Tissue-Engineered Disease Modeling of Lymphangioleiomyomatosis Exposes a Therapeutic Vulnerability to HDAC Inhibition.

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2-/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.

Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity.

Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a biosynthetic process that conjugates glycans to proteins, which plays crucial roles in mAb bioactivities including effector functions and pharmacokinetics. These glycans are heterogeneous and have diverse chemical structures whose composition is sensitive to manufacturing conditions, rendering the understanding of how specific glycan structures affect mAb bioactivity challenging. There is a need to delineate the effects of specific glycans on mAb bioactivity to determine whether changes in certain glycosylation profiles (that can occur during manufacturing) will significantly affect product quality. Using enzymatic transglycosylation with chemically-defined N-glycans, we show that galactosylation at a specific location of N-glycans in an afucosylated anti-viral mAb is responsible for FcγRIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We report a facile method to obtain purified asymmetric mono-galactosylated biantennary complex N-glycans, and their influence on bioactivity upon incorporation into an afucosylated mAb. Using ELISA, surface plasmon resonance and flow cytometry, we show that galactosylation of the α6 antenna, but not the α3 antenna, consistently increases FcγRIIIA binding affinity. We confirm its relevance in an anti-viral model of respiratory syncytial virus (RSV) using an adapted ADCC reporter assay. We further correlate this structure-function relationship to the interaction of the galactose residue of the α6 antenna with the protein backbone using 2D-1H-15N-NMR, which showed that galactosylation of at this location exhibited chemical shift perturbations compared to glycoforms lacking this galactose residue. Our results highlight the importance of identifying and quantifying specific glycan isomers to ensure adequate quality control in batch-to-batch and biosimilar comparisons.

Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell.

BACKGROUND: Extracellular vesicles (EVs) produced by human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are currently investigated for their clinical effectiveness towards immune-mediated diseases. The large amounts of stem cell-derived EVs required for clinical testing suggest that bioreactor production systems may be a more amenable alternative than conventional EV production methods for manufacturing products for therapeutic use in humans. METHODS: To characterize the potential utility of these systems, EVs from four hBM-MSC donors were produced independently using a hollow-fiber bioreactor system under a cGMP-compliant procedure. EVs were harvested and characterized for size, concentration, immunophenotype, and glycan profile at three separate intervals throughout a 25-day period. RESULTS: Bioreactor-inoculated hBM-MSCs maintained high viability and retained their trilineage mesoderm differentiation capability while still expressing MSC-associated markers upon retrieval. EVs collected from the four hBM-MSC donors showed consistency in size and concentration in addition to presenting a consistent surface glycan profile. EV surface immunophenotypic analyses revealed a consistent low immunogenicity profile in addition to the presence of immuno-regulatory CD40 antigen. EV cargo analysis for biomarkers of immune regulation showed a high abundance of immuno-regulatory and angiogenic factors VEGF-A and IL-8. CONCLUSIONS: Significantly, EVs from hBM-MSCs with immuno-regulatory constituents were generated in a large-scale system over a long production period and could be frequently harvested with the same quality and quantity, which will circumvent the challenge for clinical application.

Benchmarking to the Gold Standard: Hyaluronan-Oxime Hydrogels Recapitulate Xenograft Models with In Vitro Breast Cancer Spheroid Culture.

Many 3D in vitro models induce breast cancer spheroid formation; however, this alone does not recapitulate the complex in vivo phenotype. To effectively screen therapeutics, it is urgently needed to validate in vitro cancer spheroid models against the gold standard of xenografts. A new oxime-crosslinked hyaluronan (HA) hydrogel is designed, manipulating gelation rate and mechanical properties to grow breast cancer spheroids in 3D. This HA-oxime breast cancer model maintains the gene expression profile most similar to that of tumor xenografts based on a pan-cancer gene expression profile (comprising 730 genes) of three different human breast cancer subtypes compared to Matrigel or conventional 2D culture. Differences in gene expression between breast cancer cultures in HA-oxime versus Matrigel or 2D are confirmed for 12 canonical pathways by gene set variation analysis. Importantly, drug response is dependent on the culture method. Breast cancer cells respond better to the Rac inhibitor (EHT-1864) and the PI3K inhibitor (AZD6482) when cultured in HA-oxime versus Matrigel. This study demonstrates the superiority of an HA-based hydrogel as a platform for in vitro breast cancer culture of both primary, patient-derived cells and cell lines, and provides a hydrogel culture model that closely matches that in vivo.

Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.

Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion.

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. We demonstrate that increases in donor-derived fibers when using HAMC are attributed to increased MuSC proliferation via a CD44-independent mechanism, preventing injected cell active clearance, and supporting in vivo expansion by delaying differentiation. Furthermore, we observed a significant improvement in donor fiber dispersion when MuSCs were delivered in HAMC. Our study results suggest that HAMC is a promising muscle stem cell delivery vehicle.

Diels-Alder Click-Cross-Linked Hydrogels with Increased Reactivity Enable 3D Cell Encapsulation.

Engineered hydrogels have been extensively used to direct cell function in 3D cell culture models, which are more representative of the native cellular microenvironment than conventional 2D cell culture. Previously, hyaluronan-furan and bis-maleimide polyethylene glycol hydrogels were synthesized via Diels-Alder chemistry at acidic pH, which did not allow encapsulation of viable cells. In order to enable gelation at physiological pH, the reaction kinetics were accelerated by replacing the hyaluronan-furan with the more electron-rich hyaluronan-methylfuran. These new click-cross-linked hydrogels gel faster and at physiological pH, enabling encapsulation of viable cells, as demonstrated with 3D culture of 5 different cancer cell lines. The methylfuran accelerates Diels-Alder cycloaddition yet also increases the retro Diels-Alder reaction. Using computational analysis, we gain insight into the mechanism of the increased Diels-Alder reactivity and uncover that transition state geometry and an unexpected hydrogen-bonding interaction are important contributors to the observed rate enhancement. This cross-linking strategy serves as a platform for bioconjugation and hydrogel synthesis for use in 3D cell culture and tissue engineering.

Photo-immobilized EGF chemical gradients differentially impact breast cancer cell invasion and drug response in defined 3D hydrogels.

Breast cancer cell invasion is influenced by growth factor concentration gradients in the tumor microenvironment. However, studying the influence of growth factor gradients on breast cancer cell invasion is challenging due to both the complexities of in vivo models and the difficulties in recapitulating the tumor microenvironment with defined gradients using in vitro models. A defined hyaluronic acid (HA)-based hydrogel crosslinked with matrix metalloproteinase (MMP) cleavable peptides and modified with multiphoton labile nitrodibenzofuran (NDBF) was synthesized to photochemically immobilize epidermal growth factor (EGF) gradients. We demonstrate that EGF gradients can differentially influence breast cancer cell invasion and drug response in cell lines with different EGF receptor (EGFR) expression levels. Photopatterned EGF gradients increase the invasion of moderate EGFR expressing MDA-MB-231 cells, reduce invasion of high EGFR expressing MDA-MB-468 cells, and have no effect on invasion of low EGFR-expressing MCF-7 cells. We evaluate MDA-MB-231 and MDA-MB-468 cell response to the clinically tested EGFR inhibitor, cetuximab. Interestingly, the cellular response to cetuximab is completely different on the EGF gradient hydrogels: cetuximab decreases MDA-MB-231 cell invasion but increases MDA-MB-468 cell invasion and cell number, thus demonstrating the importance of including cell-microenvironment interactions when evaluating drug targets.

Independently Tuning the Biochemical and Mechanical Properties of 3D Hyaluronan-Based Hydrogels with Oxime and Diels-Alder Chemistry to Culture Breast Cancer Spheroids.

