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2.  A bioengineered niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy 
Nature biotechnology  2016;34(7):752-759.
A promising therapeutic strategy for diverse genetic disorders involves transplantation of autologous stem cells that have been genetically corrected ex vivo. A major challenge in such approaches is a loss of stem cell potency during stem cell culture. Here we describe a system for maintaining muscle stem cells (MuSCs) in vitro in a potent, quiescent state. Using a machine learning method, we identified a molecular signature of quiescence and used it to screen for factors that could maintain mouse MuSC quiescence, thus defining a quiescence medium (QM). We also designed artificial muscle fibers (AMFs) that mimic the native myofiber of the MuSC niche. Mouse MuSCs maintained in QM on AMFs showed enhanced potential for engraftment, tissue regeneration and self-renewal after transplantation in mice. An artificial niche adapted to human MuSCs showed similarly prolonged quiescence in vitro and enhanced potency in vivo. Our approach for maintaining quiescence may be applicable to stem cells from a range of other tissues.
doi:10.1038/nbt.3576
PMCID: PMC4942359  PMID: 27240197
3.  Adaptable Hydrogel Networks with Reversible Linkages for Tissue Engineering 
Adaptable hydrogels have recently emerged as a promising platform for three-dimensional (3D) cell encapsulation and culture. In conventional, covalently crosslinked hydrogels, degradation is typically required to allow complex cellular functions to occur, leading to bulk material degradation. In contrast, adaptable hydrogels are formed by reversible crosslinks. Through breaking and re-forming of the reversible linkages, adaptable hydrogels can be locally modified to permit complex cellular functions while maintaining their long-term integrity. In addition, these adaptable materials can have biomimetic viscoelastic properties that make them well suited for several biotechnology and medical applications. In this review, adaptable hydrogel design considerations and linkage selections are overviewed, with a focus on various cell compatible crosslinking mechanisms that can be exploited to form adaptable hydrogels for tissue engineering.
doi:10.1002/adma.201501558
PMCID: PMC4528979  PMID: 25989348
adaptable hydrogel; reversible linkage; cell encapsulation
4.  Protein-Engineered Hydrogel Encapsulation for 3-D Culture of Murine Cochlea 
Hypothesis
Elastin-like protein (ELP) hydrogel helps maintain the three-dimensional (3-D) cochlear structure in culture.
Background
Whole-organ culture of the cochlea is a useful model system facilitating manipulation and analysis of live sensory cells and surrounding nonsensory cells. The precisely organized 3-D cochlear structure demands a culture method that preserves this delicate architecture; however, current methods have not been optimized to serve such a purpose.
Methods
A protein-engineered ELP hydrogel was used to encapsulate organ of Corti isolated from neonatal mice. Cultured cochleae were immunostained for markers of hair cells and supporting cells. Organ of Corti hair cell and supporting cell density and organ dimensions were compared between the ELP and nonencapsulated systems. These culture systems were then compared with noncultured cochlea.
Results
After 3 days in vitro, vital dye uptake and immunostaining for sensory and nonsensory cells show that encapsulated cochlea contain viable cells with an organized architecture. In comparison with nonencapsulated cultured cochlea, ELP-encapsulated cochleae exhibit higher densities of hair cells and supporting cells and taller and narrower organ of Corti dimensions that more closely resemble those of noncultured cochleae. However, we found compromised cell viability when the culture period extended beyond 3 days.
Conclusion
We conclude that the ELP hydrogel can help preserve the 3-D architecture of neonatal cochlea in short-term culture, which may be applicable to in vitro study of the physiology and pathophysiology of the inner ear.
