this section.

[0042] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0043] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0044] As used herein, to “alleviate” a disease, defect, disorder or condition means reducing the severity of one or more symptoms of the disease, defect, disorder or condition.

[0045] As used herein, “autologous” refers to a biological material derived from the same individual into whom the material will later be re-introduced.

[0046] As used herein, “allogeneic” refers to a biological material derived from a genetically different individual of the same species as the individual into whom the material will be introduced.

[0047] As used here, “biocompatible” refers to any material, which, when implanted in a mammal, does not provoke a significant adverse response in the mammal. A biocompatible material, when introduced into an individual, is not toxic or injurious to that individual, nor does it induce immunological rejection of the material in the mammal.

[0048] As used herein, the terms “biocompatible polymer” and “biocompatibility” when used in relation to polymers are recognized in the art. For example, biocompatible polymers include polymers that are generally neither toxic to the host, nor degrade (if the polymer degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations in the host. In one embodiment, biodegradation generally involves degradation of the polymer in a host, e.g., into its monomeric subunits, which may be known to be effectively non-toxic.

[0049] Intermediate oligomeric products resulting from such degradation may have different toxicological properties, however, or biodegradation may involve oxidation or other biochemical reactions that generate molecules other than monomeric subunits of the polymer. Consequently, in one embodiment, toxicology of a biodegradable polymer intended for in vivo use, such as implantation or injection into a patient, may be determined after one or more toxicity analyses. It Attorney Docket No.: 047162-5388-00WO

[0050] is not necessary that any subject composition have a purity of 100% to be deemed biocompatible; indeed, it is only necessary that the subject compositions be biocompatible as set forth above. Hence, a subject composition may include polymers comprising 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or even less of biocompatible polymers, e.g., including polymers and other materials and excipients described herein, and still be biocompatible.

[0051] As used herein, a “graft” refers to a composition that is implanted into an individual, typically to replace, correct or otherwise overcome a cell, tissue, or organ defect. In some embodiments, a graft may include a scaffold. In some embodiments, a graft comprises decellularized tissue. In some embodiments, the graft comprises a cell, tissue, or organ. In some embodiments, the graft is produced by seeding cells on a biodegradable polymeric scaffold. In some embodiments, the graft comprises a scaffold and population of cells. In some embodiments, the graft comprise a scaffold and a population of cells that are seeded on the scaffold. In some embodiments, grafts comprising the scaffold and the population of cells are decellularized. The graft may comprise cells or tissue that originate from the same individual; this graft is referred to herein by the following interchangeable terms: “autograft,” “autologous transplant,” “autologous implant” and “autologous graft.” A graft comprising cells or tissue from a genetically different individual of the same species is referred to herein by the following interchangeable terms: “allograft,” “allogeneic transplant,” “allogeneic implant” and “allogeneic graft.” A graft from an individual to his identical twin is referred to herein as an “isograft,” a “syngeneic transplant,” a “syngeneic implant” or a “syngeneic graft ” A “xenograft,” “xenogeneic transplant” or “xenogeneic implant” refers to a graft from one individual to another of a different species.

[0052] As used herein, “scaffold” refers to a structure, comprising a biocompatible material that provides a surface suitable for adherence of a substance and proliferation of cells. A scaffold may further provide mechanical stability and support. A scaffold may be in a particular shape or form so as to influence or delimit a three-dimensional shape or form such as that assumed by a population of proliferating cells. Such shapes or forms include, but are not limited to, films (e.g. a form with two-dimensions substantially greater than the third dimension), ribbons, cords, sheets, flat discs, cylinders, spheres, three-dimensional amorphous shapes, etc.

[0053] The term "vascular tissue" is used herein to mean a blood vessel, a portion thereof, one or more valves dissected from a blood vessel, a valve retained within a portion of a blood vessel, an Attorney Docket No.: 047162-5388-00WO

[0054] aortic or pulmonary valve dissected and free of non- valvular tissue, an aortic or pulmonary valve retained within a dissected blood vessel or cardiac tissue, or any other vascular tissue that may be suitable for use as a prosthesis. Blood vessels may include arteries and veins, portions thereof, and vascular beds containing arteries or veins.

[0055] The term "decellularized" is used herein to mean that physical, chemical, or enzymatic means, or any combination thereof, has removed the cellular component of vascular tissue thereof. The remaining decellularized vascular tissue comprises the extracellular matrix of the native vascular tissue and may include, but is not limited to, elastin, collagen, fibrin, and other extracellular proteins or non-proteinaceous compounds found in vascular tissue, or any combination thereof known to one of ordinary skill in the art.

[0056] The terms "prosthesis", "vascular prosthesis", "vascular prostheses" or "vascular implant" are used herein to mean a surgical implant or implants derived from, or inserted into, the vascular system of a human or animal patient. The term is intended to apply to surgical implants made of synthetic or natural materials or any combination thereof including, but not limited to, decellularized vascular tissue.

[0057] The term "anti-thrombogenic agent" is used herein to mean any compound, or combination of compounds, that minimize the induction of thrombus formation, or the stability of the thrombus. Anti-thrombogenic compounds include glycosaminoglycans such as heparin, heparin sulfate, dermatan sulfate and any other glycosaminoglycan with antithrombotic activity known to one skilled in the art. Anti-thrombogenic compounds may also include dextran and derivatives thereof, hirudin and derivatives thereof, and coumarin and derivatives thereof, including but not limited to 4-hydroxycoumarin, warfarin, dicumarol, phenprocoumon and acenocoumarol, indan-1, 3-dione, anisindone, and any other related compounds known to one skilled in the art. The anti-thrombogenic compound may include thrombolytic agents including, but not limited to, proteins that dissolve blood thrombi, including urokinase, plasminogen activator, antithrombin III, and modified forms thereof. The anti-thrombogenic compound may also include any other compound that may be immobilized on a decellularized vascular prosthesis and which inhibits the formation of, or participates in the destabilization of, thrombotic occlusions of the vascular prosthesis.

[0058] The term "growth factor" is used herein to mean any protein or non-proteinaceous compound capable of inducing or promoting the growth of cells. Such cells include, but are not Attorney Docket No.: 047162-5388-00WO

[0059] limited to, endothelial cells, smooth muscle cells and fibroblasts. Growth factors may include, but are not limited to, fibroblast growth factor (FGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), heparin-binding epidermal growth factor (HBEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-0), vascular endothelial cell growth factor (VEGF), placenta growth factor (PIGF), insulin-like growth factor (IGF), or any other growth factor, fragment or derivative thereof known to one skilled in the art.

[0060] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkey and human), most preferably a human.

[0061] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0062] Description

[0063] The present invention provides tissue-engineered vascular conduits (TEVCs), methods for generating TEVCs, and methods of using TEVCs.

[0064] Tissue-Engineered Vascular Grafts

[0065] The present invention provides tissue-engineered vascular conduits (TEVCs). Exemplary TEVCs are described in U.S. nonprovisional application 17 / 781,478 and is incorporated by reference herein in its entirety. In some embodiments, the TEVC comprises a graft. In some embodiments, the graft comprises one or more of a scaffold, a biodegradable scaffold, a tissue, a decellularized tissue, a synthetic graft, or a combination thereof. In some embodiments, the graft is a polymeric graft. In some embodiments, the graft is a synthetic graft. Synthetic grafts include, Attorney Docket No.: 047162-5388-00WO

[0066] but are not limited to, grafts that comprise a scaffold and population of cells. In some embodiments, synthetic grafts comprise a scaffold and a population of cells that are seeded on the scaffold. In some embodiments, synthetic grafts comprising the scaffold and the population of cells are decellularized.

[0067] In some embodiments, the graft comprises a biodegradable scaffold. In some embodiments, the biodegradable scaffold comprises one or more synthetic polymers, one or more biopolymers, and or combinations thereof. In some embodiments, the synthetic polymer is one or more selected from the group consisting of: polyglycolic acid (PGA), polylactic acid (PLA), poly caprolactone, polyethylene glycol, polylactic-co-glycolic acid (PLGA), poly(glycerol sebacate) (PGS), fast-degrading polymers such as that comprising 87% glycolide, 7% trimethylene carbonate (TMC), and 6% polyethylene glycol and / or combinations thereof.

[0068] In some embodiments, the scaffold comprises one or more polymer mesh including for example, non-woven polymer meshes. The polymer mesh may have a thickness of 0.05 mm to about 0.1 mm, about 0.1 mm to about 0.3 mm, about 0.3 mm to about 0.5 mm, about 0.5 mm to about 0.7 mm, about 0.7 mm to about 0.9 mm, about 1 mm and the like. The polymer mesh may have a square shape with side lengths of about 1 mm to about 2 mm, about 2 mm to about 3 mm, about 3 mm to about 4 mm, about 4 mm to about 5 mm, about 5 mm to about 6 mm, about 6 mm to about 7 mm, about 7 mm to about 8 mm, about 8 mm to about 9 mm about 9 mm to about 10 mm and so on. The polymer meshes may be coated with one or more compounds for improving cell adhesion. For example, the polymer mesh may be coated with gelatin, poly-lysine, and the like. In some embodiments, the polymer mesh may be coated with one or more extracellular matrix proteins and / or fragments thereof. The extracellular matrix proteins may include one or more of gelatin, fibronectin, laminin, collagen, vitronectin, glycosaminoglycan, elastin, fibrillin, and / or combinations thereof.

[0069] In some embodiments, the graft may be seeded with one or more populations of cells. The cells can include stem cells such as embryonic stem cells, mesenchymal stem cells, bone marrow-derived stem cells, hematopoietic stem cells, and the like. The cells can include somatic cells including vascular somatic cells such as smooth muscle cells, endothelial cells, fibroblast, and the like. The cells may include stem cell-derived vascular smooth muscle cells (VSMCs), stem cell-derived vascular endothelial cells (ECs), and / or combinations thereof. The cells can include somatic cell-derived stem cells such as, for example, induced pluripotent stem cells Attorney Docket No.: 047162-5388-00WO

[0070] (hiPSCs). In some embodiments, the iPSC is obtained from a mammalian source. In some embodiments, the iPSC is a human iPSC (hiPSC). In some embodiments, the cells include vascular cells induced from hiPSCs, including for example, hiPSC-derived vascular smooth muscle cells (hiPSC-VSMCs), hiPSC-derived vascular endothelial cells (hiPSC-ECs), hiPSC-derived cardiomyocytes (hiPSC-CMs), and the like. The hiPSCs may include allogeneic stem cells, autogeneic stem cells, xenogeneic stem cells, gene-edited stem cells and / or combinations thereof.

[0071] The one or more populations of cells may include hiPSCs that are immunocompatible pluripotent stem cells. Any methods known in the art for increasing the immunocompatibility of hiPSCs may be used. In some examples, the one or more populations of hiPSCs have modulated or abrogated expression of one or more human leukocyte antigens (HLAs). In some embodiments, the one or more populations of hiPSCs have modulated expression of one or more of HLA-A alleles, HLA-B alleles, HLA-C alleles, one or more class II HLAs, and / or one or more combinations thereof. In some examples, the one or more hiPSCs have modulated, decreased, minimal, or no expression of MHC class I molecules and / or MHC class II molecules. In some embodiments, the one or more populations of hiPSCs have modulated, decreased, minimal, or no expression of 02-microglobulin (B2M). Decreases or elimination of the expression of 02-microglobulin (B2M) may inhibit assembly of MHC class I molecules and / or their transport to the plasma membrane. For example, MHC class I molecule assembly and / or MHC class I molecule transport may be modulated via modulation of B2M expression. In some embodiments, the one or more populations of hiPSCs have modulated, decreased, minimal, or no expression of class II transactivator (CIITA). Decreases or elimination of the expression of class II transactivator (CIITA) may decrease or inhibit the expression of MHC class II molecules. For example, the expression of MHC class II molecules may be modulated via modulation of CIITA expression. In some embodiments, the one or more hiPSCs may have modulated or increased CD47 expression. In some embodiments, the one or more hiPSCs are engineered to express CD47. CD47 expression may inhibit immune cell such as natural killer cell, T cell, B cell, and / or macrophage mediated killing of the hiPSCs.

[0072] Immunocompatible pluripotent stem cells may be useful for methods of making immunocompatible TEVCs optionally for use in allogeneic grafts. For example, Attorney Docket No.: 047162-5388-00WO

[0073] immunocompatible pluripotent stem cells may be derived from cells of a subject in which the TEVC is designated to be grafted or implanted.

[0074] Modulation of any gene expression in any populations of cells described herein such as hiPSCs may be achieved by any gene-editing methods known in the art. For example, any CRISPR-Cas9-mediated methods and / or any TALEN-mediated methods may be used. In some embodiments, the one or more populations of cells may include any cell type, for example vascular smooth muscle cells and vascular endothelial cells, induced from hiPSCs in which any gene expression has been modulated by any method known in the art.

[0075] The one or more populations of cells may be seeded onto the one or more biodegradable synthetic polymer scaffolds. The cells may be cultured under mechanical stimulation, biochemical stimulation, and / or combinations thereof. The mechanical stimulation may include incremental radial stretching, pulsatile radial distension, and or combinations thereof. The radial stretching may include incremental radial stretching. The incremental radial stretching may include radial strain having radial distension of up to about 0.5%, about 0.5% to about 1%, about 1% to about 1.5%, about 1.5% to about 2%, about 2% to about 2.5%, about 2.5% to about 3%, about 3% to about 3.5%, about 3.5% to about 4%, about 4% to about 4.5%, about 4.5% to about 5%, and / or greater than about 5%. In some embodiments, and incremental strain may be applied for a duration including up to about 1 day, about 1 day to about 1 week, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 5 weeks to about 6 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, or greater than about 10 weeks. The incremental strain may be gradually increased or decreased at a continuous rate over an increment of up to 1 week, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, or greater than 4 weeks. The pulsatile radial distension may include pulsatile radial distension having a pulse rate of up to about 110 bpm, about 110 bpm to about 120 bpm, about 120 bpm to about 130 bpm, about 130 bpm to about 140 bpm, about 140 bpm to about 150 bpm, about 150 bpm to about 160 bpm, about 160 to about 170 bpm, about 170 to about 180 bpm, about 180 to about 190 bpm, about 190 to about 200 bpm, and the like. The incremental strain and / or pulsatile radial distention may be applied as a regime including: 1) one or more phases having one or more particular radial strains (e.g., 0.5%, up to 5%, etc.) for one or more particular durations (e.g., 1 week, 4 weeks, etc ), 2) one or more Attorney Docket No.: 047162-5388-00WO

[0076] particular pulsatile radial distensions (e ., 110 bpm, 120 bpm, etc.) for one or more particular durations (e.g., 1 week, 4 weeks, etc.), and 3) one or more combinations thereof. Each phase may have a particular duration of about 1 day to about 1 week, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 5 weeks to about 6 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, and / or increments or combinations thereof. The regime may have a total duration of about 4 weeks, about 4 weeks to about 8 weeks, about 8 weeks to about 12 weeks, and / or increments thereof.

