THEREOF

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the field of tissue engineered blood vessels (TEBV) and capillary network generation.

[0002] A major problem in blood vessel tissue engineering is the construction of vessel grafts that possess suitable, long-lasting biomechanical properties commensurate with native vessels and will fully integrate into the circulatory system and last for many years.

[0003] The principal use of such blood vessels is for small diameter bypass grafts such as coronary artery bypass graft (CABGs), peripheral bypass grafts, or arteriovenous shunts.

[0004] Recently, a tissue engineering technique has been developed which is a radical departure from prior art techniques. This work is described in U.S. Pat. No. 5,618,718 and in an article entitled "A Completely Biological Tissue-Engineered Human Blood Vessel," L'Heureux, N., et al., FASEB J. 12:47-56, 1998, both of which are incorporated herein by reference. A fully biological and autologous human TEBV, with no synthetic materials, was made and found capable of withstanding physiological burst pressures in excess of 2000 mm Hg. These vessels were suturable and maintained patency for two weeks when xenografted into a dog. A living graft of this type is self-renewing with an inherent healing potential. The completely biological graft can be remodeled by the body according to the demands of the local mechanical environment. Moreover, the absence of synthetic materials precludes foreign body reactions, thus increasing the likelihood of long-term graft success.

[0005] Endothelial differentiaion of adipose-derived stem cells has been reported previously, e.g., "Endothelial differentiation of adipose-derived stem cells: effects of endothelial cell growth supplement and shear force ," Fischer LJ, Mcllhenny S, Tulenko T, Golesorkhi N, Zhang P, Larson R, Lombardi J, Shapiro I, DiMuzio PJ. J Surg Res. 2009 Mar;152(l): 157-66); "In vivo behavior of decellularized vein allograft, " Martin ND, Schaner PJ, Tulenko TN, Shapiro

IM, DiMatteo CA, Williams TK, Hager ES, DiMuzio PJ, J Surg Res. 2005 Nov;129(l): 17-23.

"eNOS transfection of adipose-derived stem cells yields bioactive nitric oxide production and improved results in vascular tissue engineering. "

[0006] Cell-seeded conduits have been tried that require resorbable, non-biological scaffolds to generate sufficient mechanical strength. In this approach, cells, typically smooth muscle cells, are seeded into tubular structures made from materials such as polylactic acid. These vessels are susceptible to the same thrombosis/inflammation failures typically associated with graft failure.

[0007] Furthermore, these vessels provide no mechanism to limit the proliferation of smooth muscle cells. Smooth muscle proliferation may infiltrate the lumen of the vessel and occlude it in a process called intimal hyperplasia. Finally, synthetic grafts have very different mechanical properties from natural tissues. These differences, particularly in tissue compliance, may induce adverse remodeling responses and graft failure due to localized non-physiological hemodynamic forces.

[0008] One promising solution to the problems associated with synthetic-based vascular grafts is to assemble blood vessels in vitro using only the patient's own cells and then re-implant them into the patient. This approach is called tissue engineering. In theory, tissue-engineered blood vessels (TEBVs) should provide mechanically stable vessels built only from biological tissue, therefore improving the likelihood of graft success. Another advantage to a tissue engineering approach is the ability to manipulate the vessel ends to facilitate grafting of the vessel into place. Tissue engineering has been used successfully in the past to build two-dimensional structures such as skin, but has had only limited success with three dimensional tissues and organs such as TEBVs.

[0009] Even these prior art fully autologous TEBVs have significant problems, the most significant of which are rejection, coagulation, and/or intimal hyperplasia after a few years. [0010] It is an object of the present invention to provide a TEBV which fully integrates into vascular segments indistinguishable from native vessels.

[0011] Another object of the invention is to provide a method of generating capillary networks in ischemic tissues of an individual.