doi:10.1097/MAO.0000000000000518
PMCID: PMC4905747  PMID: 25111520
Hair cells; Organ of Corti; Organotypic; Tissue architecture
5.  Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis 
Exposing myoblasts to basic fibroblast growth factor (bFGF), which is released after muscle injury, results in receptor phosphorylation, faster migration, and increased proliferation. These effects occur on time scales that extend across three orders of magnitude (100 – 103 minutes). Finite element modeling of Transwell assays, which are traditionally used to assess chemotaxis, revealed that the bFGF gradient formed across the membrane pore is short-lived and diminishes 45% within the first minute. Thus, to evaluate bFGF-induced migration over 102 minutes, we employed a microfluidic assay capable of producing a stable, linear concentration gradient to perform single-cell analyses of chemokinesis and chemotaxis. We hypothesized that the composition of the underlying extracellular matrix (ECM) may affect the behavioral response of myoblasts to soluble bFGF, as previous work with other cell types has suggested crosstalk between integrin and fibroblast growth factor (FGF) receptors. Consistent with this notion, we found that bFGF significantly reduced the doubling time of myoblasts cultured on laminin but not fibronectin or collagen. Laminin also promoted significantly faster migration speeds (13.4 μm/h) than either fibronectin (10.6 μm/h) or collagen (7.6 μm/h) without bFGF stimulation. Chemokinesis driven by bFGF further increased migration speed in a strictly additive manner, resulting in an average increase of 2.3 μm/h across all ECMs tested. We observed relatively mild chemoattraction (~ 67% of myoblast population) in response to bFGF gradients of 3.2 ng/mL/mm regardless of ECM identity. Thus, while ECM-bFGF crosstalk did impact chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. These data suggest that the main physiological effect of bFGF on myoblast migration is chemokinesis and that changes in the surrounding ECM, resulting from aging and/or disease may impact muscle regeneration by altering myoblast migration and proliferation.
doi:10.1039/c5ib00060b
PMCID: PMC4528978  PMID: 25909157
6.  Injectable Hydrogels with In Situ Double Network Formation Enhance Retention of Transplanted Stem Cells 
Advanced functional materials  2015;25(9):1344-1351.
doi:10.1002/adfm.201403631
PMCID: PMC4529129  PMID: 26273242
Injectable hydrogels; Stem cell transplantation; Molecular recognition; Self-healing; Reinforcing networks; Thermoresponsive polymer
7.  Microfluidic Gradients Reveal Enhanced Neurite Outgrowth but Impaired Guidance within 3D Matrices with High Integrin Ligand Densities 
The density of integrin-binding ligands in an extracellular matrix (ECM) is known to regulate cell migration speed by imposing a balance of traction forces between the leading and trailing edges of the cell, but the effect of cell-adhesive ligands on neurite chemoattraction is not well understood. We present a platform that combines gradient-generating microfluidic devices with three-dimensional (3D) protein-engineered hydrogels to study the effect of RGD ligand density on neurite pathfinding from chick dorsal root ganglia-derived spheroids. Spheroids are encapsulated in elastin-like polypeptide (ELP) hydrogels presenting either 3.2 or 1.6 mM RGD ligands and exposed to a microfluidic gradient of nerve growth factor (NGF). While the higher ligand density matrix enhanced neurite initiation and persistence of neurite outgrowth, the lower ligand density matrix significantly improved neurite pathfinding and increased the frequency of growth cone turning up the NGF gradient. The apparent trade-off between neurite extension and neurite guidance is reminiscent of the well-known parabolic relationship between cell adhesion and migration speed, implying that a similar matrix-mediated balance of forces regulate neurite elongation and growth cone turning. These results have implications in the design of engineered materials for in vitro models of neural tissue and in vivo nerve guidance channels.
doi:10.1002/smll.201401574
PMCID: PMC4528974  PMID: 25315156
neurite pathfinding; chemotaxis; elastin-like polypeptides; nerve growth factor; 3D migration
8.  Matrix RGD ligand density and L1CAM-mediated Schwann cell interactions synergistically enhance neurite outgrowth 
Acta biomaterialia  2014;11:48-57.
The innate biological response to peripheral nerve injury involves a complex interplay of multiple molecular cues to guide neurites across the injury gap. Many current strategies to stimulate regeneration take inspiration from this biological response, however, little is known about the balance of cell-matrix and Schwann cell-neurite dynamics required for regeneration of neural architectures. We present an engineered extracellular matrix (eECM) microenvironment with tailored cell-matrix and cell-cell interactions to study their individual and combined effects on neurite outgrowth. This eECM regulates cell-matrix interactions by presenting integrin-binding RGD ligands at specified densities. Simultaneously, the addition or exclusion of nerve growth factor (NGF) is used to modulate L1CAM-mediated Schwann cell-neurite interactions. Individually, increasing the RGD ligand density from 0.16 mM to 3.2 mM resulted in increasing neurite lengths. In matrices presenting higher RGD ligand densities, neurite outgrowth was synergistically enhanced in the presence of soluble NGF. Analysis of Schwann cell migration and colocalization with neurites revealed that NGF enhanced cooperative outgrowth between the two cell types. Interestingly, neurites in NGF-supplemented conditions were unable to extend on the surrounding eECM without the assistance of Schwann cells. Blocking studies revealed that L1CAM is primarily responsible for these Schwann cell-neurite interactions. Without NGF supplementation, neurite outgrowth was unaffected by L1CAM blocking or the depletion of Schwann cells. These results underscore the synergistic interplay between cell-matrix and cellcell interactions in enhancing neurite outgrowth for peripheral nerve regeneration.