[0077] The biochemical stimulation may include culturing the one or more populations of cells in one or more culture media, including, for example, tissue engineered vascular graft (TEVG) culture media as described elsewhere herein. The culture media may include or may exclude one or more growth factors and / or other molecules and reagents including for example, transforming growth factor-pi (TGF pi), platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factors (FGFs), bone morphogenetic factors (BMPs), vascular endothelial growth factors (VEGFs), latent TGF-beta binding proteins (LTBPs), epidermal growth factor (EGF), copper sulfate, ascorbic acid, retinoic acid, polyphenols (such as tannic acid, epigallocatechin gallate (EGCG) and pentagalloylglucose [PGG]), microRNA-29 inhibitors, proteoglycan inhibitors, fetal bovine sera, human platelet lysates, human sera, and or combinations thereof. In some embodiments, the culture media comprises TGF pi. In some embodiments, the culture media excludes PDGF-BB.

[0078] In some embodiments, the synthetic graft comprises a biodegradable scaffold and a plurality of stem cell-derived VSMCs. In some embodiments, the synthetic graft comprises a biodegradable scaffold and a plurality of stem cell-derived VSMCs, wherein the plurality of stem cell-derived VSMCs are seeded on the biodegradable scaffold. In some embodiments, the synthetic graft comprises a biodegradable scaffold and a plurality of stem cell-derived VSMCs, wherein the plurality of stem cell-derived VSMCs are seeded on the biodegradable scaffold and are cultured under mechanical and biochemical stimulation. In some embodiments, the synthetic graft is decellularized.

[0079] In some embodiments, a graft is seeded with one or more populations of cells. In some embodiments, the graft is a synthetic graft. In some embodiments, the graft comprises a tissue. In some embodiments, the graft comprises a decellularized tissue. In some embodiments, the graft Attorney Docket No.: 047162-5388-00WO

[0080] comprises a decellularized synthetic graft. Tn some embodiments, the graft comprises a human umbilical artery. In some embodiments, the graft comprises a decellularized umbilical artery.

[0081] In some embodiments, the TEVC comprises a graft seeded with one or more population of cells. The cells can include stem cells such as embryonic stem cells, mesenchymal stem cells, bone marrow-derived stem cells, hematopoietic stem cells, and the like. The cells can include somatic cells including vascular somatic cells such as smooth muscle cells, endothelial cells, fibroblast, and the like. The cells may include stem cell-derived vascular smooth muscle cells (VSMCs), stem cell-derived vascular endothelial cells (ECs), and / or combinations thereof. The cells can include somatic cell-derived stem cells such as, for example, human induced pluripotent stem cells (hiPSCs). In some embodiments, the cells include vascular cells induced from hiPSCs, including for example, hiPSC-derived vascular smooth muscle cells (hiPSC-VSMCs), hiPSC-derived vascular endothelial cells (hiPSC-ECs), and the like. The hiPSCs may include allogeneic stem cells, autogeneic stem cells, xenogeneic stem cells, gene-edited stem cells and / or combinations thereof. In some embodiments, the cells are stem-cell derived cells. In some embodiments, the cells are endothelial cells. In some embodiments, the cells are stem-cell derived endothelial cells. In some embodiments, the cells are iPSC-derived endothelial cells. In some embodiments, the cells are hiPSC-derived endothelial cells.

[0082] In some embodiments, the TEVC comprises a graft seeded with hiPSC-derived endothelial cells. In some embodiments, the TEVC comprises a tissue seeded with hiPSC-derived endothelial cells. In some embodiments, the TEVC comprises a decellularized tissue seeded with hiPSC-derived endothelial cells. In some embodiments, the TEVC comprises a synthetic graft seeded with hiPSC-derived endothelial cells. In some embodiments, the TEVC comprises a decellularized synthetic graft seeded with hiPSC-derived endothelial cells. In some embodiments, the TEVC comprises human umbilical artery seeded with hiPSC-derived endothelial cells. In some embodiments, the TEVC comprises decellularized human umbilical artery seeded with hiPSC-derived endothelial cells.

[0083] In some embodiments, the TEVC comprises hiPSC-derived endothelial cells seeded on a synthetic graft comprising a biodegradable scaffold and a plurality of stem cell-derived VSMCs. In some embodiments, the TEVC comprises hiPSC-derived endothelial cells seeded on a synthetic graft comprising a biodegradable scaffold and a plurality of stem cell-derived VSMCs, wherein the plurality of stem cell-derived VSMCs are seeded on the biodegradable scaffold. In Attorney Docket No.: 047162-5388-00WO

[0084] some embodiments, the TEVC comprises hiPSC-derived endothelial cells seeded on a synthetic graft comprising a biodegradable scaffold and a plurality of stem cell-derived VSMCs, wherein the plurality of stem cell-derived VSMCs are seeded on the biodegradable scaffold and are cultured under mechanical and biochemical stimulation.

[0085] In some embodiments, a shear stress is applied to the population of cells seeded on the graft. In some embodiments, a shear stress is applied to hiPSC-derived endothelial cells seeded on the graft. In some embodiments, the shear stress is modulated. In some embodiments, the shear stress is modulated more than one time. In some embodiments, the shear stress is modulated 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times.

[0086] The magnitude and duration of the shear stress applied to the population of cells seeded on the graft can vary over a wide range. Shear stress can be calculated based on Poiseuille’s equation: T = 4 pQ / nr3, where T is shear stress, p is fluid viscosity, Q is medium flow rate, and r is the radius of the vessel. Devices and uses thereof for measuring shear stress and modulating shear stress are known in the art.

[0087] In some embodiments, the shear stress is applied for a duration of 0 to 1 second. In some embodiments, the shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the shear stress is applied for a duration of 0 to 1 day. In some embodiments, the shear stress is applied for a duration of 0 to 1 week. In some embodiments, the shear stress is applied for a duration of 0 to 1 month. In some embodiments, the shear stress is applied continuously.

[0088] In some embodiments, the initial shear stress is a low shear stress. In some embodiments, the shear stress is subsequently increased to a high shear stress. In some embodiments, the shear stress is further reduced from the high shear stress to a final shear stress.

[0089] In some embodiments, the initial shear stress is a low shear stress. In some embodiments, the low shear stress is a shear stress that is smaller than the shear stress in a blood vessel. In some embodiments, the low shear stress is a shear stress that is equal to the shear stress in a blood vessel. In some embodiments, the low shear stress is a shear stress that is larger than the shear stress in a blood vessel. In some embodiments, the low shear stress is chosen to be smaller than, equal to, about equal to, or larger than the shear stress in any physiological blood vessel. In some embodiments, the initial shear stress ranges from 0 dynes / cm2to 10 dynes / cm2. In some embodiments, the initial shear stress is 0 dynes / cm2, 1 dynes / cm2, 2 dynes / cm2, 3 dynes / cm2, 4 Attorney Docket No.: 047162-5388-00WO

[0090] dynes / cm2, 5 dynes / cm2, 6 dynes / cm2, 7 dynes / cm2, 8 dynes / cm2, or 9 dynes / cm2. In some embodiments, the initial shear stress ranges from 0 dynes / cm2to 1 dynes / cm2. In some embodiments, the initial shear stress ranges from 0 dynes / cm2to 0.1 dynes / cm2. In some embodiments, the initial shear stress is 1 dynes / cm2.

[0091] In some embodiments, the initial shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the initial shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the initial shear stress is applied for a duration of 0 to 1 day. In some embodiments, the initial shear stress is applied for a duration of 0 to 1 week. In some embodiments, the initial shear stress is applied for a duration of 0 to 1 month. In some embodiments, the initial shear stress is applied continuously. In some embodiments, the initial shear stress is applied for a duration of 12 hours.

[0092] In some embodiments, the initial shear stress is subsequently increased to a high shear stress. In some embodiments, the high shear stress is larger than the initial shear stress. In some embodiments, the high shear stress is equivalent to the arterial shear stress. In some embodiments, the high shear stress is chosen to be equivalent to or about equivalent to the shear stress of any physiological artery. In some embodiments, the high shear stress ranges from 10 to 100 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 90 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 80 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 70 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 60 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 50 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 40 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 30 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 20 dynes / cm2. In some embodiments, the high shear stress is 10 dynes / cm2, 11 dynes / cm2, 12 dynes / cm2, 13 dynes / cm2, 14 dynes / cm2, 15 dynes / cm2, 16 dynes / cm2, 17 dynes / cm2, 18 dynes / cm2, 19 dynes / cm2, or 20 dynes / cm2. In some embodiments, the high shear stress is 15 dynes / cm2.

[0093] In some embodiments, the high shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the high shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the high shear stress is applied for a duration of 0 to 1 day. In some embodiments, the high shear stress is applied for a duration of 0 to 1 week. In some embodiments, the high shear stress is applied for a duration of 0 to 2 weeks. In some embodiments, the high shear stress Attorney Docket No.: 047162-5388-00WO

[0094] is applied for a duration of 0 to 3 weeks. Tn some embodiments, the high shear stress is applied for a duration of 0 to 4 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 5 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 6 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 7 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 8 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 1 month. In some embodiments, the high shear stress is applied for a duration of 10 to 100 years. In some embodiments, the high shear stress is applied continuously. In some embodiments, the high shear stress is applied for a duration of 24 hours. In some embodiments, the high shear stress is applied for a duration of 3.5 days. In some embodiments, the high shear stress is applied for a duration of 24 hours and maintained for an additional 3.5 days. In some embodiments, the high shear stress is applied for a duration of 4.5 days.

[0095] In some embodiments, the shear stress is subsequently decreased from the high shear stress to a final shear stress. In some embodiments, the final shear stress is smaller than the shear stress of the high shear stress. In some embodiments, the final shear stress is equal to the shear stress exerted on a cell lining a blood vessel. In some embodiments, the final shear stress is equal to the shear stress exerted on a cell lining a vein. In some embodiments, the final shear stress is equivalent to the shear stress exerted on a cell lining a blood vessel of a recipient or subject receiving the TEVC of the present invention. In some embodiments, the final shear stress is chosen to be equal to or about equal to the shear stress known to be exerted on a cell lining any physiological blood vessel such as a vein. In some embodiments, the final shear stress is equivalent to the shear stress exerted on a cell lining a vein of a recipient or subject receiving the TEVC of the present invention. In some embodiments, the final shear stress is equal to the venous shear stress. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 100 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 80 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 60 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 40 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 20 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 15 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 10 dynes / cm2. In some embodiments, the final shear stress is 0 dynes / cm2, 1 dynes / cm2, 2 Attorney Docket No.: 047162-5388-00WO

[0096] dynes / cm2, 3 dynes / cm2, 4 dynes / cm2, 5 dynes / cm2, 6 dynes / cm2, 7 dynes / cm2, 8 dynes / cm2, or 9 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 1 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 0.1 dynes / cm2. In some embodiments, the final shear stress is 5 dynes / cm2.

[0097] In some embodiments, the final shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the final shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the final shear stress is applied for a duration of 0 to 1 day. In some embodiments, the final shear stress is applied for a duration of 0 to 1 week. In some embodiments, the final shear stress is applied for a duration of 0 to 1 month. In some embodiments, the final shear stress is applied continuously. In some embodiments, the final shear stress is applied for 12 hours.

[0098] In some embodiments, the shear stress is decreased from the high shear stress, to a rampdown shear stress, then to a final shear stress. In some embodiments, the ramp-down shear stress is smaller than the shear stress of the high shear stress. In some embodiments, the ramp-down shear stress is equal to the shear stress exerted on a cell lining a blood vessel. In some embodiments, the ramp-down shear stress is equal to the shear stress exerted on a cell lining a vein. In some embodiments, the ramp-down shear stress is chosen to be equal to or about equal to the shear stress known to be exerted on a cell lining any physiological blood vessel such as a vein. In some embodiments, the ramp-down shear stress is equivalent to the shear stress exerted on a cell lining a blood vessel of a recipient or subject receiving the TEVC of the present invention. In some embodiments, the ramp-down shear stress is equivalent to the shear stress exerted on a cell lining a vein of a recipient or subject receiving the TEVC of the present invention. In some embodiments, the ramp-down shear stress is equal to the venous shear stress. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 20 dynes / cm2. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 15 dynes / cm2. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 10 dynes / cm2. In some embodiments, the ramp-down shear stress is 0 dynes / cm2, 1 dynes / cm2, 2 dynes / cm2, 3 dynes / cm2, 4 dynes / cm2, 5 dynes / cm2, 6 dynes / cm2, 7 dynes / cm2, 8 dynes / cm2, or 9 dynes / cm2. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 1 dynes / cm2. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 0.1 dynes / cm2. In some embodiments, the ramp-down shear stress is 10 dynes / cm2. Attorney Docket No.: 047162-5388-00WO

[0099] In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 day. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 week. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 month. In some embodiments, the ramp-down shear stress is applied continuously. In some embodiments, the ramp-down shear stress is applied for 12 hours.