SUMMARY OF THE INVENTION

[0012] These objects, and others which will become apparent from the following detailed description, are achieved by the present invention which comprises in one aspect a method of making a tissue engineered blood vessel (TEBV) having a lumen comprising obtaining fat cells from an individual, isolating human adipose-derived stem cells (hASC) from the fat cells, treating the hASC with endotheliogenic medium and an agent selected from the group consisting of sphingosine-1 -phosphate (SIP); 2-amino-2-[2-(4-octylphenyl)ethyl]propane-l,3-diol (FTY- 720P); 2-amino-2-(l,2,12-trihydroxy-4-octadecenyl)-l,3-propanediol; 6-Eicosene-l,3,4-triol, 2- amino-2-(hydroxymethyl)- 1 ,3-propanediol; 1 ,3 ,4, 14-Eicosanetetrol, 2-amino-2- (hydroxymethyl)- 1,3 -propanediol; and 1,3,4-Eicosanetriol, 2-amino-2-(hydroxymethyl)-l,3- propanediol, and coating the lumen with the treated hASC. The individual can be a human or an animal.

[0013] Another aspect of the invention comprises a method of generating capillary networks comprising obtaining fat or bone marrow from an individual, isolating stem cells from the fat or bone marrow, treating the stem cells with endotheliogenic medium and an agent selected from the group consisting of sphingosine-1 -phosphate (SIP); 2-amino-2-[2-(4- octylphenyl)ethyl]propane-l ,3-diol (FTY-720P); 2-amino-2-(l ,2, 12-trihydroxy-4-octadecenyl)- 1,3-propanediol; 6-Eicosene-l,3,4-triol, 2-amino-2-(hydroxymethyl)-l,3-propanediol; 1,3,4,14- Eicosanetetrol, 2-amino-2-(hydroxymethyl)- 1 ,3 -propanediol; and 1,3,4-Eicosanetriol, 2-amino- 2-(hydroxymethyl)- 1,3 -propanediol, and injecting the treated stem cells into ischemic tissues of the individual. [0014] The fat or bone marrow are preferably obtained from the individual by methods known in the art, for example from abdominal fat or other fat deposits or by withdrawing bone marrow with a needle.

[0015] In most embodiments the stem cells will be differentiated to endothelial-like cells using the procedure of the invention.

[0016] The invention also comprises a TEBV prepared by the method of the invention.

[0017] The novel TEBV can be implanted in an animal or human in need of a replacement blood vessel.

[0018] Another aspect of the invention is a method comprising annealing one of the agents with or without heparin to the lumen and throughout the scaffold wall. The resultant TEBV has many advantages.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Fig. 1 is a graph of the time course of eNOS expression in "EC-like"- ASCs. eNOS expression in EC-like ASCs without SIP (left bar) and incubated with SIP for 1 day, 2 days and 12 days. Human ECs (HUVEC) are shown as a positive control. In each experiment, ASCs were first differentiated to an EC-like phenotype using endothelial growth medium (EGM) followed by SIP where indicated.

[0020] Fig. 2 is a graph of the increase in EC marker genes CD31, vWF and eNOS in ASCs incubated first in EGM followed by addition of SIP to the EGM for 12 days. These data demonstrate the functionality of EC-like ASCs which are eNOS positive for their application to TEBVs to line the lumen, or for capillary network generation in tissues containing dysfunctional capillary networks or no capillary networks.

[0021] Fig. 3 is a graph showing SIP markedly increases the attachment of EC-like ASCs to plastic. [0022] Fig. 4 is a graph showing SIP markedly increases the attachment of EC-like ASCs to a biological scaffold (SIS).

[0023] Fig. 5 is a graph showing SIP increases the cell proliferation of EC-like ASCs in cell culture.

[0024] Fig. 6 is an ultrasound image of cell-free scaffold saturated with SIP 28 days following implantation into the dog carotid circulations demonstrating full functionality. Red indicates flowing blood.

