Michael P. Schwartz

Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells

Cell-matrix interactions have critical roles in regeneration, development and disease. The work presented here demonstrates that encapsulated human mesenchymal stem cells (hMSCs) can be induced to differentiate down osteogenic and adipogenic pathways by controlling their three-dimensional environment using tethered small-molecule chemical functional groups. Hydrogels were formed using sufficiently low concentrations of tether molecules to maintain constant physical characteristics, encapsulation of hMSCs in three dimensions prevented changes in cell morphology, and hMSCs were shown to differentiate in normal growth media, indicating that the small-molecule functional groups induced differentiation. To our knowledge, this is the first example where synthetic matrices are shown to control induction of multiple hMSC lineages purely through interactions with small-molecule chemical functional groups tethered to the hydrogel material. Strategies using simple chemistry to control complex biological processes would be particularly powerful as they could make production of therapeutic materials simpler, cheaper and more easily controlled.

Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility

Due to mild reaction conditions and temporal and spatial control over material formation, photopolymerization has become a valuable technique for the encapsulation of living cells in three dimensional, hydrated, biomimetic materials. For such applications, 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (I2959) is the most commonly used photoinitiator (by virtue of its moderate water solubility), yet this initiator has an absorption spectrum that is poorly matched with wavelengths of light generally regarded as benign to living cells, limiting the rate at which it may initiate polymerization in their presence. In contrast, acylphosphine oxide photoinitiators, generally exhibit absorption spectra at wavelengths suitable for cell encapsulation, yet commercially available initiators of this class have low water solubility. Here, a water soluble lithium acylphosphinate salt is evaluated for its ability to polymerize diacrylated poly(ethylene glycol) (PEGDA) monomers rapidly into hydrogels, while maintaining high viability during direct encapsulation of cells. Through rheometric measurements, the time to reach gelation of a PEGDA solution with the phosphinate initiator is one tenth the time for that using I2959 at similar concentrations, when exposed to 365 nm light. Further, polymerization with the phosphinate initiator at 405 nm visible light exposure is achieved with low initiator concentrations and light intensities, precluded in polymerizations initiated with I2959 by its absorbance profile. When examined 24h after encapsulation, survival rates of human neonatal fibroblasts encapsulated in hydrogels polymerized with the phosphinate initiator exceed 95%, demonstrating the cytocompatibility of this initiating system.

A Versatile Synthetic Extracellular Matrix Mimic via Thiol‐Norbornene Photopolymerization

Step-growth, radically mediated thiol-norbornene photopolymerization is used to create versatile, stimuli-responsive poly(ethylene glycol)-co-peptide hydrogels The reaction is cytocompatible and allows for the encapsulation of human mesenchymal stem cells with a viability greater than 95%. Cellular spreading is dictated via three-dimensional biochemical photopatterning.

Extracellular matrix protein adsorption to phosphate-functionalized gels from serum promotes osteogenic differentiation of human mesenchymal stem cells

One of the primary goals for tissue engineering is to induce new tissue formation by stimulating specific cell function. Human mesenchymal stem cells (hMSCs) are a particularly important cell type that has been widely studied for differentiation down the osteogenic (bone) lineage, and we recently found that simple phosphate functional groups incorporated into poly(ethylene glycol) (PEG) hydrogels could induce osteogenesis without using differentiation medium by unknown mechanisms. Here, we aimed to determine whether direct or indirect cell/materials interactions were responsible for directing hMSCs down the osteogenic lineage on phosphate (PO(4))-functionalized PEG hydrogels. Our results indicated that serum components adsorbed onto PO(4)-PEG hydrogels from medium in a presoaking step were sufficient for attachment and spreading of hMSCs, even when seeded in serum-free conditions. Blocking antibodies for collagen and fibronectin (targeted to the hydrogel), as well as β1 and β3 integrin blocking antibodies (targeted to the cells), each reduced attachment of hMSCs to PO(4)-PEG hydrogels, suggesting that integrin-mediated interactions between cells and adsorbed matrix components facilitate attachment and spreading. Outside-in signaling, and not merely shape change, was found to be required for osteogenesis, as alkaline phosphatase activity and expression of CBFA1, osteopontin and collagen-1 were each significantly down regulated upon inhibition of focal adhesion kinase phosphorylation even though the focal adhesion structure or cell shape was unchanged. Our results demonstrate that complex function (i.e. osteogenic differentiation) can be controlled using simple functionalization strategies, such as incorporation of PO(4), but that the role of these materials may be due to more complex influences than has previously been appreciated.

Wnt5a Directs Polarized Calcium Gradients by Recruiting Cortical Endoplasmic Reticulum to the Cell Trailing Edge

Wnt5a directs the assembly of the Wnt-receptor-actin-myosin-polarity (WRAMP) structure, which integrates cell-adhesion receptors with F-actin and myosin to form a microfilament array associated with multivesicular bodies (MVBs). The WRAMP structure is polarized to the cell posterior, where it directs tail-end membrane retraction, driving forward translocation of the cell body. Here we define constituents of the WRAMP proteome, including regulators of microfilament and microtubule dynamics, protein interactions, and enzymatic activity. IQGAP1, a scaffold for F-actin nucleation and crosslinking, is necessary for WRAMP structure formation, potentially bridging microfilaments and MVBs. Vesicle coat proteins, including coatomer-I subunits, localize to and are required for the WRAMP structure. Electron microscopy and live imaging demonstrate movement of the ER to the WRAMP structure and plasma membrane, followed by elevation of intracellular Ca2+. Thus, Wnt5a controls directional movement by recruiting cortical ER to mobilize a rear-directed, localized Ca2+ signal, activating actomyosin contraction and adhesion disassembly for membrane retraction.