[0100] Changing the applied shear stress, for example when transitioning between initial shear stress, high shear stress, ramp-down shear stress, final shear stress, or any other shear stress, can be done in any fashion. In some embodiments, a change in applied shear stress is abrupt. In some embodiments, a change in applied shear stress occurs gradually. For example, applied shear stress may be increased or decreased at a constant rate until a new desired applied shear stress is reached. In some examples, applied shear stress may be increased or decreased at a variable rate. In some examples, the rate of change in applied shear stress may be described by any function. In some examples, applied shear stress may be increased or decreased in a stepwise fashion to reach a new desired shear stress. For example, an increase or decrease in applied shear stress of a larger magnitude may be achieved by any number of subsequent abrupt smaller magnitude increases or decreases in shear stress.

[0101] In some embodiments, it may take at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, or at least 2 weeks, to gradually reach a new desired applied shear stress from any previous applied shear stress. In some embodiments, it may take or take about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 1 week, or 2 weeks, to gradually reach a new desired applied shear stress from any previous applied shear stress. In some embodiments, it is particularly beneficial to gradually increase shear stress when transitioning from the initial shear stress to the high shear stress. In some embodiments, it is particularly beneficial to gradually increase shear stress when transitioning from a lower applied shear stress to a higher applied shear stress. For example, more gradual increases in applied shear stress may allow for minimal disruption of endothelial cell coating compared to more abrupt increases in applied shear stress. Attorney Docket No.: 047162-5388-00WO

[0102] Methods for generating TEVCs

[0103] The present invention provides methods for generating one or more TEVCs as described herein.

[0104] The method comprises obtaining one or more populations of cells. The cells can include stem cells such as embryonic stem cells, mesenchymal stem cells, bone marrow-derived stem cells, hematopoietic stem cells, and the like. The cells can include somatic cells including vascular somatic cells such as smooth muscle cells, endothelial cells, fibroblast, and the like. The cells may include stem cell-derived vascular smooth muscle cells (VSMCs), stem cell-derived vascular endothelial cells (ECs), and / or combinations thereof. The cells can include somatic cell-derived stem cells such as, for example, induced pluripotent stem cells (hiPSCs). In some embodiments, the iPSC is obtained from a mammalian source. In some embodiments, the iPSC is a human iPSC (hiPSC). In some embodiments, the cells include vascular cells induced from hiPSCs, including for example, hiPSC-derived vascular smooth muscle cells (hiPSC-VSMCs), hiPSC-derived vascular endothelial cells (hiPSC-ECs), and the like. The hiPSCs may include allogeneic stem cells, autogeneic stem cells, xenogeneic stem cells, gene-edited stem cells and / or combinations thereof.

[0105] The hiPSCs may be obtained by way of an allograft, an autograft and / or a xenograft. In some embodiments, the hiPSCs are modulated to enhance their immunocompatibility. Any methods known in the art for increasing the immunocompatibility of hiPSCs may be used. The hiPSCs with enhanced immunocompatibility may have modulated HLA expression. For example, the hiPSCs may have modulated expression of one or more of HLA- A alleles, HLA-B alleles, one or more class II HLAs, and / or combinations thereof. The immunocompatible hiPSCs may retain HLA-C expression.

[0106] In some examples, the hiPSCs are modulated to have decreased, minimal, or no expression of MHC class I molecules and / or MHC class II molecules. In some embodiments, the hiPSCs are modulated to have decreased, minimal, or no expression of p2-microglobulin (B2M). Decreases or elimination of the expression of p2-microglobulin (B2M) may inhibit assembly of MHC class I molecules and / or their transport to the plasma membrane. For example, MHC class I molecule assembly and / or MHC class I molecule transport may be modulated via modulation of B2M expression. In some embodiments, the hiPSCs are modulated to have decreased, Attorney Docket No.: 047162-5388-00WO

[0107] minimal, or no expression of class IT transactivator (CUT A). Decreases or elimination of the expression of class II transactivator (CIITA) may decrease or inhibit the expression of MHC class II molecules. For example, the expression of MHC class II molecules may be modulated via modulation of CIITA expression. In some embodiments, the hiPSCs are modulated to express CD47 or have increased CD47 expression. CD47 expression may inhibit immune cell such as natural killer cell and / or macrophage mediated killing of the hiPSCs.

[0108] The immunocompatible hiPSCs may be used in methods of making immunocompatible TEVCs optionally for use in allogeneic grafts. For example, immunocompatible hiPSCs may be derived from cells of a subject in which the TEVC is designated to be grafted or implanted.

[0109] Modulation of any gene expression in any populations of cells described herein such as hiPSCs may be achieved by any gene-editing methods known in the art. For example, any CRISPR-Cas9-mediated methods and / or any TALEN-mediated methods may be used.

[0110] In some embodiments, the method comprises inducing hiPSCs, including any immunocompatible hiPSCs described herein, to differentiate into a population of hiPSC-derived vascular endothelial cells (hiPSC-ECs), hiPSC-derived vascular smooth muscle cells (hiPSC-VSMCs), or any other desired cell type. In some embodiments, the method comprises expanding hiPSCs before being induced to differentiate into any desired cell type. Methods of expanding hiPSCs are known in the art. As a non-limiting examples, hiPSCs may be expanded using a medium comprising Mercaptoethanol, N2, B27, CHIRON, BMP4, or a combination thereof.

[0111] The hiPSCs may be induced to differentiate into hiPSC-ECs using differentiation media. Various differentiation media are known in the art. As a non-limiting example, the differentiation media may comprise activin A, FGF-2, VEGF-A, BMP4, or a combination thereof. As another non-limiting example, the differentiation media may comprise VEGF-A, Forskolin, or a combination thereof. As another non-limiting example, the differentiation media may comprise VEGF-A. The differentiation media may contain one or more additional factors including serum, such as fetal bovine serum (FBS). The media may contain about 10% FBS. The media may contain between about 5% and about 15% FBS, about 1% to about 20% FBS, and / or increments thereof. The media may contain up to about 0.5% FBS, about 0.5% to about 1% FBS, about 1% to about 2% FBS, about 2% to about 3% FBS, about 3% to about 4% FBS, about 4% to about 5% FBS, about 5% to about 6% FBS, about 6% to about 7% FBS, about 7% to about 8% FBS, about 8% to about 9% FBS, about 9% to about 10% FBS or more than 10% FBS. Attorney Docket No.: 047162-5388-00WO

[0112] In some embodiments, the method comprises expanding the population of hiPSC-ECs. The hiPSC-ECs may be expanded using expansion media. Various expansion media are known in the art. As a non-limiting example, the expansion media may comprise FGF-2, VEGF-A, BMP4, or a combination thereof. As another non-limiting example, the expansion media may comprise VEGF-A. The expansion media may contain one or more additional factors including serum, such as fetal bovine serum (FBS). The media may contain about 10% FBS. The media may contain between about 5% and about 15% FBS, about 1% to about 20% FBS, and / or increments thereof. The media may contain up to about 0.5% FBS, about 0.5% to about 1% FBS, about 1% to about 2% FBS, about 2% to about 3% FBS, about 3% to about 4% FBS, about 4% to about 5% FBS, about 5% to about 6% FBS, about 6% to about 7% FBS, about 7% to about 8% FBS, about 8% to about 9% FBS, about 9% to about 10% FBS or more than 10% FBS. The hiPSC-ECs may express one or more phenotypic markers including, but not limited to, CD31, CD 144, eNOS, KDR, NRP-1, or a combination thereof.

[0113] In some embodiments, the method comprises seeding the one or more population of cells on a graft, thereby endothelializing the graft. In some embodiments, the method comprises seeding hiPSC-EC on a graft. The graft may include one or more grafts as described herein. In some embodiments, the graft comprises one or more of a scaffold, a biodegradable scaffold, a tissue, a decellularized tissue, an engineered graft, or a combination thereof. In some embodiments, the graft is a polymeric graft. In some embodiments, the graft is an engineeredgraft. Grafts may comprise a scaffold and population of cells. In some embodiments, grafts comprise a scaffold and a population of cells that are seeded on the scaffold. In some embodiments, grafts comprising a scaffold and the population of cells are decellularized.

[0114] In some embodiments, the graft is an engineered graft. In some embodiments, the graft comprises a biodegradable scaffold and a plurality of vascular smooth muscle cells (VSMCs). In some embodiments, the VSMCs are hiPSC-VSMC.

[0115] In some embodiments, the method comprises seeding a population of cells on a graft and then applying a shear stress. Shear stress is applied as understood in the art and / or as described herein.

[0116] In some embodiments, the method comprises applying a shear stress to the population of cells seeded on the graft. In some embodiments, the method comprises applying a shear stress to hiPSC-derived endothelial cells seeded on the graft. In some embodiments, the method Attorney Docket No.: 047162-5388-00WO

[0117] comprises modulating the shear stress. Tn some embodiments, the method comprises modulating the shear stress more than one time. In some embodiments, the method comprises modulating the shear stress 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times.

[0118] The magnitude and duration of the shear stress applied to the population of cells seeded on the graft can vary over a wide range. Shear stress can be calculated based on Poiseuille’s equation: T = 4 pQ / nr3, where T is shear stress, p is fluid viscosity, Q is medium flow rate, and r is the radius of the vessel. Devices and uses thereof for measuring shear stress and modulating shear stress are known in the art.

[0119] In some embodiments, the method comprises applying the shear stress for a duration of 0 to 1 second. In some embodiments, the method comprises applying the shear stress for a duration of 0 to 1 minute. In some embodiments, the method comprises applying the shear stress for a duration of 0 to 1 hour. In some embodiments, the method comprises applying the shear stress for a duration of 0 to 1 day. In some embodiments, the method comprises applying the shear stress for a duration of 0 to 1 week. In some embodiments, the method comprises applying the shear stress for a duration of 0 to 1 month. In some embodiments, the method comprises applying the shear stress continuously.

[0120] In some embodiments, the initial shear stress is a low shear stress. In some embodiments, the shear stress is subsequently increased to a high shear stress. In some embodiments, the shear stress is further reduced from the high shear stress to a final shear stress.

[0121] In some embodiments, the initial shear stress is a low shear stress. In some embodiments, the low shear stress is a shear stress that is smaller than the shear stress in a blood vessel. In some embodiments, the low shear stress is a shear stress that is equal to the shear stress in a blood vessel. In some embodiments, the low shear stress is a shear stress that is larger than the shear stress in a blood vessel. In some embodiments, the low shear stress is chosen to be smaller than, equal to, about equal to, or larger than the shear stress in any physiological blood vessel. In some embodiments, the initial shear stress ranges from 0 dynes / cm2to 10 dynes / cm2. In some embodiments, the initial shear stress is 0 dynes / cm2, 1 dynes / cm2, 2 dynes / cm2, 3 dynes / cm2, 4 dynes / cm2, 5 dynes / cm2, 6 dynes / cm2, 7 dynes / cm2, 8 dynes / cm2, or 9 dynes / cm2. In some embodiments, the initial shear stress ranges from 0 dynes / cm2to 1 dynes / cm2. In some embodiments, the initial shear stress ranges from 0 dynes / cm2to 0.1 dynes / cm2. In some embodiments, the initial shear stress is 1 dynes / cm2. Attorney Docket No.: 047162-5388-00WO

[0122] In some embodiments, the initial shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the initial shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the initial shear stress is applied for a duration of 0 to 1 day. In some embodiments, the initial shear stress is applied for a duration of 0 to 1 week. In some embodiments, the initial shear stress is applied for a duration of 0 to 1 month. In some embodiments, the initial shear stress is applied continuously. In some embodiments, the initial shear stress is applied for a duration of 12 hours.

[0123] In some embodiments, the method comprises increasing the initial shear stress to a high shear stress. In some embodiments, the high shear stress is larger than the initial shear stress. In some embodiments, the high shear stress is equivalent to the arterial shear stress. In some embodiments, the high shear stress is chosen to be equal or about equal to any physiological arterial shear stress. In some embodiments, the high shear stress ranges from 10 to 100 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 90 dynes / cm2In some embodiments, the high shear stress ranges from 10 to 80 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 70 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 60 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 50 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 40 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 30 dynes / cm2. In some embodiments, the high shear stress ranges from 10 to 20 dynes / cm2. In some embodiments, the high shear stress is 10 dynes / cm2, 11 dynes / cm2, 12 dynes / cm2, 13 dynes / cm2, 14 dynes / cm2, 15 dynes / cm2, 16 dynes / cm2, 17 dynes / cm2, 18 dynes / cm2, 19 dynes / cm2, or 20 dynes / cm2. In some embodiments, the high shear stress is 15 dynes / cm2.

[0124] In some embodiments, the high shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the high shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the high shear stress is applied for a duration of 0 to 1 day. In some embodiments, the high shear stress is applied for a duration of 0 to 1 week. In some embodiments, the high shear stress is applied for a duration of 0 to 2 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 3 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 4 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 5 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 6 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 7 Attorney Docket No.: 047162-5388-00WO

[0125] weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 8 weeks. In some embodiments, the high shear stress is applied for a duration of 0 to 1 month. In some embodiments, the high shear stress is applied for a duration of 10 to 100 years. In some embodiments, the high shear stress is applied continuously. In some embodiments, the high shear stress is applied for a duration of 24 hours. In some embodiments, the high shear stress is applied for a duration of 3.5 days. In some embodiments, the high shear stress is applied for a duration of 24 hours and maintained for an additional 3.5 days. In some embodiments, the high shear stress is applied for a duration of 4.5 days.

[0126] In some embodiments, the method comprises decreasing the high shear stress to a final shear stress. In some embodiments, the final shear stress is smaller than the shear stress of the high shear stress. In some embodiments, the final shear stress is equal to the shear stress exerted on a cell lining a blood vessel. In some embodiments, the final shear stress is equal to the shear stress exerted on a cell lining a vein. In some embodiments, the final shear stress is equivalent to the shear stress exerted on a cell lining a blood vessel of a recipient or subject receiving the TEVC of the present invention. In some embodiments, the final shear stress is equivalent to the shear stress exerted on a cell lining a vein of a recipient or subject receiving the TEVC of the present invention. In some embodiments, the final shear stress is chosen to be equal or about equal to the shear stress exerted on a cell lining any physiological blood vessel such as a vein. In some embodiments, the final shear stress is equal to the venous shear stress. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 100 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 80 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 60 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 40 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 20 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 15 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 10 dynes / cm2. In some embodiments, the final shear stress is 0 dynes / cm2, 1 dynes / cm2, 2 dynes / cm2, 3 dynes / cm2, 4 dynes / cm2, 5 dynes / cm2, 6 dynes / cm2, 7 dynes / cm2, 8 dynes / cm2, or 9 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 1 dynes / cm2. In some embodiments, the final shear stress ranges from 0 dynes / cm2to 0.1 dynes / cm2. In some embodiments, the final shear stress is 5 dynes / cm2. Attorney Docket No.: 047162-5388-00WO

[0127] In some embodiments, the final shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the final shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the final shear stress is applied for a duration of 0 to 1 day. In some embodiments, the final shear stress is applied for a duration of 0 to 1 week. In some embodiments, the final shear stress is applied for a duration of 0 to 1 month. In some embodiments, the final shear stress is applied continuously. In some embodiments, the final shear stress is applied for 12 hours.