doi:10.1016/j.actbio.2014.10.008
PMCID: PMC4528982  PMID: 25308870
Dorsal Root Ganglia; Nerve Growth Factor; Elastin-like peptide; L1CAM; RGD ligands
9.  Dual-stage growth factor release within 3D protein-engineered hydrogel niches promotes adipogenesis 
Biomaterials science  2014;2(11):1627-1639.
Engineered biomimetic microenvironments from hydrogels are an emerging strategy to achieve lineage-specific differentiation in vitro. In addition to recapitulating critical matrix cues found in the native three-dimensional (3D) niche, the hydrogel can also be designed to deliver soluble factors that are present within the native inductive microenvironment. We demonstrate a versatile materials approach for the dual-stage delivery of multiple soluble factors within a 3D hydrogel to induce adipogenesis. We use a Mixing-Induced Two-Component Hydrogel (MITCH) embedded with alginate microgels to deliver two pro-adipogenic soluble factors, fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) with two distinct delivery profiles. We show that dual-stage delivery of FGF-1 and BMP-4 to human adipose-derived stromal cells (hADSCs) significantly increases lipid accumulation compared with the simultaneous delivery of both growth factors together. Furthermore, dual-stage growth factor delivery within a 3D hydrogel resulted in substantially more lipid accumulation compared to identical delivery profiles in 2D cultures. Gene expression analysis shows upregulation of key adipogenic markers indicative of brown-like adipocytes. These data suggest that dual-stage release of FGF-1 and BMP-4 within 3D microenvironments can promote the in vitro development of mature adipocytes.
doi:10.1039/C4BM00142G
PMCID: PMC4188404  PMID: 25309741
10.  Avidity-controlled hydrogels for injectable co-delivery of induced pluripotent stem cell-derived endothelial cells and growth factors 
To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCH) from engineered proteins, here we develop protein-polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20 kDa and 40 kDa star-shaped PEG were investigated, and all four PEG variants were able to undergo a sol-gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C-P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20 kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40 kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.
A biomimetic avidity strategy was used to design a family a Mixing-Induced Two-Component Hydrogels for the injectable co-delivery of growth factors and human induced pluripotent stem cell-derived endothelial cells.
doi:10.1016/j.jconrel.2014.05.015
PMCID: PMC4518026  PMID: 24848744
iPSC; hydrogel; protein engineering; VEGF; endothelial cell
11.  Hybrid Elastin-like Polypeptide–Polyethylene Glycol (ELP-PEG) Hydrogels with Improved Transparency and Independent Control of Matrix Mechanics and Cell Ligand Density 
Biomacromolecules  2014;15(9):3421-3428.
Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP’s lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly cross-links with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent anti-Stokes Raman scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 °C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell–matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (>98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics.
doi:10.1021/bm500969d
PMCID: PMC4157761  PMID: 25111283
12.  Small-molecule axon-polarization studies enabled by a shear-free microfluidic gradient generator 
Lab on a chip  2014;14(12):2047-2056.
A deep understanding of the mechanisms behind neurite polarization and axon path-finding is important for interpreting how the human body guides neurite growth during development and response to injury. Further, it is of great clinical importance to identify diffusible chemical cues that promote neurite regeneration for nervous tissue repair. Despite the fast development of various types of concentration gradient generators, it has been challenging to fabricate neuron friendly (i.e. shear-free and biocompatible for neuron growth and maturation) devices to create stable gradients, particularly for fast diffusing small molecules, which typically require high flow and shear rates. Here we present a finite element analysis for a polydimethylsiloxane/polyethylene glycol diacrylate (PDMS/PEG-DA) based gradient generator, describe the microfabrication process, and validate its use for neuronal axon polarization studies. This device provides a totally shear-free, biocompatible microenvironment with a linear and stable concentration gradient of small molecules such as forskolin. The gradient profile in this device can be customized by changing the composition or width of the PEG-DA barriers during direct UV photo-patterning within a permanently bonded PDMS device. Primary rat cortical neurons (embryonic E18) exposed to soluble forskolin gradients for 72 hr exhibited statistically significant polarization and guidance of their axons. This device provides a useful platform for both chemotaxis and directional guidance studies, particularly for shear sensitive and non-adhesive cell cultures, while allowing fast new device design prototyping at a low cost.
