Poster BB9 - Viscoelasticity of Hyaluronic Acid Hydrogels Regulates Human Pluripotent Stem Cell-derived Spinal Cord Organoid Patterning and Vascularization

Introduction: Recently, it has been recognized that natural extracellular matrix (ECM) and tissues are viscoelastic but not pure elastic. Hence, how the viscoelastic matrix regulates cellular behaviors and stem cell patterning is critical for understanding stem cell-ECM mechano-transduction. The spinal cord is a complex neural organ and has important effects on locomotion, including neurotransmission of sensory input and motor output between the brain and body and coordination of central pattern creation.

Materials and

Methods: Here, based on methylacrylated (MA) hyaluronic acid (HA), this study fabricated different HA hydrogels for generation and vascularization of spinal cord organoids derived from human induced pluripotent stem cells. Four kinds of hydrogels were synthesized, including soft-elastic, soft-viscoelastic, stiff-elastic, and stiff-viscoelastic. The biocompatibility of the hydrogels was determined by cell morphology and viability assays. Meanwhile, by adding dopamine, an iron coordinated hydrogel was fabricated. In particular, the effects of viscoelasticity of the HA hydrogels on spinal cord organoid patterning was investigated.

Results, Conclusions, and Discussions: The morphogenesis, regional patterning, and vascularization of human spinal cord organoids (hSCOs) was influenced by different hydrogels. Dorsal marker expression was evaluated where, more viscoelastic hydrogels can promote dorsal marker expression, while softer hydrogels resulted in higher interneuron marker expression. This study also found differences when generating the blood-spinal cord barrier with the coculture of hSCOs and human blood vessel organoids. In addition, Yes-associated protein nuclear translocation was determined to reveal mechanism of cell-ECM mechano-transduction.

Conclusions: . The viscoelasticity of the hydrogels influenced the size and circularity. Then, by comparing the gene and protein expression of hSCOs with different hydrogels, the results reveal that the more viscoelastic hydrogels led to more dorsal marker expression and the stiff-elastic gels can promote interneuron marker expression. Furthermore, by analysis of the localization of YAP, this study found that the nuclei localization increased in the faster relaxation hydrogel groups. Lastly, by coculture of hSCOs and hBVOs, this study was able to create a fusion of the two organoids. The EC and tight junction makers were differentially promoted in different hydrogels when cultured with specific hSCOs, which enables the possibility of building in vitro blood-spinal cord barrier models. This research provides insights on viscoelastic behaviors of the hydrogels during human organoid patterning with ECM-mimicking in vitro microenvironment for applications in regenerative medicines.

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