[0128] In some embodiments, the method comprises decreasing the high shear stress, to a rampdown shear stress, then to a final shear stress. In some embodiments, the ramp-down shear stress is smaller than the shear stress of the high shear stress. In some embodiments, the ramp-down shear stress is equal to the shear stress exerted on a cell lining a blood vessel. In some embodiments, the ramp-down shear stress is equal to the shear stress exerted on a cell lining a vein. In some embodiments, the ramp-down shear stress is equivalent to the shear stress exerted on a cell lining a blood vessel of a recipient or subject receiving the TEVC of the present invention. In some embodiments, the ramp-down shear stress is equivalent to the shear stress exerted on a cell lining a vein of a recipient or subject receiving the TEVC of the present invention. In some embodiments, the ramp-down shear stress is equal to the venous shear stress. In some embodiments, the ramp-down shear stress is chosen to be equal to or about equal to a physiological venous shear stress. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2 to 20 dynes / cm2. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 15 dynes / cm2. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 10 dynes / cm2. In some embodiments, the ramp-down shear stress is 0 dynes / cm2, 1 dynes / cm2, 2 dynes / cm2, 3 dynes / cm2, 4 dynes / cm2, 5 dynes / cm2, 6 dynes / cm2, 7 dynes / cm2, 8 dynes / cm2, or 9 dynes / cm2. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 1 dynes / cm2. In some embodiments, the ramp-down shear stress ranges from 0 dynes / cm2to 0.1 dynes / cm2. In some embodiments, the ramp-down shear stress is dynes / cm2. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 minute. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 hour. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 day. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 week. In some embodiments, the ramp-down shear stress is applied for a duration of 0 to 1 month. In Attorney Docket No.: 047162-5388-00WO

[0129] some embodiments, the ramp-down shear stress is applied continuously. In some embodiments, the ramp-down shear stress is applied for 12 hours.

[0130] The method comprises changing the applied shear stress, for example when transitioning between initial shear stress, high shear stress, ramp-down shear stress, final shear stress, or any other shear stress, in any fashion. In some embodiments, a change in applied shear stress is abrupt. In some embodiments, a change in applied shear stress occurs gradually. For example, applied shear stress may be increased or decreased at a constant rate until a new desired applied shear stress is reached. In some examples, applied shear stress may be increased or decreased at a variable rate. In some examples, the rate of change in applied shear stress may be described by any function. In some examples, applied shear stress may be increased or decreased in a stepwise fashion to reach a new desired shear stress. For example, an increase or decrease in applied shear stress of a larger magnitude may be achieved by any number of subsequent abrupt smaller magnitude increases or decreases in shear stress.

[0131] In some embodiments, it may take at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, or at least 2 weeks, to gradually reach a new desired applied shear stress from any previous applied shear stress. In some embodiments, it may take or take about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 1 week, or 2 weeks, to gradually reach a new desired applied shear stress from any previous applied shear stress. In some embodiments, it is particularly beneficial to gradually increase shear stress when transitioning from the initial shear stress to the high shear stress. In some embodiments, it is particularly beneficial to gradually increase shear stress when transitioning from a lower applied shear stress to a higher applied shear stress. For example, more gradual increases in applied shear stress may allow for minimal disruption of endothelial cell coating compared to more abrupt increases in applied shear stress.

[0132] The graft may be reseeded with one or more populations of hiPSC-VSMCs, hiPSC-ECs, and / or combinations thereof.

[0133] Method of Use Attorney Docket No.: 047162-5388-00WO

[0134] The TEVC of the present invention may be used to treat any cardiovascular diseases or diseases or disorders associated with an impaired circulatory system. The disease or disorder may include but is not limited to single ventricle congenital heart defects (SVCHDs). The TEVC of the present invention may also be used in any procedure, such as a surgical procedure, that requires a graft or conduit for a blood vessel including but not limited to veins, arteries, and capillaries. The procedure may be for the purposes of repairing or replacing blood vessels. In some embodiments, the TEVC of the present invention is used in any procedure for congenital vascular repair such as congenital venous repair.

[0135] In some embodiments, the present invention is directed to methods of treating a cardiovascular disease, including but not limited to SVCHD, comprising implanting the TEVC of the present invention to a subject in need thereof. For example, the present invention may directed to methods of repairing a damaged or diseased vasculature comprising implanting the TEVC of the present invention to a subject in need thereof. For example, the present invention may directed to methods of congenital vascular repair such as congenital venous repair comprising implanting the TEVC of the present invention to a subject in need thereof. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an infant or baby.

[0136] In some embodiments, the TEVC is implanted after an operation. In some embodiments, the TEVC is implanted after a Fontan operation.

[0137] Embodiments

[0138] Embodiment 1. A tissue-engineered vascular conduit (TEVC) comprising a graft and a plurality of stem-cell derived cells, wherein the plurality of stem-cell derived cells are seeded on the graft, wherein a shear stress is applied to the plurality of stem-cell derived cells.

[0139] Embodiment 2. The TEVC of embodiment 1, wherein the shear stress is modulated.

[0140] Embodiment 3. The TEVC of any of the previous embodiments, wherein the shear stress is modulated more than once. Attorney Docket No.: 047162-5388-00WO

[0141] Embodiment 4. The TEVC of any of the previous embodiments wherein the shear stress is initially a low shear stress and subsequently increased to a high shear stress.

[0142] Embodiment 5. The TEVC of any of the previous embodiments, wherein the low shear stress is 0.01 to 10 dyne / cm2or 1 dyne / cm2.

[0143] Embodiment 6. The TEVC of any of the previous embodiments, wherein the high shear stress is an arterial shear stress, 10 to 100 dyne / cm2or 15 dyne / cm2.

[0144] Embodiment 7. The TEVC of any of the previous embodiments, wherein the shear stress is further reduced from the high shear stress to a final shear stress.

[0145] Embodiment 8. The TEVC of any of the previous embodiments, wherein the final shear stress is a venous shear stress, 0.01 to 10 dyne / cm2or 5 dyne / cm2.

[0146] Embodiment 9. The TEVC of any of the previous embodiments, wherein the shear stress is reduced from the high shear stress to a ramped-down shear stress before being reduced to a final shear stress.

[0147] Embodiment 10. The TEVC of any of the previous embodiments, wherein the ramped-down shear stress is 0.01 to 100 dyne / cm2or 10 dyne / cm2.

[0148] Embodiment 11. The TEVC of any of the previous embodiments, wherein the stem-cell derived cells are derived from human induced pluripotent stem cells (hiPSCs).

[0149] Embodiment 12. The TEVC of any of the previous embodiments, wherein the stem-cell derived cells are stem-cell derived endothelial cells.

[0150] Embodiment 13. The TEVC of any of the previous embodiments!, wherein the stem-cell derived cells are hiPSC-derived endothelial cells. Attorney Docket No.: 047162-5388-00WO

[0151] Embodiment 14. The TEVC of any of the previous embodiments, wherein the graft is one or more of a scaffold, a biodegradable scaffold, a tissue, a decellularized tissue, a decellularized human donor blood vessel, an animal derived blood vessel, a tissue engineered vascular graft based on human cells, a tissue engineered vascular graft based on animal cells, or a combination thereof.

[0152] Embodiment 15. The TEVC of any of the previous embodiments wherein the graft is generally tubular with a diameter ranging from 3 to 18 millimeters.

[0153] Embodiment 16. The TEVC of any of the previous embodiments, wherein the graft comprises a biodegradable scaffold and a plurality of stem cell-derived vascular smooth muscle cells (VSMCs), wherein the plurality of stem cell-derived VSMCs are seeded on the biodegradable scaffold and are cultured under mechanical and biochemical stimulation.

[0154] Embodiment 17. A method of generating a tissue-engineered vascular conduit (TEVC), the method comprising:

[0155] a. obtaining a plurality of stem -cell derived cells;

[0156] b. seeding the plurality of stem-cell derived cells on a graft; and

[0157] c. applying a shear stress to the plurality of stem-cell derived cells seeded on the graft.

[0158] Embodiment 18. The method of any of the previous embodiments, wherein the shear stress is modulated.

[0159] Embodiment 19. The method of any of the previous embodiments, wherein the shear stress is modulated more than once.

[0160] Embodiment 20. The method of any of the previous embodiments, wherein the shear stress is initially a low shear stress and subsequently increased to a high shear stress.

[0161] Embodiment 21. The method of claim 20, wherein the low shear stress is 0.01 to 10 dyne / cm2. Attorney Docket No.: 047162-5388-00WO

[0162] Embodiment 22. The method of any of the previous embodiments, wherein the high shear stress is an arterial shear stress, 10 to 100 dyne / cm2or 15 dyne / cm2.

[0163] Embodiment 23. The method of any of the previous embodiments, wherein the shear stress is further reduced from the high shear stress to a final shear stress.

[0164] Embodiment 24. The method of any of the previous embodiments, wherein the final shear stress is a venous shear stress, 0.01 to 10 dyne / cm2or 5 dyne / cm2.

[0165] Embodiment 25. The method of any of the previous embodiments, wherein the shear stress is reduced from the high shear stress to a ramped-down shear stress before being reduced to a final shear stress.

[0166] Embodiment 26. The method of any of the previous embodiments, wherein the ramped-down shear stress is 0.01 to 100 dyne / cm2or 10 dyne / cm2.

[0167] Embodiment 27. The method of any of the previous embodiments, wherein the stem-cell derived cells are derived from human induced pluripotent stem cells (hiPSCs).

[0168] Embodiment 28. The method of any of the previous embodiments, wherein the stem-cell derived cells are stem-cell derived endothelial cells.

[0169] Embodiment 29. The method of any of the previous embodiments, wherein the stem-cell derived cells are hiPSC-derived endothelial cells.

[0170] Embodiment 30. The method of any of the previous embodiments, wherein the graft is one or more of a scaffold, a biodegradable scaffold, a tissue, a decellularized tissue, or a combination thereof. Attorney Docket No.: 047162-5388-00WO

[0171] Embodiment 31. The method of any of the previous embodiments, wherein the graft is a synthetic graft.

[0172] Embodiment 32. The method of any of the previous embodiments, wherein the synthetic graft comprises a biodegradable scaffold and a plurality of stem cell-derived vascular smooth muscle cells (VSMCs), wherein the plurality of stem cell-derived VSMCs are seeded on the biodegradable scaffold and are cultured under mechanical and biochemical stimulation.

[0173] EXPERIMENTAL EXAMPLES

[0174] The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

[0175] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, point out specific embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0176] Example 1: Shear Stress, Stem Cell Types, and Scaffold Types on TEVCs

[0177] Tissue-engineered vascular conduits (TEVCs, 3-8 mm inner diameter) with functional endothelium to treat babies with single ventricle congenital heart defects (SVCHDs) were developed (Kathuria et al., J Ultrasound Med 2015; 34: 1091-1096). The coating of the abundantly available human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) to the luminal surface of decellularized native or tissue-engineered vascular grafts coupled with an appropriate shear stress training in a flow bioreactor was examined to determine if it provides antithrombotic function and graft patency. This was examined by coating hiPSC-ECs to the luminal surface of decellularized human umbilical arteries (dHUAs), as the diameters of dHUAs matched effectively with those of inferior vena cava (IVC) of the rats, which was planned to be used as an in vivo model. To mimic the rat’s IVC flow shear stress (i.e., ~5 Attorney Docket No.: 047162-5388-00WO

[0178] dynes / cm2), hiPSC-EC coated dHUAs were trained by shear stress in a flow bioreactor initially at around 1 dyne / cm2and then gradually increased to 5 dynes / cm2over 24 hours, which was maintained for an additional 4 or 5 days. Surprisingly, however, two of the three implanted grafts showed thrombosis and clotting 2 or 4 weeks after implantation (Figure 5 A), suggesting an inappropriate IVC shear stress training and suboptimal function of endothelium by the coated ECs, leading to clotting of two grafts.

[0179] Arterial-like shear stress (i.e., ~15 dynes / cm2) training was then employed to the hiPSC-EC coated grafts to examine if arterial-like training may effectively induce hiPSC-ECs to deposit anti -thrombotic agents leading to graft patency in vivo. To achieve this, hiPSC-ECs were coated to the luminal surface of dHUA, and shear stress was started at around 1 dyne / cm2for 12 hours. The flow shear stress training was then progressively increased towards 15 dynes / cm2over 24 hours, which was kept for an additional 3.5 days. This EC-coated graft was next implanted as an IVC interposition graft in a nude model for 4 weeks. Explant analysis revealed detectable thrombus formation in this graft (Figure 5B). These results prompted examination of a unique 15-5 dynes / cm2shear stress training, which begins with arterial-like shear stress training (15 dynes / cm2) for hiPSC-ECs followed by a ramp-down to a venous-like shear stress (5 dynes / cm2) prior to implantation into rat IVC. This 15-5 dynes / cm2flow shear stress training schedule was proven to be highly effective and led to 100% patency of more than 20 grafts generated from two independent sources of hiPSC-ECs derived based on two different EC differentiation protocols. Importantly, histological and immunofluorescence characterizations revealed that 15-5 dynes / cm2EC -trained grafts exhibited the substantial expression of antithrombotic and anticoagulants, including laminin, lectin-binding carbohydrates, heparan sulfate, tissue factor pathways inhibitor (TFPI), and tissue plasminogen activator (tPA) on the luminal surface of the grafts.