doi:10.1039/c4lc00162a
PMCID: PMC4528973  PMID: 24781157
13.  Avidity-Controlled Delivery of Angiogenic Peptides from Injectable Molecular-Recognition Hydrogels 
Tissue Engineering. Part A  2014;20(15-16):2102-2114.
Peptide mimics of growth factors represent an emerging class of therapeutic drugs due to high biological specificity and relative ease of synthesis. However, maintaining efficacious therapeutic dosage at the therapy site has proven challenging owing to poor intestinal permeability and short circulating half-lives in the blood stream. In this work, we present the affinity immobilization and controlled release of QK, a vascular endothelial growth factor (VEGF) mimetic peptide, from an injectable mixing-induced two-component hydrogel (MITCH). The MITCH system is crosslinked by reversible interactions between WW domains and complementary proline-rich peptide modules. Fusion of the QK peptide to either one or two units of the proline-rich sequence creates bifunctional peptide conjugates capable of specific binding to MITCH while preserving their angiogenic bioactivity. Presenting two repeats of the proline-rich sequence increases the binding enthalpy 2.5 times due to avidity effects. Mixing of the drug conjugates with MITCH components results in drug encapsulation and extended release at rates consistent with the affinity immobilization strength. Human umbilical vein endothelial cells (HUVECs) treated with the soluble drug conjugates exhibit morphogenetic events of VEGF receptor 2 signal transduction followed by cell migration and organization into networks characteristic of early angiogenesis. In a three-dimensional model where HUVECs were cultured as spheroids in a matrix of collagen and fibronectin, injection of drug-releasing MITCH resulted in significantly more cell outgrowth than drugs injected in saline. This ability to sustain local drug availability is ideal for therapeutic angiogenesis applications, where spatiotemporal control over drug distribution is a key requirement for clinical success.
doi:10.1089/ten.tea.2013.0357
PMCID: PMC4137330  PMID: 24490588
14.  One-pot Synthesis of Elastin-like Polypeptide Hydrogels with Grafted VEGF-Mimetic Peptides 
Biomaterials science  2014;2(5):757-765.
Immobilization of growth factors to polymeric matrices has been a common strategy in the design of tissue engineering scaffolds to promote tissue regeneration, which requires complex cell signaling events with the surrounding matrix. However, the use of large protein growth factors in polymeric scaffolds is often plagued by immunogenicity, short in vivo half-lives, and reduced bioactivity. To address these concerns, we develop a single-step, cell-compatible strategy to tether small, growth-factor-mimetic peptides into a protein-engineered hydrogel with tunable biomaterial properties. Specifically, we covalently immobilize the QK peptide, an angiogenic peptide mimicking the receptor-binding region of vascular endothelial growth factor (VEGF), within tunable elastin-like polypeptide (ELP) hydrogels that include a cell-adhesive RGD sequence. Using a cell-compatible, amine-reactive crosslinker, we conducted a one-pot synthesis to simultaneously encapsulate cells while precisely controlling the QK grafting density (10 nM – 100 μM) in the ELP hydrogels without altering other material properties. Fluorescence analysis of fluor-labeled QK peptides demonstrated that the conjugation efficiency to ELP hydrogels was >75% and that covalent immobilization effectively eliminates all QK diffusion. Compared with pristine ELP hydrogels, human umbilical vein endothelial cell (HUVEC) proliferation was significantly enhanced on ELP hydrogels immobilized with 10 nM or 1 μM QK. Moreover, upon encapsulation within tethered QK-ELP hydrogels, HUVEC spheroids maintained near 100% viability and demonstrated significantly more three-dimensional outgrowth compared to those supplemented with soluble QK peptide at the same concentration. These results encourage the further development of protein-engineered scaffolds decorated with growth-factor-mimetic peptides to provide long-term biological signals using this versatile, single-step synthesis.
doi:10.1039/C3BM60293A
PMCID: PMC3979545  PMID: 24729868
15.  Designing ECM-mimetic Materials Using Protein Engineering 
Acta biomaterialia  2013;10(4):1751-1760.