For native breast cancer cell growth to be mimicked in vitro as spheroids, a well-defined matrix that mimics the tumor microenvironment is required. Finding a biomimetic material for 3D cell culture other than Matrigel has challenged the field. Because hyaluronan is naturally abundant in the tumor microenvironment and can be chemically modified, we synthesized a hyaluronan (HA) hydrogel with independently tunable mechanical and chemical properties for 3D culture of breast cancer cells. By modifying HA with distinct bioorthogonal functional groups, its mechanical properties are controlled by chemical cross-linking via oxime ligation, and its biochemical properties are controlled by grafting bioactive peptides via Diels-Alder chemistry. A series of hydrogels were screened in terms of stiffness and peptide composition for cancer spheroid formation. In the optimal hydrogel formulation, the 3D breast cancer spheroids showed decreased drug diffusion into their core and upregulation of cellular multidrug-resistant efflux pumps similar to what is observed in drug-resistant tumors. Our results highlight the potential of these tunable and well-defined gels in drug screening assays.

Human Pluripotent Stem Cell-Derived TSC2-Haploinsufficient Smooth Muscle Cells Recapitulate Features of Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here, we generated a robust human cell model of LAM by reprogramming TSC2 mutation-bearing fibroblasts from a patient with both tuberous sclerosis complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSC), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2+/- mutation and recapitulated the molecular and functional characteristics of pulmonary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploinsufficiency at the TSC2 locus contributes to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM that can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM. Cancer Res; 77(20); 5491-502. ©2017 AACR.

Engineering Cellular Microenvironments with Photo- and Enzymatically Responsive Hydrogels: Toward Biomimetic 3D Cell Culture Models.

Conventional cell culture techniques using 2D polystyrene or glass have provided great insight into key biochemical mechanisms responsible for cellular events such as cell proliferation, differentiation, and cell-cell interactions. However, the physical and chemical properties of 2D culture in vitro are dramatically different than those found in the native cellular microenvironment in vivo. Cells grown on 2D substrates differ significantly from those grown in vivo, and this explains, in part, why many promising drug candidates discovered through in vitro drug screening assays fail when they are translated to in vivo animal or human models. To overcome this obstacle, 3D cell culture using biomimetic hydrogels has emerged as an alternative strategy to recapitulate native cell growth in vitro. Hydrogels, which are water-swollen polymers, can be synthetic or naturally derived. Many methods have been developed to control the physical and chemical properties of the hydrogels to match those found in specific tissues. Compared to 2D culture, cells cultured in 3D gels with the appropriate physicochemical cues can behave more like they naturally do in vivo. While conventional hydrogels involve modifications to the bulk material to mimic the static aspects of the cellular microenvironment, recent progress has focused on using more dynamic hydrogels, the chemical and physical properties of which can be altered with external stimuli to better mimic the dynamics of the native cellular microenvironment found in vivo. In this Account, we describe our progress in designing stimuli-responsive, optically transparent hydrogels that can be used as biomimetic extracellular matrices (ECMs) to study cell differentiation and migration in the context of modeling the nervous system and cancer. Specifically, we developed photosensitive agarose and hyaluronic acid hydrogels that are activated by single or two-photon irradiation for biomolecule immobilization at specific volumes within the 3D hydrogel. By controlling the spatial location of protein immobilization, we created 3D patterns and protein concentration gradients within these gels. We used the latter to study the effect of VEGF-165 concentration gradients on the interactions between endothelial cells and retinal stem cells. Hyaluronic acid (HA) is particularly compelling as it is naturally found in the ECM of many tissues and the tumor microenvironment. We used Diels-Alder click chemistry and cryogelation to alter the chemical and physical properties of these hydrogels. We also designed HA hydrogels to study the invasion of breast cancer cells. HA gels were chemically cross-linked with matrix metalloproteinase (MMP)-degradable peptides that degrade in the presence of cancer cell-secreted MMPs, thus allowing cells to remodel their local microenvironment and invade into HA/MMP-degradable gels.