Keratinocyte proximity and contact can play a significant role in determining mesenchymal stem cell fate in human tissue

Bone marrow‐derived human mesenchymal stem cells (hMSCs) possess multipotent differentiation capabilities and are a potent source of paracrine factors. We show how the epidermal keratinocyte can direct hMSC differentiation selectively. Keratinocytes and hMSCs were either cocultured in physical contact (contact cocultures)’ or separated without physical contact using a transwell insert (noncontact cocultures). We also delivered hMSCs into an ex vivo human excisional wound where subpopulations of the hMSCs were either in contact or were physically separated from the epidermal keratinocytes. In comparison to control hMSCs that were not cocultured’ contact cocultured hMSCs adopted an epithelial morphology and expressed kera‐tinocyte markers while noncontact coculutred hMSCs’ surprisingly’ adopted phenotypes that resembled myo‐fibroblast and early neural lineage, both of which are of dermal origin. Cell fusion was not a requirement in in vitro contact cocultures, as determined by fluorescence‐activated cell sorting (FACS) and fluorescence in situ hybridization analysis (FISH). To the best of our knowledge, this work provides the first example of hMSC differentiation into different lineages depending on their proximity to a single cell type.—Sivamani, R. K., Schwartz, M. P., Anseth, K. S., Isseroff, R. R., Keratinocyte proximity and contact can play a significant role in determining mesenchymal stem cell fate in human tissue, FASEB J. 25, 122–131 (2011). www.fasebj.org

A synthetic modular approach for modeling the role of the 3D microenvironment in tumor progression.

Here, we demonstrate the flexibility of peptide-functionalized poly(ethylene glycol) (PEG) hydrogels for modeling tumor progression. The PEG hydrogels were formed using thiol-ene chemistry to incorporate a matrix metalloproteinase-degradable peptide crosslinker (KKCGGPQG↓IWGQGCKK) permissive to proteolytic remodeling and the adhesive CRGDS peptide ligand. Tumor cell function was investigated by culturing WM239A melanoma cells on PEG hydrogel surfaces or encapsulating cells within the hydrogels, and either as monocultures or indirect (non-contact) cocultures with primary human dermal fibroblasts (hDFs). WM239A cluster size and proliferation rate depended on the shear elastic modulus for cells cultured on PEG hydrogels, while growth was inhibited by coculture with hDFs regardless of hydrogel stiffness. Cluster size was also suppressed by hDFs for WM239A cells encapsulated in PEG hydrogels, which is consistent with cells seeded on top of hydrogels. Notably, encapsulated WM239A clusters and single cells adopted invasive phenotypes in the hDF coculture model, which included single cell and collective migration modes that resembled invasion from human melanoma patient-derived xenograft tumors encapsulated in equivalent PEG hydrogels. Our combined results demonstrate that peptide-functionalized PEG hydrogels provide a useful platform for investigating aspects of tumor progression in 2D and 3D microenvironments, including single cell migration, cluster growth and invasion.

Autofluorescence multiphoton microscopy for quality control of human vascular tissue constructs (Conference Presentation)

Engineered tissues offer great promise as engrafted therapies and in vitro models, but these tissues require a vascular network to retain viability at large scales. Significant efforts are focused on optimizing these in vitro vascular constructs, yet current evaluation methods require fixation and immunostaining. These destructive evaluation methods alter vascular network morphology, and cannot non-invasively monitor vascular assembly over time. Here, we demonstrate that autofluorescence multiphoton microscopy (MPM) can quantitatively assess the morphology of living 3D vascular networks without fixation, labels, or dyes. Autofluorescence MPM was used to non-invasively monitor the effect of culture conditions on 3D vascular network formation. Human embryonic stem (ES) cell-derived endothelial cells and primary human pericytes cultured in polyethylene glycol (PEG) hydrogels self-assembled into 3D vascular networks. Autofluorescence MPM of the metabolic co-enzyme NAD(P)H (excitation/emission wavelengths of 750 nm/400-460 nm) was used to quantify morphological parameters at day 6 of culture. Specifically, vessel diameter, vascular density, branch point density, and integration of endothelial cells into the network were quantified. Dynamic culture conditions (flow at 1μL/sec) led to vascular networks with higher mean vessel diameter compared to static culture (p<0.05). Standard immunohistochemistry found that vascular networks were positive for markers of endothelial cells, pericytes, and tight junctions. Scanning electron micrographs confirmed vessel lumen formation with pericytes wrapped around vessels. Dye transit of FITC-dextran through the network confirmed leaky endothelial barrier function. Our results demonstrate that autofluorescence MPM can non-invasively evaluate in vitro 3D vascular networks, and could be used for quality control of engineered tissues.