The natural extracellular matrix (ECM), with its multitude of evolved cell-instructive and cell-responsive properties, provides inspiration and guidelines for the design of engineered biomaterials. One strategy to create ECM-mimetic materials is the modular design of protein-based engineered ECM (eECM) scaffolds. This modular design strategy involves combining multiple protein domains with different functionalities into a single, modular polymer sequence, resulting in a multifunctional matrix with independent tunability of the individual domain functions. These eECMs often enable decoupled control over multiple material properties for fundamental studies of cell-matrix interactions. In addition, since the eECMs are frequently composed entirely of bioresorbable amino acids, these matrices have immense clinical potential for a variety of regenerative medicine applications. This brief review demonstrates how fundamental knowledge gained from structure-function studies of native proteins can be exploited in the design of novel protein-engineered biomaterials. While the field of protein-engineered biomaterials has existed for over 20 years, the community is only now beginning to fully explore the diversity of functional peptide modules that can be incorporated into these materials. We have chosen to highlight recent examples that either (1) demonstrate exemplary use as matrices with cell-instructive and cell-responsive properties or (2) demonstrate outstanding creativity in terms of novel molecular-level design and macro-level functionality.
doi:10.1016/j.actbio.2013.12.028
PMCID: PMC3973542  PMID: 24365704
Protein engineering; Extracellular matrix; Modular design strategy; Tissue engineering; Recombinant protein synthesis; Three-dimensional hydrogels
16.  A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration 
Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration.
doi:10.1039/c3ib40177d
PMCID: PMC4114769  PMID: 24599496
17.  Matrix interactions modulate neurotrophin-mediated neurite outgrowth and pathfinding 
Neural Regeneration Research  2015;10(4):514-517.
Both matrix biochemistry and neurotrophic factors are known to modulate neurite outgrowth and pathfinding; however, the interplay between these two factors is less studied. While previous work has shown that the biochemical identity of the matrix can alter the outgrowth of neurites in response to neurotrophins, the importance of the concentration of cell-adhesive ligands is unknown. Using engineered elastin-like protein matrices, we recently demonstrated a synergistic effect between matrix-bound cell-adhesive ligand density and soluble nerve growth factor treatment on neurite outgrowth from dorsal root ganglia. This synergism was mediated by Schwann cell-neurite contact through L1CAM. Cell-adhesive ligand density was also shown to alter the pathfinding behavior of dorsal root ganglion neurites in response to a gradient of nerve growth factor. While more cell-adhesive matrices promoted neurite outgrowth, less cell-adhesive matrices promoted more faithful neurite pathfinding. These studies emphasize the importance of considering both matrix biochemistry and neurotrophic factors when designing biomaterials for peripheral nerve regeneration.
doi:10.4103/1673-5374.155426
PMCID: PMC4424732  PMID: 26170800
neurotrophic factors; cell-adhesive ligands; dorsal root ganglia; L1CAM; nerve growth factor; biomaterials; elastin-like proteins
18.  Presentation of BMP-2 Mimicking Peptides in 3D Hydrogels Directs Cell Fate Commitment in Osteoblasts and Mesenchymal Stem Cells 
Biomacromolecules  2014;15(2):445-455.
Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by 1H-NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.
doi:10.1021/bm401726u
PMCID: PMC3930060  PMID: 24400664
bone morphogenetic protein; peptide mimic; mesenchymal stem cell; osteogenesis
19.  Spontaneous cardiomyocyte differentiation of mouse embryoid bodies regulated by hydrogel crosslink density 
Biomaterials science  2013;1(10):1082-1090.