[0180] For luminal endothelialization of dHUAs, hiPSC-ECs that are differentiated using two previously published protocols were utilized. The first EC protocol was published by Prasain (Prasain et al., Nat Biotechnol 2014; 32: 1151-1157). It can generate cord-blood endothelial colony-forming cells (CB-ECFC) in about 20 days. Once generated, the CB-ECFCs were shown to proliferate until passage 18, and they had an excellent ability to form blood vessels in vivo. To make such cells feasible fortissue engineering that requires a large number of cells, significant changes regarding medium components and matrix coating were made. Prasain used complete Attorney Docket No.: 047162-5388-00WO

[0181] endothelial growth media (cEGM) to expand these hiPSC-ECs. However, these ECs fail to expand effectively in this media. This media was thus supplemented with an additional 8% vol / vol fetal bovine serum (FBS) and 5 ng / mL VEGF-A. Moreover, highly affordable 0.1% gelatin-coated tissue culture plates were used instead of the costly collagen-coated plates used by the Prasain. Furthermore, 30 mg / mL dextran was included in the optimized media to increase media viscosity in order to provide effective shear stress training to the coated ECs on the dHUA scaffolds in the shear flow bioreactors.

[0182] The second protocol utilized was published by Patsch (Patsch et al., Nat Cell Biol 2015; 17(8):994-1003). This protocol generates ECs in about 6 days. For a large-scale production of ECs for tissue engineering, costly plate-coating matrix fibronectin was replaced with highly affordable 0.1% gelatin. While Patsch used the StemPro-34 media supplemented with 50 ng / ml VEGF-A to expand these ECs, EGM-2 medium supplemented with an additional 8% vol / vol FBS and 50 ng / mL VEGF-A was used to expand ECs for our tissue engineering studies. Both protocols appeared to generate functional hiPSC-ECs of mesodermal origin. The 15-5 dynes / cm2robust shear stress training schedule has the ability to induce hiPSC-ECs derived from two distinct protocols to produce robust anti-thrombotic and anti-coagulant endothelium in coated grafts.

[0183] The data suggest that three days of arterial-like shear stress training (15 dynes / cm2) and ramping it down to venous-like stress (5 dynes / cm2) can provide functional endothelium, leading to 100% graft patency in more than 20 tested IVC grafts. Based on these data, longer duration of arterial-like shear stress training followed by ramping it down to the target vessel may enhance the anti -thrombotic capacity of the hiPEC-EC coated grafts, leading to even more robust efficacy in vivo.

[0184] hiPSC-ECs were coated to the luminal surface of the decellularized human umbilical artery (dHUA) as the diameter of dHUA matched effectively with that of the IVC in rats. To mimic the rat IVC flow shear stress (i.e., ~5 dynes / cm2), the hiPSC-EC coated dHUAs were shear stress trained in a flow bioreactor at around 1 dyne / cm2and then gradually increased to 5 dynes / cm2in a 24-hour period, which was continued for an additional 4 or 5 days. The data revealed that two out of the three implanted grafts showed blood clotting (Figure 5A), suggesting an inefficient EC shear training with suboptimal functional endothelium resulting in the clotting of two grafts. It was then examined if arterial-like shear stress training (i.e., ~15 dynes / cm2) of Attorney Docket No.: 047162-5388-00WO

[0185] the hiPSC-EC coated grafts could provide more beneficial endothelial function for better graft patency. To test this, hiPSC-ECs were coated to the luminal surface of dHUA, and shear stress training was initiated at about 1 dyne / cm2for 12 hours. The flow shear stress training was then gradually increased towards 15 dynes / cm2during a 24-hour period, which was continued for an additional 3.5 days. This EC-coated graft was then implanted as an IVC interposition graft in a nude rat model for 4 weeks. Explant analysis showed appreciable thrombus formation in this graft (Figure 5B). These experimental observations prompted examination of a unique 15-5 dynes / cm2shear stress training, which begins with an arterial-like shear stress training for hiPSC-ECs at 15 dynes / cm2followed by a ramp-down to a venous-like shear stress at 5 dynes / cm2prior to implantation into rat IVC. This 15-5 dynes / cm2flow shear stress training schedule has showed a great beneficial effect with an 100% patency of more than 20 grafts produced from two independent sources of hiPSC-ECs derived based on two different EC differentiation protocols. Notably, the histological and immunofluorescence data showed that 15-5 dynes / cm2EC-trained grafts exhibited an effective expression of anti -coagulants, including laminin, lectin-binding carbohydrates, heparan sulfate, tissue factor pathways inhibitor and tissue plasminogen activator on the luminal surface of the grafts.

[0186] The endothelial coating technique targets human cadaver donor native grafts or tissue engineered vascular grafts fabricated de novo using vascular smooth muscle cells (VSMCs) differentiated from hiPSCs (Luo et al., Cell Stem Cell 2020; 26:251-261) with inner diameters ranging from 3-8 mm, suitable for treating patients with SVCHDs (Kathuria et al., J Ultrasound Med 2015; 34:1091-1096). While dHUAs (~2 mm inner diameter) used in the examples herein are primarily for animal modeling in rats, such dHUAs would not be used for SVCHD patient care, as patients typically require grafts with wider diameters.

[0187] The bioreactor used for EC coating is modular, allowing each component to be adjusted independently to accommodate different graft sizes. The fixed arms that connect the graft can be adapted to fit various vascular graft diameters, and the chamber that holds the tissue and culture medium is also adjustable, enabling it to accommodate scaffolds of different sizes. Additionally, the connective tubes in this system can be adjusted to modify the flow rate, thereby creating various shear stresses. The peristaltic pump can simulate pulsatile pressure, providing ECs with training that mimics physiological conditions. This setup allows for easy loading of SVCHD grafts, which are approximately 3-8 mm in diameter, into the bioreactor for EC coating and Attorney Docket No.: 047162-5388-00WO

[0188] conditioning under physiological shear stress and pulsatile pressure.

[0189] In summary, the system and technique provided herein support the coating and conditioning of ECs on scaffolds of various sizes, making it adaptable for animal model testing as well as for different vascular replacement applications in SVCHD patients

[0190] Example 2: Fully Biologic Endothelialized Tissue-Engineered Vascular Conduits Provide Antithrombotic Function and Graft Patency

[0191] TEVCs are of great interest and promise, due to their ability to remodel and grow with patients. TEVCs generated by seeding autologous bone marrow cells within biodegradable polymeric tubular scaffolds made from poly(glycolic acid) (PGA) fibers and a copolymer of caprolactone and lactide (PCLA) have previously been in clinical trials for treating SVCHDs. (Hibino, N., et al., 2010, J Thorac Cardiovasc Surg 139:431-436; Drews, J.D., et al., 2020, Sci Transl Med 12). However, an unexpectedly high incidence of graft stenosis was reported in a prior TEVC clinical trial, leading to termination. (Drews, J.D., et al., 2020, Sci Transl Med 12). It is likely that overt inflammatory responses caused by graft material degradation and preseeded bone marrow cells may have led to over-proliferation of repopulated host cells and graft stenosis.

[0192] The example herein examines polymeric, inflammation-prone synthetic scaffolds (Drews, J.D., et al., 2020, Sci Transl Med 12) replaced with native blood vessel scaffolds that provide a more natural extracellular matrix (ECM) milieu for effective vessel remodeling and maturation. Further, inflammation-mediating bone marrow cells (Roh, J.D., et al., 2010, Proc Natl Acad Sci U S A 107:4669-4674) were replaced with vascular endothelial cells (ECs), capable of mitigating thrombosis and cellular over-proliferation (Jeremy, J.Y., et al., 1999, Cardiovasc Res 43:580-594; Triggle, C.R., et al., 2012, Can J Physiol Pharmacol 90:713-738; Neubauer, K., et al., 2022, Cell Tissue Res 387:391-398), during TEVC generation in a flow bioreactor (Figure 1 A). Human induced pluripotent stem cell (hiPSC)-derived ECs were chosen as hiPSCs can be readily derived from somatic cells (e.g. skin fibroblasts) by ectopic expression of stem cell factors, self-renew, and differentiate into any cell type in the body, providing an unlimited supply of ECs for tissue engineering. (Takahashi, K., etal., 2007, Cell 131:861-872). Furthermore, hiPSC-ECs have the potential to be a future off-the-shelf therapeutic, immunocompatible with any patient following modulation of human leukocyte antigens (HLAs). (Deuse, T., et al., 2019, Nat Biotechnol Attorney Docket No.: 047162-5388-00WO

[0193] 37:252-258). The results herein demonstrate that TEVCs endothelialized with hiPSC-ECs can provide both immediate antithrombotic function and a supportive ECM milieu for robust host EC recruitment, paving the way for using fully biologic, endothelialized conduits for single ventricle repair.

[0194] The materials and methods are described herein.

[0195] Table E Key Resources

[0196]

[0197] Attorney Docket No.: 047162-5388-00WO

[0198]

[0199] Attorney Docket No.: 047162-5388-00WO

[0200]

[0201] EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Attorney Docket No.: 047162-5388-00WO

[0202] Human Induced Pluripotent Stem Cells and Derived Endothelial Cells

[0203] Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) were endothelial colony forming cells (ECFCs) differentiated from hiPSCs. (Prasain, N., et al., 2014, Nat Biotechnol 32:1151-1157). hiPSC-ECs were also derived from an additional hiPSC line (Luo, J., et al., 2021, Acta Biomater 119: 184-196) based on a previous protocol. (Patsch, C., et al., 2015, Nat Cell Biol 17:994-1003). hiPSC-ECs were expanded in Endothelial Growth Medium (EGM-2; Lonza) supplemented with an additional 8% vol / vol fetal bovine serum (FBS) and VEGF-A (5 ng / ml).

[0204] Animal Use

[0205] NIH-Foxnlmunude rats were acquired from Charles River Laboratories. The research was conducted on male nude rats that were 8 weeks or older.

[0206] Human Umbilical Arteries

[0207] Deidentified discarded human umbilical cords were obtained and were delivered at 4°C. The cords, which were 20-30 cm in length, were immediately processed after arrival to isolate human umbilical arteries (HUAs) within 30 minutes using sharp dissection tools in a sterile manner. To remove the Wharton’s jelly surrounding the HUAs, a pair of Metzenbaum scissors were used. The newly isolated HUAs were then gently washed with Dulbecco’s Phosphate-Buffered Saline (PBS; ThermoFisher) containing penicillin 100 U / mL and streptomycin 100 mg / mL (ThermoFisher) to remove blood clots.

[0208] METHOD DETAILS

[0209] Differentiation of Endothelial Cells (ECs) from hiPSCs

[0210] ECs established from a previous study were utilized in this study. (Prasain, N., et al., 2014, Nat Biotechnol 32:1151-1157). Briefly, after being cultured in mTeSRl medium (STEMCELL) for two days (-D2), hiPSCs (DF19-9-1 IT) were differentiated into the mesodermal lineage in the presence of activin A, FGF-2, VEGF-A, and BMP4 (all at 10 ng / ml) for 24 hours. On the next day, the activin A-containing medium was replaced with Stemline II complete media (Sigma) including FGF-2 (Stemgent), VEGF-A (R&D), and BMP4 (R&D) to induce the production and expansion of endothelial cells. Medium was changed to fresh Stemline Attorney Docket No.: 047162-5388-00WO

[0211] IT differentiation medium on days 3, 5, 7, and 8, and then to Stemline IT differentiation media on day 9 and thereafter. To generate endothelial colony forming cells from the sorted population, CD31+, CD144+, KDR+, and NRP-1+cells were centrifuged and resuspended in a mixture of 50% EGM-2 and 50% complete Stemline IT differentiation media. 2,500 cells per well were then seeded on rat tail type I collagen-coated 12-well plates. After a 2-day culture, the medium was removed, and a mixture of three volumes of EGM-2 and one volume of differentiation medium was added to cells. After 7 days, endothelial colony forming cell colonies emerged as highly adherent cells with cobblestone-like morphology. In order to derive highly enriched populations of proliferative endothelial cells, cloning of endothelial cell clusters was carried out. After reaching confluency, endothelial colony forming cells were passaged by plating 10,000 cells per cm2, and cultured in complete endothelial growth media in collagen-coated plates with medium refreshed every other day. These hiPSC-ECs were then transduced with lentivirus expressing tdTomato (puc2CL7tdTOMwo, pczVSV-G and pcoNL2). Subsequently, tdTomato-expressing hiPSC-ECs were sorted by a flow cytometry (FACS Aria, BD) and maintained in complete EGM-2 medium (Lonza) supplemented with 5ng / ml VEGF-A and 10% FBS (vol / vol).

[0212] Y6 hiPSC line (Luo, J., et al., 2021, Acta Biomater 119:184-196) was used to derive vascular ECs using an independent, previously published protocol. (Patsch, C., et al., 2015, Nat Cell Biol 17:994-1003). Briefly, Y6 hiPSCs were seeded onto a Matrigel -coated 6-well plate until -20% confluency using mTeSR medium. After 24 hours of cell seeding, mTeSR medium was replaced with the N2B27 priming medium. The N2B27 is a 1 : 1 mixture of DMEM:F12 with glutamax and Neurobasal medium supplemented with 50 mM 2-Mercaptoethanol, N2, and B27 (all from Life Technologies) and further supplemented with 6 |1M CHIRON (Selleckchem) and 25 ng / mL recombinant BMP4 (PeproTech). After a 3-day culture, the priming medium was switched to an EC induction medium containing StemPro34 SFM (Life Technologies) supplemented with 200 ng / mL of VEGF-A (PeproTech) and 2 pM Forskolin (Abeam). After two days, the EC induction medium was refreshed with StemPro34 SFM medium containing only 50 ng / mL VEGF-A for an additional two days. On day 7, hiPSC-derived vascular ECs were trypsinized (0.05% trypsin-EDTA, ThermoFisher) and sorted for CD31+cells using the Dynabeads® CD31 Endothelial Cell (Life Technologies). Purified hiPSC-ECs were seeded into a 0.1% gelatin-coated tissue culture plate in EGM-2 medium supplemented with an additional 8% vol / vol FBS and 50 ng / mL VEGF-A. The derived vascular ECs were used for luminal Attorney Docket No.: 047162-5388-00WO

[0213] endothelialization of decellularized HUAs at or prior to passage 4 post differentiation.

