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.

FIG. 1.

Schematic of a layered construct that could provide both the vascular as well as dermal fibroblast component for treatment of wounds. The bilayer construct would consist of two different hydrogel matrices controlling adipose-derived mesenchymal stem cell (ASC) differentiation. ASC could be seeded on microcarriers and either migrate into the matrix or the carriers could be seeded within the matrix. Color images available online at www.liebertonline.com/tea.

FIG. 2.

Immunocytochemical analysis of ASC isolated from rats. Photomicrographs of markers expressed in third-passage ASC. Figures in each panel indicate the specific cell surface marker. All antibodies, except Stro-1, are fluorescein isothiocyanate-labeled primary antibodies. Stro-1 is identified using isotype-matched fluorescein isothiocyanate-labeled rat IgM. Color images available online at www.liebertonline.com/tea.

FIG. 3.

Light microscopic images of differentiation time course of ASC into vascular like structures. Cells began to form vascular tube-like networks in the PEGylated fibrin gel in the absence of additional soluble cytokines. The amount of network formation was related to the initial cell density. Scale bar = 100 μm.

FIG. 4.

Proliferation of ASC in PEGylated fibrin gels analyzed by MTT (3-(4,5-dimethylthiozole-2-yl)-2,5-diphenyltetrazolium bromide) assay. Viability in comparison to the initial cell seeding density and time (mean optical density [OD] value ± standard deviation, n = 3). Cell proliferation and viability increased with increasing cell seeding density.

FIG. 5.

Endothelial and pericyte-specific markers expressed by the differentiated ASC in PEGylated fibrin gels. Expression levels of endothelial cell-specific markers (CD31, von Willebrand factor [vWF]) and pericyte-specific markers (NG2 and platelet-derived growth factor receptor beta [PDGFRβ]) were analyzed using real-time polymerase chain reaction. There was significant increase in endothelial cell-specific markers, CD31 (25-fold) and vWF (42-fold), in comparison to pericyte markers, NG2 (6-fold) and PDGFRβ (9-fold), by day 11.

FIG. 6.

Confocal Z-stacked images of tube-like structures formed by ASC in PEGylated fibrin gel. ASC when seeded in PEGylated fibrin exhibit an endothelial phenotype expressing both vWF (B) and CD31 (C). (D) The merged image of (B and C) stained with Hoeschst (A) for nuclei. The formed tubes were positive for both pericyte-specific markers NG2 (G) and alpha smooth muscle actin (α-SMA) (K) and the endothelial cell-specific marker vWF (F and J). (H and L) vWF and Hoeschst (E and I) merged with NG2 and α-SMA, respectively. Color images available online at www.liebertonline.com/tea.

FIG. 7.

TEM images of ASC forming tube-like structures in PEGylated fibrin following outgrowth from chitosan microspheres (CSM). (A and B) Two separate cells designated by white arrows that are organized around a lumen (L). The nucleus is designated as nu. (C and D) A cross section of two separate cells around a lumen that is free of the PEGylated fibrin gel (FPEG). White arrow designates cells involved in lumen formation.

FIG. 8.

ASC released from CSM in vitro in PEGylated fibrin and collagen gels. Phase-contrast images of ASC migrated from CSM into PEGylated fibrin (A–C) and collagen (D–F). ASC that have migrated from CSM attached to the PEGylated fibrin shows classical sprouting (A, day 2) followed by differentiating into tube-like structures (B, day 5). Over the time course of differentiation, they migrate into the gel forming a dense multicellular network (C, day 8). ASC released from the CSM into collagen were more spindle in appearance (D, day 2) which developed filopodias (E, day 6). Over time they formed more elongated morphological structures stretching along fibril assemblies resembling cells that are associated with stromal tissues.

FIG. 9.

Quantum dot (Qdot) 565-labeled ASC tracked after migration from CSM after day 6 into PEGylated fibrin and collagen gels. Epifluorescent images of Qdot 565–labeled ASC tracked after migration from the CSM into PEGylated fibrin (A–C) and collagen (D–F) after 6 days. ASC released from CSM into both PEGylated fibrin and collagen could be tracked (A and D) over 6 days. Cells forming tubes-like structures (B) in PEGylated fibrin and striated morphologies (E) were colocalized with Qdot 565 (C and F). Color images available online at www.liebertonline.com/tea.

FIG. 10.

Bidirectional differentiation of ASC in the PEGylated fibrin–(ASC-CSM)–collagen gel constructs. ASC loaded in CSM exhibited matrix-driven phenotypic changes into a fibroblast-like morphology in the collagen layer (A, C, E, and G) and a tube-like morphology in the PEGylated fibrin layer (B, D, F, and H) simultaneously. ASC started to migrate into both the gels on day 3 (A and B) and proliferated as a fibroblast-like phenotype in collagen (C) and tube-like sprouts (D) in PEGylated fibrin on day 5. By day 7 the collagen layer showed an increase in fibroblast-like cells (E), which eventually populated the gels by day 11 (G). In the PEGylated fibrin layer the sprouts started to form long networks by day 7 (F), which formed complex networks by day 11 (H). Panels below (E–H) demonstrate lower magnification view.