[0025] Fig. 7 are two ultrasound photos showing formation of pre-capillary structures by EC-like ASCs differentiated with the SIP methodology described herein.

DETAILED DESCRIPTION

[0026] These objects, and others which will become apparent from the following detailed description, are achieved by the present invention which comprises in one aspect a method of making a tissue engineered blood vessel (TEBV) having a lumen comprising obtaining fat cells or bone marrow cells from an individual, isolating human adipose-derived stem cells (hASC) from the fat cells or human bone marrow stem cells (hBMSCs), treating the hASC with endotheliogenic medium and an agent selected from the group consisting of sphingosine-1- phosphate (SIP); 2-amino-2-[2-(4-octylphenyl)ethyl]propane-l,3-diol (FTY-720P); 2-amino-2- (1 ,2, 12-trihydroxy-4-octadecenyl)-l ,3 -propanediol; 6-Eicosene-l ,3,4-triol, 2-amino-2- (hydroxymethyl)-l ,3 -propanediol; 1 ,3,4, 14-Eicosanetetrol, 2-amino-2-(hydroxymethyl)-l ,3- propanediol; and 1,3,4-Eicosanetriol, 2-amino-2-(hydroxymethyl)- 1,3 -propanediol, and coating the lumen with the treated hASC.

[0027] In some embodiments hASCs or hBMSCs are obtained from individuals and are suspended in or cultured in endotheliogenic medium containing SIP or one of the other agents followed by infusion into the lumen and scaffold wall of a biological scaffold similar, but not limited to, an FDA-approved decellularized sheep intestinal submucosal scaffolds (SIS, Cook Technology) which is rotated slowly to allow full cellar coating of the lumen, followed by flow conditioning in a bioreactor over 4 days. The coated TEBVs will then be implanted in a dog carotid artery as an arterial interposition graft and tested over 6 months, with the contralateral side receiving a cell free control biological scaffold (e.g., SIS graft).

[0028] In some embodiments heparin and SIP are annealed to the SIS lumen, the heparin being present to prevent coagulation and the SIP being present to help differentiate and proliferate circulating EPCs to an eNOS competent phenotype. In addition, SIP is annealed to the deeper layers of the scaffold so as to enhance the differentiation of incoming host cells to a smooth muscle cell (SMC) phenotype. In this embodiment a cell free TEBV saturated or annealed with SIP is produced and is implanted.

[0029] Data suggest that EC-like hASCs differentiated with endotheliogenic SIP provides a lining to the TEBV that enables development into authentic arterial segments in vivo that are as resistant to lifelong insults similar to native arteries. Additional data suggests that SMC-like hASCs differentiated with annealed SIP within the scaffold wall provides a tunica media consisting of SMC that, in concert with the luminal surface SIP, enables development into authentic arterial segments in vivo that are as resistant to lifelong insults as native arteries.