[0214] HUA Derivation and Decellularization

[0215] To obtain HUAs, umbilical cords were delivered at 4°C, and HUAs were separated from the cords (length of 20-30 cm) using sharp dissection under aseptic conditions. The Wharton's jelly surrounding HUAs was removed using Metzenbaum scissors. Newly separated HUA segments (length of 5-8 cm, inner diameter ~2.0 mm) were next gently washed in PBS containing 100 U / mL penicillin and 100 pg / mL streptomycin (ThermoFisher) to remove blood clots prior to decellularization. To perform tissue decellularization, HUAs segments were placed in 250 mb of CHAPS buffer (8 mM CHAPS, 1 M NaCl, 25 mM EDTA in PBS) for 24 hours and then subsequently washed in PBS to remove cells. Next, HUAs were placed in 250 m of sodium dodecyl sulfate (SDS) buffer (1.8 mM SDS, 1 M NaCl, 25 mM EDTA in PBS) for 24 hours followed by washing with PBS. Finally, HUAs were incubated in PBS containing 20% (vol / vol) fetal bovine serum at 37°C for 24 hours to remove residual DNA followed by PBS washing. DNA contents were measured using Invitrogen™ Quant-iT™ PicoGreen™ kit as per the vender’s protocol. Briefly, about 2 mm segments of non-decellularized and decellularized HUAs were frozen at -80°C overnight followed by lyophilization for about 24 hours.

[0216] Lyophilized segments were weighed and digested in 5 mM cysteine-HCl and 5 mM disodium EDTA buffer containing papain enzyme (125 pg / mL, Sigma-Aldrich) at 60°C overnight.

[0217] Digested sample solution was then diluted with IX TE buffer and incubated with an equal volume of Quant-iT™ PicoGreen dsDNA reagents for five minutes at room temperature.

[0218] Fluorescence signals were next measured using a fluorometer at an excitation wavelength of 485 nm and emission wavelength of 530 nm. The lambda dsDNA (Invitrogen) was used as standard, and DNA contents were presented as pg of DNA per mg of the dry -weighed tissue.

[0219] Decellularized HUAs were stored in PBS at 4°C for endothelialization.

[0220] Histological and Mechanical Characterization of dHUAs

[0221] Histological Staining

[0222] Non-decellularized and decellularized HUAs were washed with PBS, fixed in 10% neutral -buffered formalin at 4°C for 24 hours, and washed with PBS prior to H&E staining for nuclear materials, Masson’s trichrome staining for extracellular collagen matrix, elastic van Attorney Docket No.: 047162-5388-00WO

[0223] Gieson (EVG) staining for elastin fiber, and Alizarin red staining fortissue calcification. The basement membrane was examined through immunohistochemical staining of collagen IV (Invitrogen # PA 1-28534).

[0224] Mechanical Characterization

[0225] Mechanical properties of the non-decellularized and decellularized HUAs were assessed by examining the rupture pressure and suture retention as described. (Gui, L., et al., 2016, Biomaterials 102:120-129). Briefly, suture retention strength was evaluated by threading a loop of 6-0 Prolene suture (Ethicon) through one side of HUA wall approximately 5 mm from the end of the vessel segment, and the weights were added to the loop in such a way that a force was applied axially to the HUA’s wall. The weights were increased incrementally by 10-20 g until the HUA wall was torn. The rupture pressure was measured by tying a 1-1.5 cm long HUA segment to a flow system connected with a pressure transducer. PBS was injected into the flow system until the vessel ruptured. The maximum tensile stress, failure strain, and Young’s modulus of approximately 2 mm long graft segments were evaluated using an Instron 5960 micro tester (Instron) with a 10 N load cell as described. (Luo, J., et al., 2017, Biomaterials 147: 116-132). Briefly, the graft segments were mounted between two stainless steel pins, with one attached to the load cell and the other to the actuator. The graft segments were cyclically prestretched three times at 10% strain, and then progressively stretched till failure of the graft to determine the maximum tensile stress. The tensile force was normalized to a cross-sectional area of the surface perpendicular to the applied force for each graft segment to yield the engineering stress of the tissue. The cross-sectional area of each graft segment was calculated as: A = 2 * T * L, where T and L are the wall thickness and length of the graft segment, respectively. Note that the cross-sectional area is multiplied by two to include both sides of the graft segment. The length of a graft segment was measured by electronic caliper (Uline, USA). Wall thicknesses of vessel segment samples were measured using optical coherence tomography (OCT) imaging (Callisto OCT, Thorlabs, Inc., Newton, NJ). Vessel segments were placed horizontally in a bath of PBS at room temperature. Segments were then imaged vertically through the entire thickness of the wall to obtain three generated cross-sectional images along each vessel. Wall thickness was calculated by sampling three representative locations from each cross-section with three generated sections using Image J software (Version 2.14.0 / 1.54f). To derive the inner diameter Attorney Docket No.: 047162-5388-00WO

[0226] of decellularized HUA, a 1 cm segment of the graft was connected to the FlexCell FX-6000T™ Tension System and Tissue-Tek O.C.T. Compound was injected to achieve a luminal pressure of 50 mmHg. The O.C.T.-fdled segments were embedded in O.C.T., frozen, and cryosectioned to obtain 10 pm sections for H&E staining. The inner diameter was calculated by measuring the luminal perimeter of each graft via NIH ImageJ (Version 2.14.0 / 1.541).

[0227] EC Coating in Flow Bioreactors

[0228] The approach used to endothelialize decellularized HUAs was developed following methods in a previous study. (Kural, M.H., et al., 2018, Cell Transplant). Both ends of the decellularized vessels were sutured to glass arms and assembled to the flow bioreactor chamber with a medium circulatory circuit (Figure 1A). Next, the luminal surface of decellularized vessels were coated with 1.5 mL human fibronectin at 100 pg / mL for overnight at 37°C. Two consecutive rounds of seeding of hiPSC-ECs resuspended in medium (Endothelial Growth Medium 2 from Lonza supplemented with 8% vol / vol FBS, 5 ng / mL VEGF-A, and 30 mg / mL dextran) were performed to coat the luminal surface of dHUAs (-2.0x106cells / cm2) in flow bioreactors for 4 hours under static conditions, followed by rotating the vessels 180 degrees and seeding with the second round of hiPSC-ECs for 4 hours. Then, the flow rate was gradually increased by the pump assembled with Masterflex L / S BioPharm Platinum-Cured Silicone Pump Tubing, L / S 14, L / S 16 (Cole-Parmer), Masterflex L / S PharMed BPT Tubing, L / S #16 (Cole-Parmer), and 3 -way stopcock (Air-Tite) connected to an Injection Site Interlink® (McKesson Medical) (for EC suspension injection) to establish the desired shear stress. Dynamic medium flow shear stress was calculated based on Poiseuille’s equation: T = 4 pQ / nr3, where r is shear stress, p is fluid viscosity, Q is medium flow rate, and r is the radius of the vessel. Shear stress was initially maintained at around 1 dyne / cm2for 12 hours and gradually elevated towards 15 dynes / cm2over 24 hours, which was then maintained for another 3.5 days. Shear stress was next decreased to 10 dynes / cm2for 12 hours and then to around 5 dynes / cm2for 12 hours in order to match the shear stress in the inferior vena cava in nude rats where the endothelialized dHUAs would be employed as an interposition graft.

[0229] Fibrinogen Adsorption Assay

[0230] Grafts prepared in the bioreactor were cut into 5 mm long segments around the mid Attorney Docket No.: 047162-5388-00WO

[0231] region and immediately immersed in a solution of 50 pg / mL human plasma fibrinogen conjugated with 647 Alexa Fluorophore (ThermoFisher, F35200) for 1 hour at room temperature. Segments were washed with PBS three times for 5 minutes each. Segments were then fixed with 10% formalin and cut into several imageable flat sheets. The luminal surface coated with hiPSC-ECs were imaged using a confocal fluorescence microscope (LEICA TCS SP8). The mean fluorescence intensity of the adsorbed fibrinogen was quantified using ImageJ from five independent biological replicates.

[0232] Scanning Electron Microscopy (SEM)

[0233] Decellularized human blood vessels endothelialized with hiPSC-ECs were analyzed using scanning electron microscopy (Hitachi SU7000), following the protocol described previously. (Dimitri evska, S., et al., 2020, Adv Funct Mater 30). Briefly, the samples were fixed with 2.5% glutaraldehyde overnight and then dehydrated via a series of graded alcohol solutions (50%, 70%, 90%, and 100% alcohol) for 15 minutes each. After being air-dried overnight, the samples were cut longitudinally to expose the inner surface of the conduits. Finally, the samples were sputter-coated with carbon and viewed under the SEM at an accelerating voltage of 20 kV. Representative images at 500x magnification were captured.

[0234] Whole Blood Incubation Assay of hiPSC-EC -coated Grafts

[0235] Blood was drawn from human donors. Bioreactor-prepared, five-millimeter segments of grafts around the mid-region were fully immersed in whole blood and incubated for 30 minutes at 37°C and 5% CO2. Segments were then washed three times with PBS for 5 minutes each washing. The thrombus aggregates were observed using SEM as described above.

[0236] Implantation of hiP SC -EC-Coated dHUAs into Nude Rats

[0237] hiPSC-EC-coated dHUAs (15-5 dynes / cm2) were implanted using an end-to-end anastomosis configuration to the inferior vena cava (JVC) of male NIH-Foxnlmunude rats (8 weeks or older; Charles River Laboratories). Under standard sterile conditions, nude rats were anesthetized with isoflurane and their infrarenal abdominal IVC was exposed via a midline abdominal incision. Cross-clamps were applied to assist in the removal of an IVC segment between the renal artery and the iliac artery. An EC-coated dHUA, approximately 6-10 mm in Attorney Docket No.: 047162-5388-00WO

[0238] length, was then implanted into the TVC using a 10-0 monofilament nylon suture in an "end-to-end" manner. Once blood flow and hemostasis were confirmed following the clamp's removal, the wound area was then closed. After the surgery, the animals were allowed to recover and were closely monitored for a specific duration of either 1, 7, 14, 30, or 60 days. Non-EC coated dHUAs (with or without shear flow training) and statically hiPSC-EC-coated grafts were also implanted as controls for hiPSC-EC-coated dHUAs trained under a gradual shear flow (15-5 dynes / cm2).

[0239] Micro-Computed Tomography Methodology

[0240] Micro-computed tomography (micro-CT) was performed on rats one month after implantation of hiPSC-EC-coated dHUAs (15-5 dynes / cm2) as the interposition IVC grafts. Briefly, rats were induced and maintained under mild anesthesia with isoflurane (1-2%) with oxygen. Following induction, 750-800 pL of CT contrast (Exitron 12000, Miltenyl Biotech) was injected via the lateral tail vein. A respiratory -gated CT scan was acquired on a hybrid SPECT / CT scanner (U-SPECT4CT MILabs, Utrecht, Netherlands) using the total body CT system protocol (50kVp, 0.48 mA) for assessment of vena cava structure. Images were reconstructed with filtered back projection with a voxel size of 160 pm. Vessel diameter and 3D quantification of the vena cava were performed with Slicer (www(dot)slicer(dot)org / ) and ITK-Snap.

[0241] Immunofluorescence Staining and Immunohistochemistry

[0242] Bioreactor-prepared or explanted graft tissues were washed with PBS once and fixed in 10% formalin at 4°C for 24 hours. Tissues were then washed with PBS three times prior to incubation in 30% sucrose in PBS at 4°C overnight gently. One volume of 30% sucrose and one volume of O.C.T. were mixed and used to incubate the tissues with gentle shaking at room temperature for 30 minutes, followed by tissue embedding in O.C.T. on dry ice and storage at -80°C. Frozen tissue blocks were sectioned into 7-10 pm thick sections using a cryostat. Sections were mounted onto glass slides and allowed to air dry for 10-20 minutes. Regarding en face staining for EC-coated grafts in Figure 4A, grafts were fixed and washed as described above, but the O.C.T. embedding part was skipped. Tissues were then permeabilized with 0.1% Triton X-100 (Fisher) for 20 minutes. After washing with PBS, sections were incubated with 1% SDS for Attorney Docket No.: 047162-5388-00WO

[0243] antigen retrieval for 1 minute and then blocked with 5% normal goat serum in PBS. Sections were then incubated with primary antibodies diluted in the blocking solution overnight at 4°C in a humidified chamber. Sections were washed with PBS three times with 5-minute intervals between washings, incubated with appropriate secondary antibodies diluted 1:500 in blocking solution for 1 hour at room temperature, and finally washed three times with PBS. Images were acquired using a confocal microscope (Leica SP8). Primary antibodies include: CD31 (1:50, Abeam, ab28364), eNOS (1:100, Abeam, ab5589), HLA-A (1:50, Abeam, ab52922), a-Smooth Muscle antibody (1:100, Sigma, A5228), CD68 (1:100, Abeam, ab31680), smooth muscle myosin heavy chain 11 (1:50, Abeam, ab224804), laminin (1:50, Sigma, L-9393), heparan sulfate (1:100, U.S. Biological, H1890), TFPI (1:100, GenuIN Biotech, 32350), tPA (1:100, and Proteintech, 10147-1-AP). Carbohydrates (glycosaminoglycans) are detected by lectin (1:250, Vector Labs, DL-1178-1). Secondary antibodies include goat anti-mouse IgG conjugated with Alexa 488 goat anti-mouse IgG (1:500, ThermoFisher, A-l 1029), Alexa 647 fluorophore (1:500, ThermoFisher, A-21235) or goat anti-rabbit IgG conjugated with Alexa 647 fluorophore (1 :500, ThermoFisher, A-21245).

