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A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation.

ABSTRACT: This work describes the differentiation of adipose-derived mesenchymal stem cells (ASC) in a composite hydrogel for use as a vascularized dermal matrix. Our intent is that such a construct could be utilized following large-surface-area burn wounds that require extensive skin grafting and that are limited by the availability of uninjured sites. To develop engineered skin replacement constructs, we are pursuing the use of ASC. We have established that a PEGylated fibrin gel can provide a suitable environment for the proliferation of ASC over a 7 day time course. Further, we have demonstrated that PEGylated fibrin can be used to control ASC differentiation toward vascular cell types, including cells characteristic of both endothelial cells and pericytes. Gene expression analysis revealed strong upregulation of endothelial markers, CD31, and von Willebrand factor, up to day 11 in culture with corresponding evidence of protein expression demonstrated by immunocytochemical staining. ASC were not only shown to express endothelial cell phenotype, but a subset of the ASC expressed pericyte markers. The NG2 gene was upregulated over 11 days with corresponding evidence for the cell surface marker. Platelet-derived growth factor receptor beta gene expression decreased as the multipotent ASC differentiated up to day 7. Increased receptor expression at day 11 was likely due to the enhanced pericyte gene expression profile, including increased NG2 expression. We have also demonstrated that when cells are loaded onto chitosan microspheres and sandwiched between the PEGylated fibrin gel and a type I collagen gel, the cells can migrate and proliferate within the two different gel types. The matrix composition dictates the lineage specification and is not driven by soluble factors. Utilizing an insoluble bilayer matrix to direct ASC differentiation will allow for the development of both vasculature as well as dermal connective tissue from a single population of ASC. This work underscores the importance of the extracellular matrix in controlling stem cell phenotype. It is our goal to develop layered composites as wound dressings or vascularized dermal equivalents that are not limited by nutrient diffusion.

SUBMITTER: Natesan S

PROVIDER: S-EPMC3117235 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation.

Natesan Shanmugasundaram S Zhang Ge G Baer David G DG Walters Thomas J TJ Christy Robert J RJ Suggs Laura J LJ

Tissue engineering. Part A 20110104 7-8

This work describes the differentiation of adipose-derived mesenchymal stem cells (ASC) in a composite hydrogel for use as a vascularized dermal matrix. Our intent is that such a construct could be utilized following large-surface-area burn wounds that require extensive skin grafting and that are limited by the availability of uninjured sites. To develop engineered skin replacement constructs, we are pursuing the use of ASC. We have established that a PEGylated fibrin gel can provide a suitable ...[more]

PMID: 21083419

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Project description:BackgroundRetinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy.Methodology/principal findingsWe found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection).Conclusions/significanceASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.

Project description:BackgroundAdipose tissue is a readily available source of multipotent adult stem cells for use in tissue engineering/regenerative medicine. Various growth factors have been used to stimulate acquisition of endothelial characteristics by adipose-derived stem cells (ASC). Herein we study the effects of endothelial cell growth supplement (ECGS) and physiological shear force on the differentiation of ASC into endothelial cells.Materials and methodsHuman ASC (CD13(+)29(+)90(+)31(-)45(-)) were isolated from periumbilical fat, cultured in ECGS media (for up to 3 wk), and exposed to physiological shear force (12 dynes for up to 8 d) in vitro. Endothelial phenotype was defined by cord formation on Matrigel, acetylated-low density lipoprotein (acLDL) uptake, and expression of nitric oxide synthase (eNOS), von Willebrand factor (vWF), and CD31 (platelet endothelial cell adhesion molecule, PECAM). Additionally, cell thrombogenicity was evaluated by seeding canine autologous ASC onto vascular grafts implanted within the canine arterial circulation for 2 wk.ResultsWe found that undifferentiated ASC did not display any of the noted endothelial characteristics. After culture in ECGS, ASC formed cords in Matrigel but failed to take up acLDL or express the molecular markers. Subsequent exposure to shear resulted in stem cell realignment, acLDL uptake, and expression of CD31; eNOS and vWF expression was still not observed. Grafts seeded with cells grown in ECGS (+/- shear) remained patent (six of seven) at 2 wk but had a thin coat of fibrin along the luminal surfaces.ConclusionsThis study suggests that (1) ECGS and shear promote the expression of several endothelial characteristics in human adipose-derived stem cells, but not eNOS or vWF; (2) their combined effects appear synergistic; and (3) stem cells differentiated in ECGS appear mildly thrombogenic in vitro, possibly related, in part, to insufficient eNOS expression. Thus, while the acquisition of several endothelial characteristics by adult stem cells derived from adipose tissue suggests these cells are a viable source of autologous cells for cardiovascular regeneration, further stimulation/modifications are necessary prior to using them as a true endothelial cell replacement.

Dataset Information

A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation.

Publications

A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation.

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