Influence of adult mesenchymal stem cells on in vitro vascular formation

Abstract

The effective delivery of bioactive molecules to wound sites hasten repair. Cellular therapies provide a means for the targeted delivery of a complex, multiple arrays of bioactive factors to wound sites. Thus, the identification of ideal therapeutic populations is an essential aspect of this approach. In vitro assays can provide an important first step toward this goal by selecting populations that are likely suitable for more expensive and time-consuming in vivo assays. In this study, bone marrow-derived mesenchymal stem cells (BM-MSCs) were integrated into a three-dimensional coculture system that supports the development and stabilization of vascular tube-like structures. The presence of a limited number of BM-MSCs resulted in their coalignment with vascular structures, and it further resulted in increased tubule numbers and complexity. Thus, these studies suggest that BM-MSCs functionally interacted with and were attracted to in vitro formed vascular structures. Further, these cells also provided sufficient bioactive factors and matrix molecules to support the formation of tubular arrays and the stabilization of these arrays. This in vitro system provides a means for assessing the function of BM-MSCs in aspects of the angiogenic component of wound repair.

FIG. 1.

Fibroblast–HUVEC coculture sections. Frozen sections were cut from cocultures lifted intact from culture dishes. (A) PECAM-1 immunostaining shows tube-like structures embedded in fibroblast lawns. (B) DAPI staining of nuclei emphasizes the three-dimensional nature of the fibroblast lawns. (C) Type IV collagen immunostaining demonstrates the formation of basement membrane around tube-like structures (arrowhead). (D) Laminin immunostaining demonstrates the formation of basement membrane around tube-like structures (arrowhead). Scale bars: (A, C, D) 200 μm and (B) 500 μm.

FIG. 2.

Microenvironment of tube-like structures. (A) Frozen sections of cocultures were immunostained using an antibody against FGF-2. (B) Intact cultures were immunostained with an antibody that recognizes all splice-variants of CD44. (C) Intact cocultures were immunostained with an antibody that recognizes the small proteoglycan decorin (DCN). Scale bars: (A) 40 μm, (B) 300 μm, and (C) 200 μm.

FIG. 3.

BM-MSCs and HUVECs seeded together at day 0. (A) This photograph was taken using combined phase contrast and fluorescent optics on day 11. Arrows indicate CM-DiI–labeled MSCs aligned with tube-like structures. The circled cell is an example of a nonaligned BM-MSC. (B) This is a merged image taken at day 14, which shows CM-DiI (red)–labeled MSCs (arrows) aligned with PECAM-1 immunostained tube-like structures (green). Scale bars: (A) 500 μm and (B) 200 μm.

FIG. 4.

BM-MSCs seeded on day 5. (A) Combined phase and fluorescent optics show aligned MSCs on day 9 (arrows). (B) The merged image for CM-DiI (red) BM-MSCs and PECAM-1 (green) immunostained tube-like structures shows alignment of BM-MSCs (arrows). Scale bars: (A) 500 μm and (B) 200 μm.

FIG. 5.

Effect of BM-MSCs on the formation of vascular tube-like structures. (A) Tube-like structures formed in the absence of BM-MSCs (day 13). (B) Increased numbers of tube-like structure formed when BM-MSCs were coplated with HUVECs. There is also increased branching of tube-like structures. (C) The merged image shows the presence of CM-DiI–labeled MSCs in the culture photographed in (B). Scale bars: (A, B) 500 μm and (C) 200 μm.

FIG. 6.

BM-MSC lawns support the formation and stability of tube-like structures. (A–C) Tube-like structures at day 13 on BM-MSC lawns immunostained with PECAM-1. (A) Donor 1490; (B) donor 1494; (C) donor 1496. (A′–C′) Tube-like structures formed at day 13 on papillary dermal fibroblast lawns seeded with mixtures of HUVECs and the same BM-MSC populations used for (A–C). Panel (D) shows tube-like structures formed at day 13 when HUVECs were seeded alone onto papillary dermal fibroblast lawns. Scale bars: 2 mm.

FIG. 6.

BM-MSC lawns support the formation and stability of tube-like structures. (A–C) Tube-like structures at day 13 on BM-MSC lawns immunostained with PECAM-1. (A) Donor 1490; (B) donor 1494; (C) donor 1496. (A′–C′) Tube-like structures formed at day 13 on papillary dermal fibroblast lawns seeded with mixtures of HUVECs and the same BM-MSC populations used for (A–C). Panel (D) shows tube-like structures formed at day 13 when HUVECs were seeded alone onto papillary dermal fibroblast lawns. Scale bars: 2 mm.

FIG. 7.

BM-MSCs stabilize tubules formed on Matrigel. (A) Adult HDMVECs spontaneously form tubules on Matrigel when cultured in the presence of medium that contains proangiogenic factors, phase contrast optics, day 2 postseeding. (B) AD-MSCs coseeded with HDMVECs failed to stabilize tubules on Matrigel in the absence of exogenous proangiogenic factors, day 2 postseeding. The MSCs formed dense clusters on Matrigel (circled region) and failed to align. Combined phase contrast and fluorescence optics. (C) BM-MSCs coseeded with HDMVECs aligned with tubules and stabilized these structures when cultured in medium that lacked proangiogenic factors, day 10 postseeding. Arrows indicate BM-MSCs at sites of tubule branching. Combined phase contrast and fluorescence optics. (D) Multiple branches occurred at sites of higher concentrations of BM-MSCs, day 10 postseeding (circled region). Combined phase contrast and fluorescence optics. Scale bars: (A, B) 2 mm and (C, D) 500 μm.