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Injectable polypeptide hydrogels via methionine modification for neural stem cell delivery.

ABSTRACT: Injectable hydrogels with tunable physiochemical and biological properties are potential tools for improving neural stem/progenitor cell (NSPC) transplantation to treat central nervous system (CNS) injury and disease. Here, we developed injectable diblock copolypeptide hydrogels (DCH) for NSPC transplantation that contain hydrophilic segments of modified l-methionine (Met). Multiple Met-based DCH were fabricated by post-polymerization modification of Met to various functional derivatives, and incorporation of different amino acid comonomers into hydrophilic segments. Met-based DCH assembled into self-healing hydrogels with concentration and composition dependent mechanical properties. Mechanical properties of non-ionic Met-sulfoxide formulations (DCHMO) were stable across diverse aqueous media while cationic formulations showed salt ion dependent stiffness reduction. Murine NSPC survival in DCHMO was equivalent to that of standard culture conditions, and sulfoxide functionality imparted cell non-fouling character. Within serum rich environments in vitro, DCHMO was superior at preserving NSPC stemness and multipotency compared to cell adhesive materials. NSPC in DCHMO injected into uninjured forebrain remained local and, after 4 weeks, exhibited an immature astroglial phenotype that integrated with host neural tissue and acted as cellular substrates that supported growth of host-derived axons. These findings demonstrate that Met-based DCH are suitable vehicles for further study of NSPC transplantation in CNS injury and disease models.

SUBMITTER: Wollenberg AL

PROVIDER: S-EPMC6054810 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Injectable polypeptide hydrogels via methionine modification for neural stem cell delivery.

Wollenberg A L AL O'Shea T M TM Kim J H JH Czechanski A A Reinholdt L G LG Sofroniew M V MV Deming T J TJ

Biomaterials 20180405

Injectable hydrogels with tunable physiochemical and biological properties are potential tools for improving neural stem/progenitor cell (NSPC) transplantation to treat central nervous system (CNS) injury and disease. Here, we developed injectable diblock copolypeptide hydrogels (DCH) for NSPC transplantation that contain hydrophilic segments of modified l-methionine (Met). Multiple Met-based DCH were fabricated by post-polymerization modification of Met to various functional derivatives, and in ...[more]

PMID: 29657091

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Project description: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 (MITCHs) 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 20kDa and 40kDa star-shaped PEG variants were investigated, and all four PEG-peptide 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 20kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40kDa 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.

Project description:Biomaterial-based scaffolds are promising tools for controlled immunomodulation. They can be applied as three dimensional (3D) culture systems in vitro, whereas in vivo they may be used to dictate cellular localization and exert spatiotemporal control over cues presented to the immune system. As such, scaffolds can be exploited to enhance the efficacy of cancer immunotherapies such as adoptive T cell transfer, in which localization and persistence of tumor-specific T cells dictates treatment outcome. Biomimetic polyisocyanopeptide (PIC) hydrogels are polymeric scaffolds with beneficial characteristics as they display reversible thermally-induced gelation at temperatures above 16°C, which allows for their minimally invasive delivery via injection. Moreover, incorporation of azide-terminated monomers introduces functional handles that can be exploited to include immune cell-modulating cues. Here, we explore the potential of synthetic PIC hydrogels to promote the in vitro expansion and in vivo local delivery of pre-activated T cells. We found that PIC hydrogels support the survival and vigorous expansion of pre-stimulated T cells in vitro even at high cell densities, highlighting their potential as 3D culture systems for efficient expansion of T cells for their adoptive transfer. In particular, the reversible thermo-sensitive behavior of the PIC scaffolds favors straightforward recovery of cells. PIC hydrogels that were injected subcutaneously gelated instantly in vivo, after which a confined 3D structure was formed that remained localized for at least 4 weeks. Importantly, we noticed no signs of inflammation, indicating that PIC hydrogels are non-immunogenic. Cells co-delivered with PIC polymers were encapsulated within the scaffold in vivo. Cells egressed gradually from the PIC gel and migrated into distant organs. This confirms that PIC hydrogels can be used to locally deliver cells within a supportive environment. These results demonstrate that PIC hydrogels are highly promising for both the in vitro expansion and in vivo delivery of pre-activated T cells. Covalent attachment of biomolecules onto azide-functionalized PIC polymers provides the opportunity to steer the phenotype, survival or functional response of the adoptively transferred cells. As such, PIC hydrogels can be used as valuable tools to improve current adoptive T cell therapy strategies.

Project description:Rationale: Injectable matrices are highly desirable for stem cell delivery. Previous research has highlighted the benefit of scaffold macroporosity in enhancing stem cell survival and bone regeneration in vivo. However, there remains a lack of injectable and in situ crosslinkable macroporous matrices for stem cell delivery to achieve fast bone regeneration in immunocompetent animal model. The goal of this study is to develop an injectable gelatin-based μRB hydrogel supporting direct cell encapsulation that is available in clinics as macroporous matrices to enhance adipose-derived stromal cell (ASC) survival, engraftment and accelerate bone formation in craniofacial defect mouse. Methods: Injectable and in situ crosslinkable gelatin microribbon (μRB)-based macroporous hydrogels were developed by wet-spinning. Injectability was optimized by varying concentration of glutaraldehyde for intracrosslinking of μRB shape, and fibrinogen coating. The efficacy of injectable μRBs to support ASCs delivery and bone regeneration were further assessed in vivo using an immunocompetent mouse cranial defect model. ASCs survival was evaluated by bioluminescent imaging and bone regeneration was assessed by micro-CT. The degradation and biocompatibility were determined by histological analysis. Results: We first optimized injectability by varying concentration of glutaraldehyde used to fix gelatin μRBs. The injectable μRB formulation were subsequently coated with fibrinogen, which allows in situ crosslinking by thrombin. Fluorescence imaging and histology showed majority of μRBs degraded by the end of 3 weeks. Injectable μRBs supported comparable level of ASC proliferation and bone regeneration as implantable prefabricated μRB controls. Adding low dosage of BMP2 (100 ng per scaffold) with ASCs substantially accelerated the speed of mineralized bone regeneration, with 90% of the bone defect refilled by week 8. Immunostaining showed M1 (pro-inflammatory) macrophages were recruited to the defect at day 3, and was replaced by M2 (anti-inflammatory) macrophages by week 2. Adding μRBs or BMP2 did not alter macrophage response. Injectable µRBs supported vascularization, and BMP-2 further enhanced vascularization. Conclusions: Our results demonstrated that µRB-based scaffolds enhanced ASC survival and accelerated bone regeneration after injection into critical sized cranial defect mouse. Such injectable µRB-based scaffold can provide a versatile biomaterial for delivering various stem cell types and enhancing tissue regeneration.

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Injectable polypeptide hydrogels via methionine modification for neural stem cell delivery.

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Injectable polypeptide hydrogels via methionine modification for neural stem cell delivery.

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