Cellular therapies have great potential to provide alternative treatment options for those suffering from heart disease. In order to optimize cell delivery for therapeutic efficacy, a greater understanding of parameters that impact stem cell differentiation, survival, growth, and development are needed. In this study, we examine the role of hydrogel crosslink density on spontaneous cardiomyocyte (CM) differentiation of murine embryoid bodies (EBs). CM differentiation was accelerated in hydrogels of low crosslink density, where 100% of the hydrogels were positive for CM differentiation compared to only 53% in the high crosslink density group after 8 days of culture. DNA microarray data suggests that enhanced CM differentiation in the low crosslink density hydrogels was not tissue specific but rather a result of favoured EB development and cell proliferation. Additionally, enhanced EB growth and differentiation in low crosslink density hydrogels was independent of RGD ligand density and not a consequence of enhanced diffusion. We also demonstrate that matrix metalloproteinase activity is required for spontaneous CM differentiation in 3D hydrogels. Low hydrogel crosslink density regulates spontaneous EB differentiation by promoting EB growth and development. Elucidating the effects of microenvironmental cues on cell differentiation can aid in the optimization of stem cell-based therapies for tissue regeneration.
doi:10.1039/C3BM60139K
PMCID: PMC3987919  PMID: 24748962
20.  Microfluidic Investigation of BDNF Enhanced Neural Stem Cell Chemotaxis in CXCL12 Gradients 
In vivo studies have suggested that gradients of CXCL12 (aka stromal cell-derived factor 1α) may be a critical guidance cue for neural stem cell (NSC) migration during both brain development and neural tissue regeneration. However, traditional in vitro chemotaxis tools are typically limited by unstable concentration gradients and the inability to decouple cell migration directionality and speed. These limitations have restricted the reproducible and quantitative analysis of neuronal migration, which is a requirement for mechanism-based studies that may guide the development of new therapeutic strategies for neural regeneration. Using a microfluidic gradient generator, we quantified nestin and Sox-2 positive human embryonic NSC chemotaxis within a linear and stable CXCL12 gradient. While untreated NSCs were not able to chemotax within CXCL12 gradients, pre-treatment of the cells with brain-derived neurotrophic factor (BDNF) resulted in significant chemotactic, directional migration. BDNF pre-treatment had no effect on cell migration speed, which averaged about 1 μm min−1. Quantitative analysis determined that CXCL12 concentrations above 72 ng ml−1 (9.0 nM) are above the minimum activation threshold, while concentrations below 117 ng ml−1 (14.7 nM) are below the saturation threshold. Interestingly, although inhibitor studies with AMD 3100 revealed that CXCL12 chemotaxis requires receptor CXCR4 activation, BDNF pre-treatment was found to have no profound effects on the mRNA levels or surface presentation of CXCR4 or the putative CXCR7 scavenger receptor. The microfluidic study of NSC migration within stable chemokine concentration profiles provided quantitative analysis as well as new insight into the migratory mechanism underlying BDNF-induced chemotaxis towards CXCL12. These data will provide quantitative guidance in the development of new strategies to enhance the homing of endogenous and exogenous NSCs for clinical tissue regeneration therapies.
doi:10.1002/smll.201202208
PMCID: PMC3984949  PMID: 23109183
microfluidic gradient generator; neural stem cell; CXCL12; BDNF; chemotaxis
21.  Complex chemoattractive and chemorepellent Kit signals revealed by direct imaging of murine mast cells in microfluidic gradient chambers† 
Besides its cooperating effects on stem cell proliferation and survival, Kit ligand (KL) is a potent chemotactic protein. While transwell assays permit studies of the frequency of migrating cells, the lack of direct visualization precludes dynamic chemotaxis studies. In response, we utilize microfluidic chambers that enable direct observation of murine bone marrow-derived mast cells (BMMC) within stable KL gradients. Using this system, individual Kit+ BMMC were quantitatively analyzed for migration speed and directionality during KL-induced chemotaxis. Our results indicated a minimum activating threshold of ~3 ng ml−1 for chemoattraction. Analysis of cells at KL concentrations below 3 ng ml−1 revealed a paradoxical chemorepulsion, which has not been described previously. Unlike chemoattraction, which occurred continuously after an initial time lag, chemorepulsion occurred only during the first 90 minutes of observation. Both chemoattraction and chemorepulsion required the action of G-protein coupled receptors (GPCR), as treatment with pertussis toxin abrogated directed migration. These results differ from previous studies of GPCR-mediated chemotaxis, where chemorepulsion occurred at high ligand concentrations. These data indicate that Kit-mediated chemotaxis is more complex than previously understood, with the involvement of GPCRs in addition to the Kit receptor tyrosine kinase and the presence of both chemoattractive and chemorepellent phases.
doi:10.1039/c3ib40025e
PMCID: PMC3973540  PMID: 23835699
22.  Protein-engineered injectable hydrogel to improve retention of transplanted adipose-derived stem cells 
Advanced healthcare materials  2012;2(3):428-432.
