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Stem cells with the potential for neural differentiation are a promising therapeutic avenue both for treating neurological disease and as a system to advance our fundamental understanding of disease biology in vitro. Precisely controlled extracellular environments that recapitulate critical aspects of embryonic development or the adult stem cell niche are necessary to ensure effective differentiation into the desired cell type. Biomaterials in particular have enabled new avenues for directing stem cell differentiation through the precise presentation of biochemical and biophysical cues. Furthermore, as translation of stem cell technologies necessitates the need for scalable cultures, biomaterials will continue to be valuable tools for guiding stem cell behavior in scalable, complex, three-dimensional cultures. In this review, we highlight the critical signals that guide neurogenesis and how biomaterials can be used to control and direct the neural differentiation of pluripotent and adult stem cells. In addition, we discuss recent new technologies that are further advancing material-based regulation of stem cells. Finally, we highlight the current state of the field and how next-generation biomaterials can enable scalable stem cell culture for cell replacement therapies as well as emerging advanced tissue models for studying tissue morphogenesis and disease pathology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cell replacement therapies have broad biomedical potential; however, low cell survival and poor functional integration post-transplantation are major hurdles that hamper clinical benefit. For example, following striatal transplantation of midbrain dopaminergic (mDA) neurons for the treatment of Parkinson's disease (PD), only 1-5% of the neurons typically survive in preclinical models and in clinical trials. In general, resource-intensive generation and implantation of larger numbers of cells are used to compensate for the low post-transplantation cell-survival. Poor graft survival is often attributed to adverse biochemical, mechanical, and/or immunological stress that cells experience during and after implantation. To address these challenges, we developed a functionalized hyaluronic acid (HA)-based hydrogel for in vitro maturation and central nervous system (CNS) transplantation of human pluripotent stem cell (hPSC)-derived neural progenitors. Specifically, we functionalized the HA hydrogel with RGD and heparin (hep) via click-chemistry and tailored its stiffness to encourage neuronal maturation, survival, and long-term maintenance of the desired mDA phenotype. Importantly, ∼5 times more hydrogel-encapsulated mDA neurons survived after transplantation in the rat striatum, compared to unencapsulated neurons harvested from commonly used 2D surfaces. This engineered biomaterial may therefore increase the therapeutic potential and reduce the manufacturing burden for successful neuronal implantation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Anti-VEGF drugs that are used in conjunction with laser ablation to treat patients with diabetic retinopathy suffer from short half-lives in the vitreous of the eye resulting in the need for frequent intravitreal injections. To improve the intravitreal half-life of anti-VEGF drugs, such as the VEGF decoy receptor sFlt-1, we developed multivalent bioconjugates of sFlt-1 grafted to linear hyaluronic acid (HyA) chains termed mvsFlt. Using size exclusion chromatography with multiangle light scattering (SEC-MALS), SDS-PAGE, and dynamic light scattering (DLS), we characterized the mvsFlt with a focus on the molecular weight contribution of protein and HyA components to the overall bioconjugate size. We found that mvsFlt activity was independent of HyA conjugation using a sandwich ELISA and in vitro angiogenesis assays including cell survival, migration and tube formation. Using an in vitro model of the vitreous with crosslinked HyA gels, we demonstrated that larger mvsFlt bioconjugates showed slowed release and mobility in these hydrogels compared to low molecular weight mvsFlt and unconjugated sFlt-1. Finally, we used an enzyme specific to sFlt-1 to show that conjugation to HyA shields sFlt-1 from protein degradation. Taken together, our findings suggest that mvsFlt bioconjugates retain VEGF binding affinity, shield sFlt-1 from enzymatic degradation, and their movement in hydrogel networks (in vitro model of the vitreous) is controlled by both bioconjugate size and hydrogel network mesh size. These results suggest that a strategy of multivalent conjugation could substantially improve drug residence time in the eye and potentially improve therapeutics for the treatment of diabetic retinopathy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We have generated a bioinspired tunable system of hyaluronic acid (HyA)-based hydrogels for Matrix-Assisted Cell Transplantation (MACT). With this material, we have independently evaluated matrix parameters such as adhesion peptide density, mechanical properties, and growth factor sequestering capacity, to engineer an environment that imbues donor cells with a milieu that promotes survival and engraftment with host tissues after transplantation. Using a versatile population of Sca-1(+)/CD45(-) cardiac progenitor cells (CPCs), we demonstrated that the addition of heparin in the HyA hydrogels was necessary to coordinate the presentation of TGFβ1 and to support the trophic functions of the CPCs via endothelial cell differentiation and vascular like tubular network formation. Presentation of exogenous TGFβ1 by binding with heparin improved differentiated CPC function by sequestering additional endogenously-produced angiogenic factors. Finally, we demonstrated that TGFβ1 and heparin-containing HyA hydrogels can promote CPC survival when implanted subcutaneously into murine hind-limbs and encouraged their participation in the ensuing neovascular response, which included blood vessels that had anastomosed with the host's blood vessels., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering., (2010 Elsevier Ltd. All rights reserved.)