Combination of a peptide-modified gellan gum hydrogel with cell therapy in a lumbar spinal cord injury animal model.

Spinal Cord Injury (SCI) is a highly incapacitating condition for which there is still no cure. Current clinical approaches are mainly based on palliative care, so there is a need to find possible treatments to SCI. Cellular transplantation is regarded with great expectation due to the therapeutic potential of cells such as Adipose tissue-derived Stromal/Stem Cells (ASCs) or Olfactory Ensheathing Cells (OECs). Both are accessible sources and present positive paracrine and cell-to-cell interactions, previously reported by our group. Additionally, biomaterials such as hydrogels have been applied in SCI repair with promising results. We propose to combine a GRGDS-modified gellan gum hydrogel with ASCs and OECs in order to promote SCI regeneration. In vitro, ASCs and OECs could be co-cultured within GG-GRGDS hydrogels inducing a more robust neurite outgrowth when compared to controls. In vivo experiments in a hemisection SCI rat model revealed that the administration of ASCs and OECs encapsulated in a GG-GRGDS hydrogel led to significant motor improvements when compared to both control (SCI) and hydrogel alone (GG-GRGDS) groups. This was accompanied by a decreased infiltration of inflammatory cells and astrocytes, and by an increased intensity of neurofilament. These results suggest evident gains induced by the encapsulation of ASCs and OECs in GG-GRGDS based hydrogels.

6-Bromo-7-hydroxy-3-methylcoumarin (mBhc) is an efficient multi-photon labile protecting group for thiol caging and three-dimensional chemical patterning.

The photochemical release of chemical reagents and bioactive molecules provides a useful tool for spatio-temporal control of biological processes. However, achieving this goal requires the development of highly efficient one- and two-photon sensitive photo-cleavable protecting groups. Thiol-containing compounds play critical roles in biological systems and bioengineering applications. While potentially useful for sulfhydryl protection, the 6-bromo-7-hydroxy coumarin-4-ylmethyl (Bhc) group can undergo an undesired photoisomerization reaction upon irradiation that limits its uncaging efficiency. To address this issue, here we describe the development of 6-bromo-7-hydroxy-3-methylcoumarin-4-ylmethyl (mBhc) as an improved group for thiol-protection. One- and two-photon photolysis reactions demonstrate that a peptide containing a mBhc-caged thiol undergoes clean and efficient photo-cleavage upon irradiation without detectable photoisomer production. To test its utility for biological studies, a K-Ras-derived peptide containing an mBhc-protected thiol was prepared by solid phase peptide synthesis using Fmoc-Cys(mBhc)-OH for the introduction of the caged thiol. Irradiation of that peptide using either UV or near IR light in presence of protein farnesyltransferase (PFTase), resulted in generation of the free peptide which was then recognized by the enzyme and became farnesylated. To show the utility of this caging group in biomaterial applications, we covalently modified hydrogels with mBhc-protected cysteamine. Using multi-photon confocal microscopy, highly defined volumes of free thiols were generated inside the hydrogels and visualized via reaction with a sulfhydryl-reactive fluorophore. The simple synthesis of mBhc and its efficient removal by one- and two-photon processes make it an attractive protecting group for thiol caging in a variety of applications.

Innovative use of the taxol binding peptide overcomes key challenges of stable and high drug loading in polymeric nanomicelles.

