Injectable Supramolecular Hydrogels to Promote Oligodendrocyte Progenitor Cell Survival and Remyelination in the Central Nervous System

Introduction: In demyelinating diseases such as Multiple Sclerosis (MS), the inflammatory tissue environment in chronic lesions prevents efficient progenitor recruitment, leading to failure in oligodendrocyte regeneration and remyelination. Stem/progenitor cell transplantation of highly myelinogenic fetal or iPSC-derived human oligodendrocyte progenitor cells (hOPCs) into the demyelinating disease remains an attractive means to achieve therapeutic remyelination. While significant advances have been made in the preparation of hOPCs capable of mediating widespread myelination, poor survival of donor cells and maintenance of OPC fate are major barriers to the successful translation of this approach to clinical treatment. Indeed, current cell injection approaches are associated with substantial cell death with viability as low as 1 – 32% post-transplantation, primarily due to cell membrane damage from shear stress. As a result, about 20% of transplanted cells survive after one week, and only 5% of cells remain viable one-month post-injection. Some speculated that the extensional flow that is generated as cells move from the syringe to the much smaller diameter needle, results in substantial deformation and stretching of cells, reducing cell viability. Importantly, extensive cell death exacerbates local inflammation, increasing the likelihood of graft failure. Driven by the above considerations, shear-thinning hydrogels (STHs) have emerged as promising candidates for injection of Schwann cells, oligodendrocytes, or their stem-cell precursors with minimal death. Here, we report the development of an innovative STH with tunable bioactivity and viscoelastic properties to enhance the survival of transplanted hOPCs into a mouse model of hypomyelinating disease.

Materials and

Methods: The building units employed for the development of STHs are hyaluronic acid-thiol (HA-SH), heparin-thiol (Hep-SH), beta-cyclodextrin maleimide (βCD-Mal), and 8-arm polyethylene glycol adamantane (PEG-Ad). The development of STHs involves two steps. The first step is the engineering of multi-arm maleimide-terminated supramolecular polymer (SMP-Mal; Fig. 1Ai) through host-guest complexation between 8-arm PEG-Ad and mono-functionalized βCD-Mal at 1:1 βCD/Ad stoichiometric ratio. The second step relies on Michael addition reaction between the 8-arm SMP-Mal and HA-SH (Fig. 1Aii) at pH 7.4 to form the 3D network (Fig. 1Aiii). The hydrogel compositions were: STH-1 (HA2wt%SMP3.3wt%, stiff), STH-2 (HA1.3wt%SMP6.6wt%, intermediate stiffness), and STH-3 (HA0.66wt%SMP10wt%, soft). For immobilization of pro-survival cues, Hep-SH (Fig. 1Aii) was pre-mixed with heparin-binding bidomain peptides or platelet-derived growth factor (PDGF)-AA that were bound to HA-SH prior to crosslinking with SMP-Mal. We assessed cell death by passing hOPCs through a pulled glass needle (internal diameter: 100–200 µm, equivalent to 28–32 G needle) in the absence or presence of STH at an injection rate of 0.1 µl/ 5 sec using a live-dead assay. Then, we investigated hOPC differentiation within each hydrogel in 3D, in the absence of mitogens for 4 days. The in vivo survival of hOPCs after transplantation was evaluated in an immunodeficient NSG mouse (NOD scid gamma mouse, lacking mature T, B, and NK cells). The myelination potential of STH-transplanted hOPC cells was assessed in a shiverer/rag2 mouse, a model of congenital hypomyelinating disease that has become the gold standard for the assessment of myelinating cell preparations.

Results, Conclusions, and Discussions: We leveraged βCD-adamantane assembly to engineer state-of-art shear-thinning hydrogels (STHs) with tunable viscoelasticity and bioactivity for minimally invasive delivery of hOPCs to the corpus callosum of a shiverer/rag2 mouse (Fig. B), a model of congenital hypomyelinating disease. The attractive features of this design include: (1) the hydrogel capacity to flow under shear, thereby protecting hOPCs from mechanical damage; (2) the ability to conjugate pro-survival signals such as fibronectin and laminin-derived peptides under mild physiological conditions through Michael addition at neutral pH; (3) as well as heparin-binding GFs, such as PDGF, which may be necessary for hOPCs survival and proliferation; and (4) the ability to tune their stiffness to match that of the host tissue. The chemical structures of the STH components were confirmed by 1H NMR and ESI MS analysis, while the viscoelasticity and shear-thinning characteristic were demonstrated by rheological measurements. Assessment of the in vivo survivability of hOPCs post-transplantation in NSG mouse model showed significantly (p < 0.0001) higher number of hNA+ hOPCs as compared to the control (HBSS) group. Immobilization of PDGF-AA and HBD-RGD5 (STH-2+PDGF-AA+HBD-RGD5 group) further increased the number of hNA+ cells (p < 0.0001) in the mouse brain compared to STH-2 alone (Fig. C). Following hOPC-STH-2 transplantation in shiverer/rag2 mouse, immunostaining for MBP identified widespread myelination that was primarily restricted to the corpus callosum (Fig. D). Mature myelin was observed in both control and hydrogel groups across a region spanning approximately 650  160 µm along the rostrocaudal axis from the site of injection. Quantification of transplanted cell-mediated myelination via immunofluorescence for myelin basic protein (MBP) indicated significant enhancement following delivery of hOPCs in STH compared to the HBSS control group. In the STH-2+PDGF-AA+HBD-RGD5 group, we observed increased MBP+ area towards the rostral (6-10%, p< 0.005) and caudal (5-7%, p< 0.05) directions from the point of injection. Consistent with improved survival afforded by administration with STH, the total number of engrafted hNA+ hOPCs was significantly higher when injected with STH-2 complexed with PDGF-AA and HBD-RGD5; p< 0.005, one tailed unpaired t-test). Our results demonstrate the potential of STHs loaded with biological cues to improve cell therapies for the treatment of devastating myelopathies.

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