Improved retention of transplanted stem cells is achieved through minimally invasive delivery in MITCH, a Mixing-Induced Two-Component Hydrogel engineered to possess shear-thinning and self-healing thixotropic properties. MITCH, an ideal injectable cell-delivery vehicle, supports 3D stem cell culture, resulting in high cell viability and physiologically relevant cell morphology.
doi:10.1002/adhm.201200293
PMCID: PMC3926432  PMID: 23184882
adipose-derived stem cells; cell injections; cell transplantations; hydrogels; protein engineering
23.  Design of 3D engineered protein hydrogels for tailored control of neurite growth 
Acta biomaterialia  2012;9(3):5590-5599.
The design of bioactive materials allows for tailored studies probing cell-biomaterial interactions; however, relatively few studies have examined effects of ligand density and material stiffness on neurite growth in 3D. Elastin-like proteins (ELPs) have been designed with modular bioactive and structural regions to enable the systematic characterization of design parameters within 3D materials. To promote neurite outgrowth and better understand the effects of common biomaterial design parameters on neuronal cultures, we here focused on cell-adhesive ligand density and hydrogel stiffness as design variables for ELP hydrogels. With the inherent design freedom of engineered proteins, these 3D ELP hydrogels enabled decoupled investigation into the effects of biomechanics and biochemistry on neurite outgrowth from dorsal root ganglia (DRG). Increasing the cell-adhesive RGD ligand density from 0 to 1.9 × 107 ligands/μm3 led to a significant increase in the rate, length, and density of neurite outgrowth, as quantified by a high-throughput algorithm developed for dense neurite analysis. An approximately two-fold improvement in total neurite outgrowth was observed in materials with the higher ligand density at all time-points through 7 days. ELP hydrogels with initial elastic moduli of 0.5, 1.5, or 2.1 kPa and identical RGD ligand densities revealed that the most compliant materials led to the greatest outgrowth, with some neurites extending over 1800 μm by day 7. Given the ability of ELP hydrogels to efficiently promote neurite outgrowth within defined and tunable 3D microenvironments, these materials may be useful in developing therapeutic nerve guides and the further study of basic neuron-biomaterial interactions.
doi:10.1016/j.actbio.2012.10.033
PMCID: PMC3926440  PMID: 23128159
Hydrogel; Neuron Neurite Stiffness; Three-dimensional; Engineered protein
25.  Tetrakis (hydroxylmethyl) phosphonium chloride as a covalent crosslinking agent for cell encapsulation within protein-based hydrogels 
Biomacromolecules  2012;13(12):3912-3916.
Native tissues provide cells with complex, three dimensional (3D) environments comprised of hydrated networks of extracellular matrix proteins and sugars. By mimicking the dimensionality of native tissue while deconstructing the effects of environmental parameters, protein-based hydrogels serve as attractive, in vitro platforms to investigate cell-matrix interactions. For cell encapsulation, the process of hydrogel formation through physical or covalent crosslinking must be mild and cell compatible. While many chemical crosslinkers are commercially available for hydrogel formation, only a subset are cytocompatible; therefore, the identification of new and reliable cytocompatible crosslinkers allows for greater flexibility of hydrogel design for cell encapsulation applications. Here, we introduce tetrakis (hydroxymethyl) phosphonium chloride (THPC) as an inexpensive, amine-reactive, aqueous crosslinker for 3D cell encapsulation in protein-based hydrogels. We characterize the THPC-amine reaction by demonstrating its ability to react with primary and secondary amines of various amino acids. In addition, we demonstrate the utility of THPC to tune hydrogel gelation time (6.7 ± 0.2 to 27 ± 1.2 min) and mechanical properties (storage moduli ~250 Pa to ~2200 Pa) with a recombinant elastin-like protein. Lastly, we show cytocompatibility of THPC for cell encapsulation of two cell types, embryonic stem cells and neuronal cells, where cells exhibited the ability to differentiate and/or grow in elastin-like protein hydrogels.
doi:10.1021/bm3015279
PMCID: PMC3556456  PMID: 23151175
Crosslinker; Hydrogel; Cell Encapsulation; Amine-reactive

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