Despite widespread clinical use, delivery of taxane chemotherapeutics remains a challenge due to poor solubility and lack of selectively. Polymeric nanomicelle strategies have been pursued to overcome these issues; however current formulations are often limited by low drug loading and poor serum stability. To achieve a drug delivery system that addresses these issues, poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-g-poly(ethylene glycol) was covalently modified with the taxol binding peptide ­ a peptide from the ß-tubulin-taxane binding site. This modification resulted in drug loadings five times higher than unmodified polymers, which is significantly higher than typical hydrophobic modifications, including with benzyl and docetaxel functionalization. Unlike many formulations with high drug loading, these nanomicelles were stable in serum for up to 24 h and maintained docetaxel cytotoxicity. By incorporating the taxane binding peptide into the polymer chemistry, a new twist was applied to an old problem, which is broadly applicable to other polymeric micelle systems and drug-peptide combinations in general.

Regenerative therapies for central nervous system diseases: a biomaterials approach.

The central nervous system (CNS) has a limited capacity to spontaneously regenerate following traumatic injury or disease, requiring innovative strategies to promote tissue and functional repair. Tissue regeneration strategies, such as cell and/or drug delivery, have demonstrated promising results in experimental animal models, but have been difficult to translate clinically. The efficacy of cell therapy, which involves stem cell transplantation into the CNS to replace damaged tissue, has been limited due to low cell survival and integration upon transplantation, while delivery of therapeutic molecules to the CNS using conventional methods, such as oral and intravenous administration, have been limited by diffusion across the blood-brain/spinal cord-barrier. The use of biomaterials to promote graft survival and integration as well as localized and sustained delivery of biologics to CNS injury sites is actively being pursued. This review will highlight recent advances using biomaterials as cell- and drug-delivery vehicles for CNS repair.

Modulation of bone marrow mesenchymal stem cell secretome by ECM-like hydrogels.

It has been demonstrated that bone marrow mesenchymal stem cell (BM-MSCs) transplantation has beneficial effects on several central nervous system (CNS) debilitating conditions. Growing evidence indicate that trophic factors secreted by these cells are the key mechanism by which they are acting. These cells are frequently used in combination with 3D artificial matrices, for instance hydrogels, in tissue engineering-based approaches. However, so far, no study has been reported on the influence of such matrices, namely the presence or absence of extracellular matrix motifs, on BM-MSCs secretome and its effects in neuronal cell populations. In this sense, we herein studied the impact of a hydrogel, gellan gum, on the behavior and secretome of BM-MSCs, both in its commercial available form (commonly used in tissue engineering) and in a fibronectin peptide-modified form. The results showed that in the presence of a peptide in the gellan gum hydrogel, BM-MSCs presented higher proliferation and metabolic activity than in the regular hydrogel. Moreover, the typical spindle shape morphology of BM-MSCs was only observed in the modified hydrogel. The effects of the secretome of BM-MSCs were also affected by the chemical nature of the extracellular matrix. BM-MSCs cultured in the modified hydrogel were able to secrete factors that induced higher metabolic viabilities and neuronal cell densities, when compared to those of the unmodified hydrogel. Thus adding a peptide sequence to the gellan gum had a significant effect on the morphology, activity, proliferation and secretome of BM-MSCs. These results highlight the importance of mimicking the extracellular matrix when BM-MSCs are cultured in hydrogels for CNS applications.

Repair of the injured spinal cord by transplantation of neural stem cells in a hyaluronan-based hydrogel.

Traumatic injury to the spinal cord causes cell death, demyelination, axonal degeneration, and cavitation resulting in functional motor and sensory loss. Stem cell therapy is a promising approach for spinal cord injury (SCI); however, this strategy is currently limited by the poor survival and uncontrolled differentiation of transplanted stem cells. In an attempt to achieve greater survival and integration with the host tissue, we examined the survival and efficacy of adult brain-derived neural stem/progenitor cells (NSPCs) injected within a hydrogel blend of hyaluronan and methyl cellulose (HAMC) into a subacute, clinically relevant model of rat SCI. Prior to use, HAMC was covalently modified with recombinant rat platelet-derived growth factor-A (rPDGF-A) to promote oligodendrocytic differentiation. SCI rats transplanted with NSPCs in HAMC-rPDGF-A showed improved behavioral recovery compared to rats transplanted with NSPCs in media. Rats with NSPC/HAMC-rPDGF-A transplants had a significant reduction in cavitation, improved graft survival, increased oligodendrocytic differentiation, and sparing of perilesional host oligodendrocytes and neurons. These data suggest that HAMC-rPDGF-A is a promising vehicle for cell delivery to the injured spinal cord.

