Bioadhesive hydrogel microenvironments to modulate epithelial morphogenesis

Abstract

Epithelial cells polarize and differentiate into organotypic cell aggregates in response to cell–cell and cell–matrix interactions. For example, Madin–Darby Canine Kidney (MDCK) cells form spherical cell aggregates (cysts) with distinct apical and basolateral polarity when cultured three dimensionally (embedded) in type I collagen gels. To investigate the effects of individual extracellular factors on epithelial morphogenesis, we engineered fast degrading protease-responsive polyethylene glycol (PEG) hydrogels functionalized with controlled densities of various bioligands (RGD peptide, laminin-1 (LN)) to allow 3D culturing of MDCK cells, cyst expansion, and morphogenesis/polarization. Cysts formed after 15 days of culture in these hydrogels were analyzed with multiphoton fluorescence microscopy for markers of apical and basolateral membrane domains. Epithelial cysts formed in bioadhesive ligand-functionalized PEG gels exhibited a higher frequency of central lumen and interior apical pole formation as well as basolateral polarization compared to those of unmodified PEG hydrogels. These results demonstrate that incorporation of specific bioadhesive motifs into synthetic hydrogels provides 3D culture environments that support epithelial morphogenesis. These microenvironments provide a flexible and controlled system for systematic investigations into normal and pathologic morphogenic behaviours as well as synthetic environments for promoting tissue morphogenesis for regenerative medicine applications.

Introduction

Epithelial morphogenesis plays a central role in developmental biology by directing the organization of tissues and organs as well as producing the diversity of body shapes found in multicellular organisms [1]. Epithelial morphogenesis is a highly complex multistep process that requires coordinated cell–cell and cell–extracellular matrix (ECM) interactions [2], [3], [4], [5], [6], [7] and cellular behaviours over space and time to create functional 3D structures [1], [8], [9]. First, patterns of different cell populations undergoing particular morphogenetic movements are established by a group of genes that control germ layer fates [10]. Second, cell–cell and cell–matrix interactions activate epithelial morphogenetic events and generate cell polarity through the reorganization of proteins in the cytoplasm and on the plasma membrane [11], [12], [13]. Finally, a combination of cellular processes including proliferation, adhesion, migration and apoptosis contributes to the eventual epithelial tissue architecture [4], [9], [14], [15].

Epithelia are coherent sheets of cells that cover the external surface of the body and line all its internal cavities [16], [17]. Most internal epithelial organs consist of monolayers of cells that adhere to each other through cell–cell junctions. These monolayers are arranged in spherical (cysts) or tubular (tubules) structures that enclose a central lumen and are surrounded by a basement membrane [9]. The key functions of epithelia are to control tissue architecture, create impervious and selective permeability fluid barriers between biological compartments, and perform vectorial transport functions (for example, absorption, secretion, ion transport and transcytosis) which are crucial for the survival of multicellular organisms [17], [18], [19]. In order to carry out these specialized tasks, epithelial cells must polarize internally to create biochemically different surfaces by segregating their plasma membrane proteins into apical (facing lumen), lateral (facing neighbouring cells) and basal (facing the underlying ECM) domains [20]. Since many proteins (such as β-catenin) localize to both the basal and lateral domains, these domains are often collectively referred to as ‘basolateral’ [9].

Bissell and colleagues have pioneered the use of 3D collagen gels to study how ECM microenvironments regulate epithelial morphogenesis and functions since the early 1980s [21], [22], [23], [24]. It is well established that many epithelial cells, including Madin–Darby Canine Kidney (MDCK) cells, form tissue-like cysts with classical apical and basolateral polarity when cultured three dimensionally (embedded) in type I collagen gels [8], [9], [24]. The organization of these cysts closely resembles that of epithelia in vivo, and thus cyst development provides an ideal model system for the formation of a rudimentary epithelial suborgan [22]. Recent studies in epithelial developmental systems have demonstrated the important roles of cell–cell and cell–ECM interactions in the establishment of cell polarity, while the ECM has been implicated as a potential link between polarity and tissue organization [4], [21], [22], [24], [25], [26], [27], [28]. For example, when MDCK cells are grown in suspension culture (i.e. without exogenous ECM), they will attempt to compensate for the lack of ECM in their culture environment by creating internal cavities and filling them with secreted basement membrane to generate basal surfaces de novo, resulting in the formation of hollow cysts with opposite polarity (apical surface on the outside, basolateral surface on the inside) compared to the collagen gel-grown cysts [8].

To date, most epithelial morphogenesis studies have been cell-based (i.e. utilizing genetic modification techniques to control cellular expressions and investigate the resultant effects), while the 3D culture environments employed have always been limited to collagen gels [8], [17], [26], [28]. Although collagen gels are part of the components of the natural ECM, they do not provide controlled presentation of specific bioligands or degradation motifs, making it difficult to isolate the effects of a specific bioligand of interest on the epithelial morphogenetic behaviour. As such, no studies have yet been carried out to systematically investigate the effects of individual extracellular factors on epithelial morphogenesis largely due to the lack of suitable ECM mimetics for this kind of work. Furthermore, the construction of in vitro cell culture systems that reconstitute the 3D polarized organization and structure of native tissues and organs constitutes a major challenge in tissue engineering and regenerative medicine applications.

To this end, we have engineered biomimetic hydrogels incorporating tethered bioligands to provide signals for modulating morphogenesis and crosslinkers with protease-sensitive degradation sites to allow cells to proteolytically create space to expand inside the gel and form cysts. Specifically, PEG was selected as the inert main structural component due to its well-established cytocompatibility and resistance to protein adsorption [29], [30], allowing only the biological signal from the incorporated peptides or proteins to be exhibited to the surrounding cells with minimal biochemical background. These synthetic hydrogels were originally developed by Hubbell and colleagues and have recently been successfully employed as an alternative ECM model for guiding cellular behaviour in 3D cell migration research [31], [32], [33], [34], [35]. The hydrogels are based on end-functionalized 4-arm PEG macromers, reacted via Michael-type addition reaction with cysteine-containing peptides or proteins, then crosslinked with bis-cysteine oligopeptides under near physiological conditions in the presence of cells, resulting in a 3D hybrid network that encapsulates cells. In the present study, we examined the suitability of proteolytically degradable PEG hydrogels functionalized with bioadhesive ligands as a bioartificial ECM model system for investigating and directing epithelial morphogenesis.