Directing stem cell fate on hydrogel substrates by controlling cell geometry, matrix mechanics and adhesion ligand composition

Abstract

There is a dynamic relationship between physical and biochemical signals presented in the stem cell microenvironment to guide cell fate determination. Model systems that modulate cell geometry, substrate stiffness or matrix composition have proved useful in exploring how these signals influence stem cell fate. However, the interplay between these physical and biochemical cues during differentiation remains unclear. Here, we demonstrate a microengineering strategy to vary single cell geometry and the composition of adhesion ligands — on substrates that approximate the mechanical properties of soft tissues — to study adipogenesis and neurogenesis in adherent mesenchymal stem cells. Cells cultured in small circular islands show elevated expression of adipogenesis markers while cells that spread in anisotropic geometries tend to express elevated neurogenic markers. Arraying different combinations of matrix protein in a myriad of 2D and pseudo-3D geometries reveals optimal microenvironments for controlling the differentiation of stem cells to these “soft” lineages without the use of media supplements.

Introduction

Cells adhering to the extracellular matrix (ECM) can sense the mechanical properties through specific interactions of cell surface integrins with adhesion ligands [1], [2], [3], [4], [5]. Traction forces exerted by the cell through these interactions influence cytoskeletal tension and lead to changes in cell shape and associated signaling cascades that ultimately regulate gene expression [6], [7], [8], [9], [10]. This process of mechanotransduction has emerged as an important aspect of stem cell differentiation and is dependent on both the mechanics and the composition of the microenvironment. For example, Datta et al. revealed the importance of the mechanical and biochemical microenvironment by culturing osteoprogenitor cells on a decellularized osteoblast matrix leading to increased expression of osteogenic markers [11]. Work in the Schaffer and Healey groups has demonstrated that mechanical properties can guide neurogenesis in neural stem cells where softer matrices promote dendritic process extension [12]. A study by Engler, Discher and colleagues demonstrated the importance of matrix mechanics in guiding MSC fate by studying cells adherent to collagen-coated polyacrylamide hydrogels of variable stiffness [8]. MSCs were found to commit to lineages based on the similarity to the committed cells' native matrix; soft polyacrylamide gels (<1 kPa) promote neurogenesis, intermediate stiffness gels (∼10 kPa) promote myogenesis and stiff gels (>30 kPa) promote osteogenesis.

In addition to stiffness, the composition and presentation of adhesion ligands on a substrate have been shown to influence MSC differentiation [1], [2], [3], [13], [14], [15]. Cooper-White and co-workers demonstrated that different matrix proteins — collagen, fibronectin and laminin — grafted to hydrogel substrates of different stiffness will significantly influence the expression of myogenic and osteogenic markers. This work suggests that the identity of adhesion ligand and its presentation to the cell can play an important role in promoting competing differentiation outcomes. Kilian and Mrksich recently showed how the density and affinity of surface bound adhesion peptides could modulate the expression of markers associated with neurogenesis, myogenesis and osteogenesis, further confirming the importance of the type and presentation of ligand in guiding stem cell differentiation [3].

Another important physical parameter that has emerged as an important cue in guiding the differentiation of stem cells, and is influenced by stiffness and the presentation of adhesion ligands, is cell shape [4], [16], [17], [18], [19]. For instance, Chen and colleagues demonstrated that MSCs captured on small islands tended to prefer adipocyte differentiation when exposed to a mixture of osteogenic and adipogenic soluble cues while cells captured on large islands developed a higher degree of cytoskeletal tension and preferred to adopt an osteoblast outcome [16]. In a related study, Kilian et al. demonstrated that MSCs patterned in geometries with subcellular concave regions and moderate aspect ratios increase the actomyosin contractility of the cell and promote osteogenesis [17]. In both of these studies, keeping cell shape the same across a population of MSCs was shown to normalize the differentiation outcome when compared to unpatterned cells that take on a host of different geometries.

An important lesson that has emerged from these studies is that there is clearly interplay between matrix mechanics, adhesion ligand presentation and cell geometry during differentiation [4], [5], [20]. The majority of research efforts have focused on varying one physical cue while exploring the influence on biological activity. However, in vivo cell fate is likely influenced by a combination of geometry, mechanics and ECM composition [21], [22]. Thus we reasoned that combining these cues would prove useful in designing materials that more closely emulate the in vivo microenvironment and “fine-tune” a desired differentiation outcome.

In this paper, we use soft lithography to micropattern multiple matrix proteins — alone and in combinations — on hydrogel substrates with the mechanical properties of soft tissue to explore the physical and biochemical cues that guide MSCs towards adipogenesis and neurogenesis outcomes. Immunofluorescence staining and real-time PCR are employed to assess the expression of key markers during differentiation. We explore the translation of our findings to a pseudo-3D hydrogel format that more closely represents the in vivo environment.