The effects of peptide modified gellan gum and olfactory ensheathing glia cells on neural stem/progenitor cell fate.

The regenerative capacity of injured adult central nervous system (CNS) tissue is very limited. Specifically, traumatic spinal cord injury (SCI) leads to permanent loss of motor and sensory functions below the site of injury, as well as other detrimental complications. A potential regenerative strategy is stem cell transplantation; however, cell survival is typically less than 1%. To improve cell survival, stem cells can be delivered in a biomaterial matrix that provides an environment conducive to survival after transplantation. One major challenge in this approach is to define the biomaterial and cell strategies in vitro. To this end, we investigated both peptide-modification of gellan gum and olfactory ensheathing glia (OEG) on neural stem/progenitor cell (NSPC) fate. To enhance cell adhesion, the gellan gum (GG) was modified using Diels-Alder click chemistry with a fibronectin-derived synthetic peptide (GRGDS). Amino acid analysis demonstrated that approximately 300 nmol of GRGDS was immobilized to each mg of GG. The GG-GRGDS had a profound effect on NSPC morphology and proliferation, distinct from that of NSPCs in GG alone, demonstrating the importance of GRGDS for cell-GG interaction. To further enhance NSPC survival and outgrowth, they were cultured with OEG. Here NSPCs interacted extensively with OEG, demonstrating significantly greater survival and proliferation relative to monocultures of NSPCs. These results suggest that this co-culture strategy of NSPCs with OEG may have therapeutic benefit for SCI repair.

Solution conformation of C-linked antifreeze glycoprotein analogues and modulation of ice recrystallization.

Antifreeze glycoproteins (AFGPs) are a unique class of proteins that are found in many organisms inhabiting subzero environments and ensure their survival by preventing ice growth in vivo. During the last several years, our laboratory has synthesized functional C-linked AFGP analogues (3 and 5) that possess custom-tailored antifreeze activity suitable for medical, commercial, and industrial applications. These compounds are potent inhibitors of ice recrystallization and do not exhibit thermal hysteresis. The current study explores how changes in the length of the amide-containing side chain between the carbohydrate moiety and the polypeptide backbone in 5 influences ice recrystallization inhibition (IRI) activity. Analogue 5 (n = 3, where n is the number of carbons in the side chain) was a potent inhibitor of ice recrystallization, while 4, 6, and 7 (n = 4, 2, and 1, respectively) exhibited no IRI activity. The solution conformation of the polypeptide backbone in C-linked AFGP analogues 4-7 was examined using circular dichroism (CD) spectroscopy. The results suggested that all of the analogues exhibit a random coil conformation in solution and that the dramatic increase in IRI activity observed with 5 is not due to a change in long-range solution conformation. Variable-temperature (1)H NMR studies on truncated analogues 26-28 failed to elucidate the presence of persistent intramolecular bonds between the amide in the side chain and the peptide backbone. Molecular dynamics simulations performed on these analogues also failed to show persistent intramolecular hydrogen bonds. However, the simulations did indicate that the side chain of IRI-active analogue 26 (n = 3) adopts a unique short-range solution conformation in which it is folded back onto the peptide backbone, orienting the more hydrophilic face of the carbohydrate moiety away from the bulk solvent. In contrast, the solution conformation of IRI-inactive analogues 25, 27, and 28 had fully extended side chains, with the carbohydrate moiety being exposed to bulk solvent. These results illustrate how subtle changes in conformation and carbohydrate orientation dramatically influence IRI activity in C-linked AFGP analogues.