[0244] RNA Isolation and qPCR Analysis

[0245] Total RNA was isolated from the EC-coated grafts using TRIzol RNA Isolation Kit (ThermoFisher), and cDNA synthesis was carried out using the iScript cDNA synthesis Kit (BioRad Laboratories). Quantitative PCR (qPCR) was carried out using iQ SYBR Green Supermix (Bio-Rad Laboratories) and CFX96™ Real-Time System (Bio-Rad Laboratories). mRNA fold change of each gene in the statically EC-coated grafts with an abrupt flow was calculated after normalization to the GAPDH housekeeping gene and then to the gene expression in the 15-5 dynes / cm2EC -coated grafts, and were presented on a log 2 scale. Primer’s sequences are provided in Table 1.

[0246] Table 2: qRT-PCR Primers (Related to Table 1)

[0247]

[0248] Attorney Docket No.: 047162-5388-00WO

[0249]

[0250] QUANTIFICATION AND STATISTICAL ANALYSIS

[0251] All graphic illustrations and statistical analyses were completed using GraphPad Prism 9. Numerical data were reported in the format of mean ± S.E.M. from at least three or more independent experiments. A nonparametric Mann-Whitney test was used to compare the two groups, and a p-value of less than 0.05 was considered statistically significant. The sample size (n) for each analysis stands for the number of biological replicates and can be found in the figure legends.

[0252] The results are described herein.

[0253] To develop a proof-of-principle hiPSC-endothelialized TEVC for procedures such as Fontan operations, hiPSC-ECs were derived based on a monolayer differentiation approach through which the mesodermal lineage was induced via the treatment of hiPSCs with activin A, FGF-2, VEGF-A, and BMP4, followed by the expansion of ECs with FGF-2, VEGF-A, and BMP4. (Prasain, N., et al., 2014, Nat Biotechnol 32:1151-1157). These hiPSC-ECs were then coated on the luminal surface of decellularized human umbilical arteries (dHUAs), suitable for supporting blood flow, under shear stress (in bioreactors) to generate an endothelialized TEVC (Figure 1A).

[0254] The hiPSC-ECs were gradually trained towards an arterial-like shear stress of 15 dynes / cm2, as shear stress promotes maturation and anti -thrombotic activity in hiPSC-ECs. (Sivarapatna, A., et al., 2015, Biomaterials 53:621-633). Before implanting endothelialized TEVCs as inferior vena cava (IVC) interposition grafts, a validated model for investigating grafts for Fontan procedures (Watanabe, M., et al., 2001, Tissue Eng 7:429-439), in nude rats, shear Attorney Docket No.: 047162-5388-00WO

[0255] stress was gradually reduced to around 5 dynes / cm2to mimic IVC flow. Grafts showed an efficient coverage of ECs, which were positive for CD31 (96.93±1.51%), eNOS (95.71±2.55%) and human leukocyte antigen type A (HLA-A; 97.32±1.61%), aligned to the direction of flow, and presented a typical elongated morphology under pulsatile, unidirectional flow (Figure 1B-Figure ID). These endothelialized grafts were implanted for four weeks, remained patent without thrombus formation (Figure IE), and were functional as venous conduits (Figure 3A). Further, the luminal surface of endothelialized grafts displayed full coverage of ECs (eNOS+) that were negative for HLA-A (Figure IF), suggesting the replacement of hiPSC-ECs by host ECs.

[0256] To investigate the requirement of shear flow training for ECs in preventing thrombosis on dHUA scaffolds, the luminal surface of grafts were coated with hiPSC-ECs statically in bioreactors and subsequently implanted into nude rats as interposition IVC grafts. dHUA scaffolds alone in the presence or absence of shear were employed as additional controls for implantation. All three groups of grafts showed evidence of thrombosis two-weeks postimplantation (Figures IE, Figure 3B, and Figure 3C), suggesting both EC coating and shear flow prior to implantation are necessary for preventing graft thrombosis.

[0257] Potential antithrombotic action of hiPSC-endothelialized grafts were then determined. The endothelial glycocalyx, a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium, plays an important role in vascular permeability and blood coagulation. (Reitsma, S., et al., 2007, Pflugers Arch 454:345-359). Glycosaminoglycans (GAGs) linked to core proteins in proteoglycans can mitigate thrombosis via interactions with various anticoagulants (Figure 3D). (Reitsma, S., et al., 2007, Pflugers Arch 454:345-359;

[0258] Neubauer, K., et al., 2022, Cell Tissue Res 387:391-398). As shown in Figure 3E, binding of lectins, commonly used to label carbohydrates, and expression of heparan sulfate (HS), the most abundant GAGs in the endothelial glycocalyx (Reitsma, S., et al., 2007, Pflugers Arch 454:345-359), were detected on the luminal surface of both statically coated and shear-trained, endothelialized grafts prior to implantation. Moreover, the presence of tissue factor pathway inhibitor (TFPI), which prevents tissue factor-mediated blood coagulation, and tissue plasminogen activator (tPA), which maintains vessel patency by removing hidden deposition of fibrin via fibrinolysis, were confirmed on the luminal surface of endothelialized grafts (Figure 3E). In contrast, non-endothelialized grafts did not display evidence of lectin-binding or expression of HS, TFPI, or tPA (Figure 3E), consistent with the notion that the lack of EC- Attorney Docket No.: 047162-5388-00WO

[0259] derived antithrombotic factors may contribute to blood clotting in dHUA alone grafts.

[0260] Contributing factors to thrombus formation in grafts coated with hiPSC-ECs statically were then determined. As statically endothelialized grafts experience an abrupt increase in shear stress due to blood flow after IVC implantation, they were exposed to around 5 dynes / cm2abruptly for 6 hours in bioreactors to mimic in vivo flow to investigate molecular alterations promoting thrombosis. Compared to gradually shear-trained, endothelialized grafts, statically EC-coated grafts with an abrupt flow showed a reduced cellular coverage (Figure 2A and Figure 2B). Additionally, after 30-minute exposure to human plasma-derived fibrinogen, a larger amount of fibrinogen was adsorbed onto the luminal surface of grafts coated with ECs statically than that onto the gradually shear-trained, endothelialized grafts (Figure 2A and Figure 2C). Moreover, graft thrombus formation was evaluated by incubating them with human whole blood. After 1-hour exposure to whole blood, there was an appreciable amount of polymerized fibrin and aggregated red blood cells on the surface of grafts with statically coated ECs (Figure 2D). In contrast, almost no fibrin strands with aggregated red blood cells were observed on the shear-trained, endothelialized surface (Figure 2D). Consistently, the coverages of anticoagulants, such as lectin-binding carbohydrates, HS, TFPI, and tPA, were lower on the surface of statically EC-coated grafts compared to the shear-trained, endothelialized surface (Figure 4A and Figure 4B). Furthermore, gene expression analyses revealed that shear-trained, endothelialized grafts maintained higher expression of antithrombotic markers (eNOS, KLF2, KLF4) and lower expression of thrombogenic markers (E-selectin, P-selectin, ICAM-1, VCAM-1) compared to statically EC-coated grafts (Figure 4C). These results suggest the requirement of shear flow for ECs in maintaining the expression of antithrombotic factors, thereby preventing thrombogenesis.

[0261] As the luminal surface of shear-trained, endothelialized grafts exhibited full coverage of host ECs (HLA-AeNOS+) 1-month post-implantation (Figure IF), the timecourse replacement of hiPSC-ECs by host ECs was then determined. hiPSC-ECs gradually disappeared starting from 1-day post-implantation, with a majority of hiPSC-ECs undetected on the luminal surface of grafts after 7 days (Figure 2E and Figure 2F). Importantly, lectin-binding and expression of HS, TFPI, tPA, and laminin, a basement membrane protein critical for EC proliferation and migration (Yousif, L.F., et al., 2013, Cell Adh Migr 7:101-110; Fujiwara, H., etal., 2004, Exp Cell Res 292:67-77; Kick, K., et al., 2016, Arterioscler Thromb Vase Biol 36:2346-2357), were observed on the luminal surface of these explanted grafts (Figure 4D) as well as on pre-implant grafts Attorney Docket No.: 047162-5388-00WO

[0262] coated with ECs under shear (Figure 3E). By 2-weeks post-implantation, the luminal surface of grafts was effectively covered by rat host ECs (Figure 2E and 2F). As GAGs, anticoagulants, and laminin were readily detectable in shear- trained, endothelialized grafts 7-days post-implantation, when the majority of hiPSC-ECs had been replaced in the grafts, these results demonstrate that these endothelial factors may play crucial roles in preventing blood coagulation and promoting host cell endothelialization.

[0263] Moreover, a substantial number of rat smooth muscle cells, which were positive for alpha smooth muscle actin (aSMA) and negative for HLA-A, repopulated the graft walls 2-weeks postimplantation (Figure 2E and Figure 2F). Notably, these smooth muscle cells underwent maturation (observed via expression of a mature smooth muscle marker myosin heavy chain 11 -MYH11) 2-months post-implantation. Additionally, a few cells positive for CD68 (macrophage marker) were observed in grafts 1-day to 2-weeks post-implantation but disappeared after 2 months, suggesting limited presence of macrophages in grafts (Figure 2E). In summary, the results herein demonstrate that shear-trained, endothelialized TEVCs promote efficient recellularization of host smooth muscle cells and effective reendothelialization via host EC recruitment.

[0264] The results herein illustrate the generation of robust TEVCs with functional luminal endothelium using hiPSC-ECs via effective shear stress training in flow bioreactors.

[0265] Endothelialized TEVCs provided immediate antithrombotic function and expedited host EC recruitment after implantation as interposition IVC grafts in nude rats. Moreover, TEVCs supported effective recellularization of host smooth muscle cells, which underwent maturation post-implantation. Further, the necessity of both hiPSC-EC coating and shear flow training in preventing thrombus formation after TEVC implantation were demonstrated. Developing endothelialized vascular conduits as an innovative therapy for the treatment of patients with SVCHDs is thus possible (Figure 2G).

[0266] Though TEVCs are of great interest due to their ability to remodel and grow with the patient, and have been made by seeding autologous patient bone marrow cells onto the polymeric PGA / PCLA scaffold, clinical trials for treating SVCHDs were terminated due to an unexpectedly high incidence of graft stenosis. (Drews, J.D., et al., 2020, Sci Transl Med 12). It is likely that overt infiltration of monocytes / macrophages into the PGA / PCLA scaffold, due to a strong foreign body response may have contributed to the over-proliferation of repopulated host smooth Attorney Docket No.: 047162-5388-00WO

[0267] muscle cells, leading to inflammation-mediated TEVC stenosis. (Drews, J.D., et al., 2020, Sci Transl Med 12). To overcome this issue, the PGA / PCLA scaffold was replaced with a natural blood vessel milieu provided by dHUAs for more effective graft remodeling. Histological analysis revealed the removal of cells, maintenance of collagen, retention of basement membrane collagen IV, and preservation of mechanical strength in dHUA scaffolds (Figure 4E). Notably, CD68+macrophages appeared to be involved in early graft remodeling, but disappeared 2-months post-implantation (Figure 2E). In contrast, CD68+macrophages still exist in the PGA / PCLA grafts 1-year post-implantation (Drews, J.D., et al., 2020, Sci Transl Med 12), suggesting a prolonged proinflammatory nature of this polymeric scaffold and the necessity for a natural blood vessel such as a dHUA as vascular support during TEVC generation and for future clinical care.

[0268] The second unique feature of the TEVCs is to replace inflammation-mediating bone marrow cells (Roh, J.D., et al., 2010, Proc Natl Acad Sci U S A 107:4669-4674) with hiPSC-ECs coupled with robust shear stress training in bioreactors to mimic IVC flow, positing an innovative paradigm of TEVCs that provide an immediate functional endothelium, required for preventing blood coagulation and cellular over-proliferation, after implantation. This is in contrast to TEVCs in a recent clinical trial (Drews, J.D., et al., 2020, Sci Transl Med 12), in which both the polymeric scaffold and seeded bone marrow cells induced macrophage recruitment, leading to marked inflammation, over-proliferation of repopulated host cells, and graft stenosis. The results herein also demonstrate that anticoagulants and laminin are readily deposited on the luminal surface of endothelialized TEVCs, and that their expression persists following hiPSC-ECs replacement in the grafts post-implantation. Endothelialized TEVCs can thus provide both immediate antithrombotic function and a supportive laminin-enriched basement membrane for rapid host EC recruitment. Additionally, there is limited presence of macrophages in the grafts, likely due to the natural blood vessel milieu provided by dHUAs and the presence of a functional endothelium. Notably, TEVCs coated with ECs differentiated from an additional hiPSC line (Luo, J., et al., 2021, Acta Biomater 119:184-196) also prevented thrombosis after IVC implantation (Figure 4F-Figure 41), revealing a more general efficacy of this approach. The unique arterial-like shear stress training for hiPSC-ECs followed by a rampdown to a venous-like shear stress in flow bioreactors (Figure lA-Figure ID) may have relevance to the treatment of many other venous disorders requiring vascular replacement. Attorney Docket No.: 047162-5388-00WO

[0269] Further, in contrast to TEVCs in previous clinical trials that lack immediate endothelial function and require prolonged use of anticoagulants to prevent blood clotting (Hibino, N., et al., 2010, J Thorac Cardiovasc Surg 139:431-436; Drews, J.D., et al., 2020, Sci Transl Med 12), the endothelialized TEVCs do not need anticoagulation after implantation. Notably, cumulative anticoagulant exposure may lead to additional cardiovascular problems (Pollak, U., et al., 2018, Congenital Heart Disease 13:16-25), highlighting the importance of TEVCs with immediate endothelial function post-implantation. Future endeavors will be made to unravel potential tissue distribution(s) of hiPSC-ECs after displacement by host ECs, via genetic labeling and live imaging, to engineer a safety switch to remove hiPSC lineages if needed, and to dissect the mechanism by which host ECs re-endothelialize grafts with larger sample sizes. To make TEVCs immunocompatible with any patient, ECs derived from universal hiPSCs generated through modulating HLA genes (Deuse, T., et al., 2019, Nat Biotechnol 37:252-258) can be cryopreserved in large scales for TEVC generation, thus providing promptly available, allogeneic grafts for treatments (Figure 2G). In summary, the results herein illustrate endothelialized hiPSC-TEVCs that provide immediate endothelial function and expedite host EC recruitment via robust shear stress training, setting the stage for generating readily available, universal conduits for future treatment of patients with SVCHDs.

