Endothelial differentiation of adipose-derived stem cells from elderly patients with cardiovascular disease.

Adipose-derived stem cells (ASCs) possess significant therapeutic potential for tissue engineering and regeneration. This study investigates the endothelial differentiation and functional capacity of ASCs isolated from elderly patients. Isolation of ASCs from 53 patients (50-89 years) revealed that advanced age or comorbidity did not negatively impact stem cell harvest; rather, higher numbers were observed in older donors (>70 years) than in younger. ASCs cultured in endothelial growth medium-2 for up to 3 weeks formed cords upon Matrigel and demonstrated acetylated-low-density lipoprotein and lectin uptake. Further stimulation with vascular endothelial growth factor and shear stress upregulated endothelial cell-specific markers (CD31, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin). Inhibition of the PI(3)K but not mitogen-activated protein kinase pathway blocked the observed endothelial differentiation. Shear stress promoted an anti-thrombogenic phenotype as demonstrated by production of tissue-plasminogen activator and nitric oxide, and inhibition of plasminogen activator inhibitor-1. Shear stress augmented integrin α(5)ß(1) expression and subsequently increased attachment of differentiated ASCs to basement membrane components. Finally, ASCs seeded onto a decellularized vein graft resisted detachment despite application of shear force up to 9 dynes. These results suggest that (1) advanced age and comorbidity do not negatively impact isolation of ASCs, and (2) these stem cells retain significant capacity to acquire key endothelial cell traits throughout life. As such, adipose tissue is a practical source of autologous stem cells for vascular tissue engineering.

Availability of adipose-derived stem cells in patients undergoing vascular surgical procedures.

BACKGROUND: Most research evaluating adipose-derived stem cells (ASC) uses tissue obtained from young, healthy patients undergoing plastic surgical procedures. Given the propensity of other adult stem cell lines to diminish with increasing patient age and co-morbidities, we assess the availability of ASC in elderly patients undergoing vascular surgical procedures, and evaluate their acquisition of endothelial cell (EC) traits to define their potential use in vascular tissue engineering. METHODS AND METHODS: Adipose tissue obtained by liposuction from patients undergoing vascular procedures (n = 50) was digested with collagenase and centrifuged to remove mature adipocytes. The resultant number of cells, defined as the stromal-vascular (SV) pellet, was quantified. Following a 7-d culture period and negative selection for CD31 and CD45, the resultant number of ASC was quantified. After culture in differentiating media (EMG-2), ASCs were tested for the acquisition of endothelial-specific traits (expression of CD31, realignment in shear, cord formation on Matrigel). RESULTS: The SV pellet contained 2.87 ± 0.34 × 10(5) cells/g fat, and the resultant number of ASCs obtained was 1.41 ± 0.18 × 10(5) cells/g fat. Flow cytometry revealed a homogeneous ASC population (>98% positive for CD13, 29, 90). Advanced age or co-morbidity (obesity, diabetes, renal or peripheral vascular disease) did not significantly alter yield of ASC. After culture in differentiating media (EMG-2), ASCs acquired each of the endothelial-specific traits. CONCLUSION: ASC isolation appears independent of age and co-morbidities, and ASCs harvested from patients with vascular disease retain their ability to differentiate into endothelial-like cells. Adipose tissue, therefore, is a practical source of autologous, adult stem cells for vascular tissue engineering.

Linear shear conditioning improves vascular graft retention of adipose-derived stem cells by upregulation of the alpha5beta1 integrin.

Use of adult adipose-derived stem cells (ASCs) as endothelial cell substitutes in vascular tissue engineering is attractive because of their availability. However, when seeded onto decellularized vascular scaffolding and exposed to physiological fluid shear force, ASCs are physically separated from the graft lumen. Herein we have investigated methods of increasing initial ASC attachment using luminal precoats and a novel protocol for the gradual introduction of shear stress to optimize ASC retention. Fibronectin coating of the graft lumen increased ASC attachment by nearly sixfold compared with negative controls. Gradual introduction of near physiological fluid shear stress using a novel bioreactor whereby flow rate was increased every second at a rate of 1.5 dynes/cm(2) per day resulted in complete luminal coverage compared with near complete cell loss following conventional daily abrupt increases. An upregulation of the alpha(5)beta(1) integrin was evinced following exposure to shear stress, which accounts for the observed increase in ASC retention on the graft lumen. These results indicated a novel method for seeding, conditioning, and retaining of adult stem cells on a decellularized vein scaffold within a high-shear stress microenvironment.

Development of a tissue-engineered bypass graft seeded with stem cells.

The gold standard conduit for bypass of diseased small-diameter arteries remains autologous vascular tissue. In the absence of such tissue, patients are offered bypass with prosthetic material, with far less durable results. Vascular tissue engineering, the creation of a vascular conduit by seeding a tubular scaffold with various cells, may offer an alternative approach to this difficult situation. Herein we review some of the significant challenges that remain in designing an ideal vascular conduit and outline potential solutions offered by a graft created by seeding natural vascular tissue (decellularized vein allograft) with readily available autologous cells (adipose-derived stem cells).

In vivo behavior of decellularized vein allograft.

BACKGROUND: We are investigating decellularized vein allograft as a scaffold to engineer a non-synthetic, small-diameter vascular graft. This study examines the in vivo behavior of this scaffolding after implantation into the arterial circulation. MATERIALS AND METHODS: Canine animals underwent bilateral carotid interposition grafting using jugular vein implanted as either: 1) fresh autograft, 2) fresh allograft, or 3) decellularized allograft. Decellularization was achieved using sodium dodecyl sulfate. Grafts were examined with duplex ultrasound biweekly to determine luminal diameter, thrombosis, stenosis, or anastomotic breakdown. After perfusion fixation at 2 or 8 weeks, grafts underwent histological, morphometric, and immunohistochemical examination. RESULTS: All animals survived without neurological or hemorrhagic complication. No deterioration of graft integrity (rupture, aneurysm) was observed in any group. Luminal narrowing was observed in both allograft groups, but secondary to different pathology. Fresh allografts had significant mononuclear cell infiltrate, intimal hyperplasia, and intramural hemorrhage consistent with rejection. Conversely, decellularized allografts had minimal evidence of rejection but instead had a compact fibrin layer formed along their lumen. This fibrin layer was absent in the peri-anastomotic regions where endothelium had migrated from the native artery. By 8 weeks, decellularized grafts had repopulated with cells staining positive for smooth muscle alpha-actin. CONCLUSIONS: After 8 weeks of arterial flow, decellularized vein allograft exhibits satisfactory strength, reduced antigenicity compared to fresh allograft, and supports cellular repopulation. These characteristics make it satisfactory for further tissue engineering; combined with luminal vascular cell seeding, it may prove useful as a small-diameter arterial bypass graft.