Peptide-functionalized materials show promise in controlling stem cell behavior by mimicking cell-matrix interactions. Supported lipid bilayers are an excellent platform for displaying peptides due to their ease of fabrication and low non-specific interactions with cells. In this paper, we report on the behavior of adult hippocampal neural stem cells (NSCs) on phospholipid bilayers functionalized with different RGD-containing peptides: either GGGNGEPRGDTYRAY ('bsp-RGD(15)') or GRGDSP. Fluid supported bilayers were prepared on glass surfaces by adsorption and fusion of small lipid vesicles incorporating synthetic peptide amphiphiles. NSCs adhered to bilayers with either GRGDSP or bsp-RGD(15) peptide. After 5 days in culture, NSCs formed neurosphere-like aggregates on GRGDSP bilayers, whereas on bsp-RGD(15) bilayers a large fraction of single adhered cells were observed, comparable to monolayer growth seen on laminin controls. NSCs retained their ability to differentiate into neurons and astrocytes on both peptide surfaces. This work illustrates the utility of supported bilayers in displaying peptide ligands and demonstrates that RGD peptides may be useful in synthetic culture systems for stem cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

There has been an increasing interest in understanding how the mechanical properties of the microenvironment influence stem cell fate. We describe studies of the proliferation and differentiation of neural stem cells (NSCs) encapsulated within three-dimensional scaffolds--alginate hydrogels--whose elastic moduli were varied over two orders of magnitude. The rate of proliferation of neural stem cells decreased with increase in the modulus of the hydrogels. Moreover, we observed the greatest enhancement in expression of the neuronal marker beta-tubulin III within the softest hydrogels, which had an elastic modulus comparable to that of brain tissues. To our knowledge, this work represents the first demonstration of the influence of modulus on NSC differentiation in three-dimensional scaffolds. Three-dimensional scaffolds that control stem cell fate would be broadly useful for applications in regenerative medicine and tissue engineering.

We describe a method for creating alginate hydrogels with adjustable degradation rates that can be used as scaffolds for stem cells. Alginate hydrogels have been widely tested as three-dimensional constructs for cell culture, cell carriers for implantation, and in tissue regeneration applications; however, alginate hydrogel implants can take months to disappear from implantation sites because mammals do not produce endogenous alginases. By incorporating poly(lactide-co-glycolide) (PLGA) microspheres loaded with alginate lyase into alginate hydrogels, we demonstrate that alginate hydrogels can be enzymatically degraded in a controlled and tunable fashion. We demonstrate that neural progenitor cells (NPCs) can be cultured and expanded in vitro in this degradable alginate hydrogel system. Moreover, we observe a significant increase in the expansion rate of NPCs cultured in degrading alginate hydrogels versus NPCs cultured in standard, i.e. non-degrading, alginate hydrogels. Degradable alginate hydrogels encapsulating stem cells may be widely applied to develop novel therapies for tissue regeneration.