[0270] Example 3: Immunocompatible Endothelialized Vascular Graft for Vascular Replacement in Patients with Single Ventricle Congenital Heart Defects

[0271] Generation of immunocompatible universal hiPSC-ECs for vascular engineering

[0272] To create a universally compatible cell source for vascular regeneration, immunoevasive human induced pluripotent stem cells (hiPSCs) were developed through manifold genome editing to eliminate MHC class I and II expression while preserving pluripotency and endothelial differentiation potential. Using CRISPR-Cas9, guide RNAs were designed for targeting the coding sequences of p2-microglobulin (B2M), a structural component of MHC -I protein and class II transactivator (CIITA), a master regulator of MHC-II gene expression (Fig. 6A).

[0273] CRISPR-Cas9-mediated editing introduced deletions and premature stop codons at both loci, as confirmed by PCR amplification, Zero Blunt vector cloning, and Sanger sequencing approaches, Attorney Docket No.: 047162-5388-00WO

[0274] resulting in complete loss of gene function. To further enhance immune tolerance against natural killer cell attack, we introduced human CD47 cDNA, encoding the natural killer cells and macrophage inhibitory “don’t eat me” signal, into the AAVS1 safe harbor locus using TALEN-mediated targeted integration (Fig. 1A).

[0275] This strategy ensured controlled and stable ectopic expression of CD47 without disrupting endogenous genes. To validate the immunogenicity profde, edited hiPSCs were differentiated into endothelial cells (hiPSC-ECs) using an optimized vascular differentiation protocol (Fig. 6B). Following stimulation with interferon-y (25-100 ng / mL, 48 hours) to induce MHC expression, flow cytometry analysis confirmed complete abrogation of MHC-I and MHC-II surface proteins and robust overexpression of CD47 in B2M / CIITA / CD47 hiPSC-ECs compared with unedited wild-type controls (Fig. 6C-6D). This B2M7CIITA7CD47+hiPSC line thus represents a universal hiPSC platform capable of generating immunocompatible cardiovascular derivatives for allogeneic applications.

[0276] To assess whether multiple gene editing steps affected pluripotency, immunofluorescence staining was performed for canonical markers OCT4, NANOG, and SSEA-4, as well as TRA-1-60, revealing expression levels comparable to those of unedited wild-type hiPSCs (Fig. 9A). Directed differentiation protocols further demonstrated that B2M7CIITA7CD47+hiPSCs retained the ability to generate cardiomyocytes (hiPSC-CMs), vascular smooth muscle cells (hiPSC-vSMCs), and endothelial cells (hiPSC-ECs), confirming maintenance of trilineage cardiovascular potential (Fig. 9B-9E).

[0277] Comprehensive phenotypic and functional characterization of B2M / CIITA / CD47 hiPSC-ECs revealed strong expression of endothelial markers, including CD31, VE-cadherin, and eNOS (Fig. 9D-9E). Functionally, B2M / CIITA / CD47+hiPSC-ECs exhibited robust capillary-like network formation on Matrigel (Fig. 6E-6G) and efficient uptake of Dil-acetylated LDL, consistent with mature endothelial identity (Fig. 6H-6I). Moreover, they demonstrated physiological nitric oxide (NO) responsiveness under dynamic culture conditions with shear flow (Fig. 6J-6L). Using DAF-FM DA fluorescence imaging and the Measure-iT™ High-Sensitivity Nitrite Assay, shear stress-induced NO production was detected at levels comparable to wildtype hiPSC-ECs (Fig. 6J-6L).

[0278] Together, these results demonstrate the successful generation of B2M / CIITA / CD47' hiPSCs that preserve pluripotency, multilineage differentiation capacity, and endothelial Attorney Docket No.: 047162-5388-00WO

[0279] functionality while exhibiting complete MHC-I and II ablation and CD47 overexpression. These universal hiPSCs represent a genetically stable, immunoevasive platform for allogeneic vascular and cardiovascular tissue engineering.

[0280] In vitro evaluation of immune responses by co-culturing universal hiP SC-derived endothelial cells with T cells and natural killer cells

[0281] To assess the immune evasive potential of universal hiPSC-ECs (B2M / CIITA / CD47+hiPSC-ECs), B2M / CIITA / CD47+hiPSC-ECs were co-cultured with allogeneic human CD4+and CD8+effector memory T cells (Tem) and NK-92 natural killer (NK) cells.

[0282] When co-cultured with CD4+and CD8+Tem cells, universal hiPSC-ECs elicited markedly reduced T-cell activation compared with unedited control ECs (Fig. 7A-7B).

[0283] Specifically, TNFa secretion from both T-cell subsets was substantially lower in the presence of universal hiPSC-ECs (Fig. 7C-7D). Moreover, flow cytometric analysis revealed a significant decrease in HLA-DR expression on CD4+T cells co-cultured with universal hiPSC-ECs (Fig. 7B). In contrast, unedited wild-type hiPSC-ECs strongly upregulated this activation marker (Fig.

[0284] 7B). Importantly, TNFa production and HLA-DR expression in T cells exposed to universal hiPSC-ECs were comparable to baseline levels observed in T cells cultured alone (Fig. 7A-7D), indicating a near-complete suppression of T-cell activation.

[0285] To evaluate the response of innate immune effectors, B2M / CIITA / CD47+hiPSC-ECs were co-cultured with NK-92 cells, alongside wild-type and B2M / CIITA (double knockout) hiPSC-EC controls. Lactate dehydrogenase (LDH) release assays after 48 hours demonstrated minimal NK-mediated cytotoxicity against universal hiPSC-ECs, comparable to wild-type ECs, whereas B2M / CIITA ECs exhibited approximately 20% cytolysis (Fig. 7E).

[0286] Together, these results demonstrate that universal hiPSC-ECs effectively evade both adaptive and innate immune activation in vitro, exhibiting strong resistance to T-cell and NK-cell-mediated cytotoxicity.

[0287] Generation of universal hiPSC-EC-coated, shear-trained grafts and assessment of immune responses in an immune-humanized rat model. Attorney Docket No.: 047162-5388-00WO

[0288] The in vivo endothelial function and immunological compatibility of universal human induced pluripotent stem cell-derived endothelial cells (universal hiPSC-ECs) were next investigated using an established immune-humanized RRGS rat model (Menoret et al, 2020). These rats lack Ragl and I12rg genes, rendering them deficient in T, B, and NK cells, and express human SIRPa, which inhibits macrophage-mediated phagocytosis of human cells, thereby permitting human peripheral blood mononuclear cell (PBMC) engraftment (Menoret et al, 2020).

[0289] To evaluate endothelialization and antithrombotic function, universal hiPSC-ECs were coated onto the luminal surface of decellularized human umbilical arteries (dHUA) and subjected to arterial-like shear stress (15 dyne / cm2) in a dynamic flow bioreactor. This approach achieved >95% luminal coverage (Fig. 10A-10B, 10D). En-face fibrinogen adsorption and fibrin formation assays demonstrated that universal hiPSC-EC-coated grafts markedly suppressed fibrinogen deposition and prevented thrombus formation following incubation with fibrinogen and human whole blood, respectively (Fig. 10C-10D). These data indicate that universal hiPSC-ECs confer potent antithrombotic properties and preserve endothelial functionality ex vivo.

[0290] To assess vascular patency and physiological compatibility, both universal and wild-type hiPSC-EC-coated grafts were transplanted as inferior vena cava (IVC) interposition grafts in non-humanized, immunodeficient rats (Fig. 11). Both graft types remained patent and free of thrombus formation, confirming the functional integrity and antithrombotic competence of universal hiPSC-ECs comparable to wild-type ECs in vivo.

[0291] Next, the immunological performance of universal hiPSC-ECs in immune-humanized RRGS rats was evaluated. Human PBMCs (1 x 108cells / kg) were intravenously injected, and FACS-based profiling confirmed successful reconstitution with ~1% human CD45+immune cells prior to transplantation (Fig. 8A-8B). At this reconstitution level, either wild-type or universal hiPSC-EC-coated grafts were transplanted into the IVC and explanted after one week to capture early immune events. No macroscopic thrombosis was observed in either group (Fig.

[0292] 8C).

[0293] However, histological and immunofluorescence analyses revealed substantial CD3+T-cell infiltration in the vessel wall of wild-type grafts, whereas universal hiPSC-EC-coated grafts exhibited minimal immune cell infiltration (Fig. 8D-8E). Further staining for CD41helper and CD8+cytotoxic T cells showed that wild-type grafts contained comparable proportions of both Attorney Docket No.: 047162-5388-00WO

[0294] subsets (Fig. 12A-12B), while universal grafts displayed substantially reduced infdtration of both subsets with near absence of CD8+T cells and minimal presence of CD4+T cells (Fig. 12C-12D). This T cell infiltration pattern indicates that universal hiPSC-ECs highly effectively evade cytotoxic T-cell-mediated injury in vivo.

[0295] Consistent with this, granzyme B expression, a hallmark of cytotoxic activation, was significantly reduced in universal EC-coated grafts compared to wild-type controls (Fig. 8F-8G). Notably, wild-type grafts exhibited reduced endothelial coverage, indicative of immune-mediated endothelial damage, whereas universal hiPSC-EC-coated grafts maintained a continuous, densely packed endothelium (Fig. 8H-8J).

[0296] Together, these findings demonstrate that universal hiPSC-EC-coated grafts exhibit robust vascular functionality, resistance to immune rejection, and stable endothelial integration within immune-humanized hosts, supporting their potential as immunocompatible vascular grafts for allogeneic applications.

[0297] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attorney Docket No. 047162-5359-00USCLAIMSWhat is claimed is:

1. A tissue-engineered vascular conduit (TEVC) comprising a graft and a plurality of stem-cell derived cells, wherein the plurality of stem-cell derived cells are seeded on the graft, wherein a shear stress is applied to the plurality of stem-cell derived cells.

2. The TEVC of claim 1, wherein the shear stress is modulated.

3. The TEVC of claim 2, wherein the shear stress is modulated more than once.

4. The TEVC of claim 3, wherein the shear stress is initially a low shear stress and subsequently increased to a high shear stress.

5. The TEVC of claim 4, wherein the low shear stress is 0.01 to 10 dynes / cm2or 1 dyne / cm2.

6. The TEVC of claim 4, wherein the high shear stress is an arterial shear stress, 10 to 100 dynes / cm2or 15 dynes / cm2.

7. The TEVC of claim 4, wherein the shear stress is further reduced from the high shear stress to a final shear stress.

8. The TEVC of claim 7, wherein the final shear stress is a venous shear stress, 0.01 to 10 dyne / cm2or 5 dyne / cm2.

9. The TEVC of claim 7, wherein the shear stress is reduced from the high shear stress to a ramped-down shear stress before being reduced to a final shear stress.Attorney Docket No.: 047162-5388-00WO10. The TEVC of claim 9, wherein the ramped-down shear stress is 0.01 to 100 dyne / cm2or 10 dyne / cm2.

11. The TEVC of claim 1, wherein the stem-cell derived cells are derived from human induced pluripotent stem cells (hiPSCs).

12. The TEVC of claim 1, wherein the stem-cell derived cells are stem-cell derived endothelial cells.

13. The TEVC of claim 1, wherein the stem-cell derived cells are hiPSC-derived endothelial cells.

14. The TEVC of claim 1, wherein the graft is one or more of a scaffold, a biodegradable scaffold, a tissue, a decellularized tissue, a decellularized human donor blood vessel, an animal derived blood vessel, a tissue engineered vascular graft based on human cells, a tissue engineered vascular graft based on animal cells, or a combination thereof.

15. The TEVC of claim 1 wherein the graft is generally tubular with a diameter ranging from 3 to 18 millimeters.

16. The TEVC of claim 14, wherein the graft comprises a biodegradable scaffold and a plurality of stem cell-derived vascular smooth muscle cells (VSMCs), wherein the plurality of stem cell-derived VSMCs are seeded on the biodegradable scaffold and are cultured under mechanical and biochemical stimulation.

17. A method of generating a tissue-engineered vascular conduit (TEVC), the method comprising:a. obtaining a plurality of stem-cell derived cells;b. seeding the plurality of stem-cell derived cells on a graft; andAttorney Docket No.: 047162-5388-00WOc. applying a shear stress to the plurality of stem-cell derived cells seeded on the graft.

18. The method of claim 17, wherein the shear stress is modulated.

19. The method of claim 18, wherein the shear stress is modulated more than once.

20. The method of claim 19, wherein the shear stress is initially a low shear stress and subsequently increased to a high shear stress.

21. The method of claim 20, wherein the low shear stress is 0.01 to 10 dyne / cm2.

22. The method of claim 20, wherein the high shear stress is an arterial shear stress, 10 to 100 dynes / cm2or 15 dynes / cm2.

23. The method of claim 20, wherein the shear stress is further reduced from the high shear stress to a final shear stress.

24. The method of claim 23, wherein the final shear stress is a venous shear stress, 0.01 to 10 dyne / cm2or 5 dyne / cm2.

25. The method of claim 23, wherein the shear stress is reduced from the high shear stress to a ramped-down shear stress before being reduced to a final shear stress.

26. The method of claim 25, wherein the ramped-down shear stress is 0.01 to 100 dyne / cm2or 10 dyne / cm2.

27. The method of claim 17, wherein the stem-cell derived cells are derived from human induced pluripotent stem cells (hiPSCs).Attorney Docket No.: 047162-5388-00WO28. The method of claim 17, wherein the stem-cell derived cells are stem-cell derived endothelial cells.

29. The method of claim 17, wherein the stem-cell derived cells are hiPSC-derived endothelial cells.

30. The method of claim 17, wherein the graft is one or more of a scaffold, a biodegradable scaffold, a tissue, a decellularized tissue, or a combination thereof.

31. The method of claim 30, wherein the graft is a synthetic graft.

32. The method of claim 31, wherein the synthetic graft comprises a biodegradable scaffold and a plurality of stem cell-derived vascular smooth muscle cells (VSMCs), wherein the plurality of stem cell-derived VSMCs are seeded on the biodegradable scaffold and are cultured under mechanical and biochemical stimulation.

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