EXAMPLES

[0030] Ex. 1 - Generation of EC phenotype from hASC

[0031] The use of SIP to aid in the generation of an endothelial cell (EC) phenotype from hASCs is demonstrated by the following experiment. Culturing hASCs in endothelial inductive medium such as EGM or EGM2 helps to differentiate the hASCs to an CE-like cell, but the EC- like cell that results from endothelial induction media generates little to no eNOS mRNA, thus preventing these cells from being able to fully replace authentic ECs in which eNOS is an essential gene that underlies the ability of these cells to make NO, not to mention participate in the orchestration of a more complete EC genome within the ASCs. Hence, EC-like ASCs without sufficient eNOS do not succeed in replacing authentic EC or provide the full function of authentic ECsl . Accordingly, we set out to find a simple methodology that might increase eNOS expression in hASCs. We found experimentally that the small molecule SIP, or its analogues, can increase eNOS expression in hASCs that have been previously, or simultaneously, incubated in endothelial inductive medium. Fig. 1 demonstrates that very little eNOS is expressed in ASCs that have been incubated only in EGM. However, adding SIP (1 μΜ) increase eNOS expression in only 1 day, and considerably further in 2 days and still further in 12 days. Moreover, after 12 days in EGM containing SIP, marked increases in additional important EC markers CD31 and vWF (von Willibrand Factor) were observerd as shown in Fig. 2. These data demonstrate that fully functional ECs can be generated by differentiating ASCs in endothelial induction medium that also contains SIP. Fully functional ECs, i.e., those that are eNOS competent, are useful for lining TEBVs to protect the TEBV from coagulation and subsequent intimal hyperplasia or other alterations that might prevent the fabrication of a TEBV that performs well over the long term in vivo. Supporting this application of EC-like ASCs differentiated from hASC is the discovery that SIP also increases EC-like ASC cell adhesion to plastic (Fig. 3) as well as a biologic scaffold, similar to, but not limited to SIS (Fig. 4). In addition, SIP also increases the cell proliferation of eNOS competent EC-like ASCs prepared with SIP as shown in Fig. 5.

[0032] These experimental findings demonstrate that SIP promotes eNOS expression, thus generating eNOS competent EC-like ASCs. SIP saturated in the scaffold therefore will also promote cell attachment of circulating endothelial progenitor cells to the scaffold lumen, aid in their ability to promote proliferation, and cover the luminal surface.

[0033] Ex. 2 - Potency Testing

[0034] The inventors observed that, when tested in vivo, the SIP saturated cell-free SIS scaffold remained potent for at least 28 days as shown in Fig. 6. This ultrasound image shows fully normal blood flow through the SIP saturated SIS scaffold segment that was implanted as an interpositional arterial graft in the dog carotid circulation. Regarding TEBVs, these data strongly support the concept that SIP is a novel treatment protocol that is a critical ingredient enabling the differentiation of hASCs or circulating endothelial progenitor cells to eNOS competent ECs.

[0035] Ex. 3 - Generation of functional capillary networks [0036] In addition using SIP to create eNOS competent EC for use in constructing TEBVs, this methodology of creating eNOS competent ECs can be used to generate functional capillary networks when injected into either ischemic tissue or tissues with dysfunctional capillaries or no capillaries. Numerous clinical conditions are associated with tissues made ischemic by the loss of functional capillary networks including, but not limited to diabetic vasculopathies, myocardial ischemias, chronic wound healing, chronic ulcers, etc. In this scenario, ASCs freshly isolated and incubated in endothelial induction medium containing SIP will differentiate into eNOS competent ECs either in culture, or following injection directly into the capillary deficient tissue with or without any pre -incubation whatsoever. That these eNOS competent cells can form capillaries is suggested by their ability to form pre-capillary structures as shown in Fig. 7.

[0037] SIP appears to be unique in its ability to induce eNOS expression in hASCs. The resultant eNOS competent ECs can thus be used to replace, generate or regenerate capillary endothelial cells for any application requiring this specific cell type, including, but not limited to the construction of TEBVs and the generation of functional capillary networks in tissues in which capillary networks are limited or absent and thus compromise tissue and organ function. Regenerating functional capillary networks will thus restore tissue function, and therefore also improved nutritional support, and provide significant therapeutic relief in patients with this type of circulatory deficiency.

[0038] The present invention, therefore, is well adapted to carry out the objects and attain the ends and advantages mentioned, as well as others inherent therein. While the invention has been depicted and described and is defined by reference to particular preferred embodiments of the invention, such references do not imply a limitation on the invention, and no such limitation is to be inferred. The invention is capable of considerable modification, alteration and equivalents in form and function, as will occur to those ordinarily skilled in the pertinent arts. The depicted and described preferred embodiments of the invention are exemplary only and are not exhaustive of the scope of the invention. Consequently, the invention is intended to be limited only by the spirit and scope of the appended claims, giving full cognizance to equivalents in all respects.