Key Points mRNA transfection is an effective tool to simultaneously engineer MSCs for enhanced homing and improved secretome. MSCs can be systemically targeted to sites of inflammation to achieve therapeutically relevant concentrations of biological agents.

Traumatic spinal cord injury (SCI) results in an immediate loss of motor and sensory function below the injury site and is associated with a poor prognosis. The inhibitory environment that develops in response to the injury is mainly due to local expression of inhibitory factors, scarring and the formation of cystic cavitations, all of which limit the regenerative capacity of endogenous or transplanted cells. Strategies that demonstrate promising results induce a change in the microenvironment at- and around the lesion site to promote endogenous cell repair, including axonal regeneration or the integration of transplanted cells. To date, many of these strategies target only a single aspect of SCI; however, the multifaceted nature of SCI suggests that combinatorial strategies will likely be more effective. Biomaterials are a key component of combinatorial strategies, as they have the potential to deliver drugs locally over a prolonged period of time and aid in cell survival, integration and differentiation. Here we summarize the advantages and limitations of widely used strategies to promote recovery after injury and highlight recent research where biomaterials aided combinatorial strategies to overcome some of the barriers of spinal cord regeneration.

A big challenge in cell culture is the non-natural environment in which cells are routinely screened, making in vivo phenomena, such as cell invasion, diffi cult to understand and predict. To study cancer cell invasion, extracellular matrix (ECM) analogs with decoupled mechanical and chemical properties are required. Hyaluronic acid (HA)-based hydrogels crosslinked with matrix-metalloproteinase (MMP)-cleavable peptides are developed to study MDA-MB-231 breast cancer cell invasion. Hydrogels are synthesized by reacting furan-modifi ed HA with bismaleimide peptide crosslinkers in a Diels-Alder click reaction. This new hydrogel takes advantage of the biomimetic properties of HA, which is overexpressed in breast cancer, and eliminates the use of nonadhesive crosslinkers, such as poly(ethylene glycol) (PEG). The crosslink (mechanical) and ligand (chemical) densities are varied independently to evaluate the effects of each parameter on cell migration. Increased crosslink density correlates with decreased MDA-MB-231 cell invasion whereas incorporation of MMP-cleavable sequences within the peptide crosslinker enhances invasion. Increasing the ligand density of pendant GRGDS groups induces cell proliferation, but has no signifi cant impact on invasion. By independently tuning the mechanical and chemical environment of ECM mimetic hydrogels, a platform is provided that recapitulates variable tissue properties and elucidates the role of the microenvironment in cancer cell invasion.

The lack of tissue regeneration after traumatic spinal cord injury in animal models is largely attributed to the local inhibitory microenvironment. To overcome this inhibitory environment while promoting tissue regeneration, we investigated the combined delivery of chondroitinase ABC (chABC) with human induced pluripotent stem cell-derived neuroepithelial stem cells (NESCs). ChABC was delivered to the injured spinal cord at the site of injury by affinity release from a crosslinked methylcellulose (MC) hydrogel by injection into the intrathecal space. NESCs were distributed in a hydrogel comprised of hyaluronan and MC and injected into the spinal cord tissue both rostral and caudal to the site of injury. Cell transplantation led to reduced cavity formation, but did not improve motor function. While few surviving cells were found 2 weeks post injury, the majority of live cells were neurons, with only few astrocytes, oligodendrocytes, and progenitor cells. At 9 weeks post injury, there were more progenitor cells and a more even distribution of cell types compared to those at 2 weeks post injury, suggesting preferential survival and differentiation. Interestingly, animals that received cells and chABC had more neurons than animals that received cells alone, suggesting that chABC influenced the injury environment such that neuronal differentiation or survival was favoured.

Stem cell transplantation is a promising strategy for brain tissue regeneration; yet, despite some success, cell survival following transplantation remains low. In this study, we demonstrate that cell viability is enhanced by control over maturation of neuronal precursor cells, which are delivered in an injectable blend of hyaluronan and methylcellulose. We selected three subpopulations of human neuronal precursor cells derived from a cortically specified neuroepithelial stem cell (cNESC) population based on differences in expression of multipotent and neuron-specific proteins: early-, mid-, and late-differentiated neurons. These cells were transplanted into an endothelin-1 stroke-injured rat brain and their survival and fate were investigated 1 week later. Significantly, more cells were found in the brain after transplanting early- or mid- differentiated cNESCs compared to the late-differentiated population. The mid-differentiated population also had significantly more β-III tubulin-positive cells than either the early- or late-differentiated populations. These results suggest that maturity has a significant impact on cell survival following transplantation and cells with an intermediate maturity differentiate to neurons.

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Organoids are becoming widespread in drug-screening technologies but have been used sparingly for cell therapy as current approaches for producing selforganized cell clusters lack scalability or reproducibility in size and cellular organization. We introduce a method of using hydrogels as sacrificial scaffolds, which allow cells to form self-organized clusters followed by gentle release, resulting in highly reproducible multicellular structures on a large scale. We demonstrated this strategy for endothelial cells and mesenchymal stem cells to self-organize into blood-vessel units, which were injected into mice, and rapidly formed perfusing vasculature. Moreover, in a mouse model of peripheral artery disease, intramuscular injections of blood-vessel units resulted in rapid restoration of vascular perfusion within seven days. As cell therapy transforms into a new class of therapeutic modality, this simple method-by making use of the dynamic nature of hydrogels-could offer high yields of self-organized multicellular aggregates with reproducible sizes and cellular architectures.

AbstractOrganoids, by promoting self-organization of cells into native-like structures, are becoming widespread in drug-screening technologies, but have so far been used sparingly for cell therapy as current approaches for producing self-organized cell clusters lack scalability or reproducibility in size and cellular organization. We introduce a method of using hydrogels as sacrificial scaffolds, which allow cells to form self-organized clusters followed by gentle release, resulting in highly reproducible multicellular structures on a large scale. We demonstrated this strategy for endothelial cells and mesenchymal stem cells to self-organize into blood-vessel units, which were injected into mice using hypodermic needles, and observed in real time to rapidly form perfusing vasculature. As cell therapy transforms into a new class of therapeutic modality, this simple method – by making use of the dynamic nature of hydrogels – could offer high yields of self-organized multicellular aggregates with reproducible sizes and cellular architectures.