In this study, we have demonstrated that a relatively simple microfluidic platform supports the formation of a stable and mature vascular network in 3D, and effectively recapitulates the perivascular localization of mesenchymal stem cells (MSCs) ex vivo
. Endothelial cells co-cultured with either stromal fibroblasts (NHLFs) or bone marrow-derived mesenchymal stem cells (MSCs) in this microfluidic platform undergo a vasculogenic program to yield stable, pericyte-invested capillary networks with hollow, well-defined lumens confirmed via confocal microscopy. These results recapitulate the formation of capillary structures observed in larger 3D gel cultures (Ghajar et al. 2006
; Ghajar et al. 2008
), demonstrating that this complex morphogenetic process can easily be scaled down to study within a MFD.
Microfluidic systems have long been touted as ideal tools with which to study multi-factor regulation of cell biological phenomena, especially given their ability to deliver multiple soluble factors with precise spatial and temporal control (Mosadegh et al. 2007
) and to conserve reagents based on their small size. However, the promise of such approaches has not yet been fully realized in part because most microfluidic systems involve rather cumbersome methodologies, and because their ability to support 3D cell cultures has only recently been demonstrated (Gillette et al. 2008
). A promising recent study similarly utilized MFDs to develop vascular networks within 3D epithelial tissues in vitro
(Sudo et al. 2009
), but the quality, stability, and physiological relevance of these vessel networks lacking mature pericytes was not clear. In our study, confocal images of HUVEC-MSC and HUVEC-NHLF co-cultures seeded within 3D fibrin gels in MFDs unambiguously confirm the presence of hollow lumens (see Supplementary Movies
). Moreover, both MSCs and fibroblasts occupy perivascular locations and expressed pericyte markers when cultured with HUVECs within our MFD model.
We have previously shown that both fibroblasts and MSCs are capable of supporting capillary morphogenesis in 3D fibrin gels and in vivo
, and that both are capable of acting as pericytes that express α-SMA (Ghajar et al. 2010
). The capillary networks formed in the presence of these two different stromal populations within our MFD also possess similar morphological characteristics. However, the vessels generated from NHLF-HUVEC co-cultures formed at a significantly faster rate than in the MSC-HUVEC co-cultures. Some distinctions in the mechanisms by which these two cell types promote capillary morphogenesis have recently been identified (Ghajar et al. 2010
), and these differences may also account for the differential rates observed here as well. Although these experiments do not directly validate the utility of this platform as a tool for studying perivascular niches per se, it may be possible to utilize the small reagent volumes and amenability to high-resolution imaging offered by the MFD to identify additional mechanistic distinctions between MSCs and fibroblasts in future studies.
Many 3D culture systems already exist to study capillary morphogenesis in vitro
, including those that serve as models of vasculogenesis (Chung et al. 2009
) as well as those intended to model angiogenesis (Koh et al. 2008
). However, the key new contribution provided by this study is the recapitulation of the perivascular niche ex vivo
to mechanistically explore how MSCs interact with the vasculature. It is already widely recognized that MSCs facilitate angiogenesis in part by acting as stabilizing perictyes (Crisan et al. 2008
), and that much of their potential therapeutic benefit is based on their capacity to secrete pro-regenerative (including pro-angiogenic) factors (Wagner et al. 2009
). MSCs also facilitate capillary development in part by influencing the expression levels of critical matrix remodeling enzymes (Ghajar et al. 2006
). However, several recent studies suggest that the perivascular location of MSCs and other adult stem cells may acts as a critical anatomic cue that maintains their multilineage potential, in part due to their direct and indirect interactions with endothelial cells. Neural stem cells (NSCs), like MSCs, have also been shown to reside in perivascular niches in vivo
(Shen et al. 2008
; Tavazoie et al. 2008
). NSCs interact with capillaries in part through the binding of their α6
integrin to EC-deposited laminin, and this interaction appears to be critical for maintaining their quiescence (Shen et al. 2008
). In this study, we were able to explore the interaction between MSCs and EC-deposited basement membrane in our artificial perivascular niche. We report for the first time that the α6β1 integrin receptor is required for the perivascular interactions between MSCs and capillaries, as shown by our data indicating that treating MSCs with an anti- α6
integrin antibody prevented their perivascular association. When the antibody was added to intact vessels with perivascular MSCs, the MSCs moved away from the vascular surface in a manner similar to that observed for neural progenitor cells in mice infused with the same anti-α6
integrin antibody in their lateral ventricle (Shen et al. 2008
). Collectively, these data confirmed our hypothesis that MSCs’ perivascular location requires the interaction between the laminin rich basement membrane of the capillaries and the α6
integrin adhesion receptor on MSCs for their pericytic association. Because other adult stem cells may also localize to perivascular niches in vivo
via similar mechanisms, our system may facilitate efforts to dissect the consequences of this association.
Many different approaches to engineer artificial stem cell niches based on biomaterials, drug delivery, and microfluidic approaches are currently being explored (Lutolf et al. 2009
). However, a common anatomic feature of many adult stem cell niches, i.e. its proximity to the vasculature, may in and of itself be instructive in a way that cannot be recapitulated by the presentation of soluble and insoluble biochemical cues or by endothelial-conditioned media. Furthermore, endothelial cells may also enhance the regenerative potential of progenitor cells independent of their ability to form functional connections to the host vasculature (Kaigler et al. 2005
). By leveraging the microfluidic channels within our system to present soluble biochemical cues in gradient fashion and to spatially pattern discrete biomaterials and cell types (Huang et al. 2009
), the method and the supporting data presented here provide a novel way to recapitulate and study perivascular niches ex vivo
, and suggest a new approach to explore the regulation of adult stem cells.