P14 - Polysialic Acid–Functionalized Microporous Annealing Particle Scaffolds Modulate Neural Progenitor Cell Differentiation and Morphology

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Hannah P. Newman

Student
Duke University
Chevy Chase, Maryland, United States

Co-Author(s)

SC

Sanchuan Che

Master's Student
Duke University, United States
YO

Yunxin Ouyang

PhD Candidate
Duke University, United States

Primary Investigator(s)

TS

Tatiana Segura

Professor of Biomedical Engineering
Duke University, United States

Introduction: : Ischemic stroke remains a major cause of long-term disability due to the brain's inability to naturally regenerate necrotic tissue, limiting current treatments to rehabilitation [1]. This study investigates a novel therapy involving injectable hyaluronic acid (HA)-based microporous annealing particles (MAP) functionalized with polysialic acid (PSA), a homopolymer of sialic acid residues. PSA, naturally associated with neural cell adhesion molecules (NCAM), is crucial in neurodevelopment, promoting cell motility, axonal growth, neural progenitor cell (NPC) differentiation, and synaptic plasticity [2,3,4]. After a stroke, NPC migration towards infarct areas increases naturally, yet differentiation into functional cell types remains limited.
We evaluated the impact of PSA-functionalized MAP hydrogels on NPC morphology and differentiation using an in vitro 3D scaffold system. NPCs were seeded in PSA-MAP scaffolds with varying PSA concentrations and cultured over different time points. Results showed PSA concentration and culture duration significantly influenced NPC morphology and differentiation state. These findings underscore PSA's potential as a bioactive component for enhancing neural regeneration in MAP-based hydrogels.

Materials and

Methods: : HA-µgels were synthesized using HA-norbornene, di-thiol MMP-sensitive crosslinkers, and 0.5 mM RGD via flow-focusing microfluidic devices, then functionalized with varying PSA concentrations: low (0.625 mM), medium (2.5 mM), and high (20 mM). These µgels were combined with NPCs and HA-tetrazine annealing polymer at 12k cells/µL density, loaded into PDMS molds bonded to glass coverslips, and incubated on ice. After a 30-minute annealing incubation, ~80 µL of N5 media was added per well. Each condition had three biological replicates and two technical replicates, with experiments conducted over 4 and 7 days. Media changes occurred every two days. Post-culture, cells were fixed and stained for Tuj1 (neuronal differentiation), Sox2 (stemness), DAPI (nuclei), and Phalloidin (actin cytoskeleton), then imaged at 20x magnification via confocal microscopy. IMARIS software rendered 3D surfaces around Tuj1, Sox2, DAPI, and Phalloidin fluorescence signals, and co-localization was quantified by determining the percentage volume overlap between Tuj1 or Sox2 and Phalloidin or DAPI, respectively.

Results, Conclusions, and Discussions:: In this study, PSA-MAP scaffolds influenced NPC behavior in a manner dependent on PSA functionalization level and culture duration. On Day 4, NPCs exhibited co-expression of stemness marker Sox2 and early neuronal marker Tuj1 across all PSA concentrations. Notably, increased PSA functionalization proportionally enhanced Tuj1 co-localization with Phalloidin, indicating a higher initiation of neuronal differentiation. However, Sox2 expression remained moderately elevated even at high PSA functionalization, suggesting NPCs existed in a transitional developmental state, maintaining stem characteristics while initiating neuronal commitment.
By Day 7, a distinct temporal shift was evident. PSA-functionalized MAP scaffolds induced decreased Sox2 expression and increased Tuj1 expression. Specifically, Sox2 co-localization with DAPI decreased, while Tuj1 co-localization with Phalloidin increased, both trends correlating positively with higher PSA functionalization levels. These observations indicate PSA’s progressive promotion of neuronal differentiation and reduction in NPC stemness over extended culture periods.
Mechanistically, PSA's temporal effects likely involve its inhibition of neural cell adhesion molecule (NCAM)-mediated cell adhesion during early culture stages. This inhibition initially sustains Sox2 expression and stemness by reducing downstream differentiation signaling pathways. As cultures mature, PSA transitions to facilitating neurite outgrowth and axonal extension, thus actively supporting NPC differentiation and maturation. The temporal regulation may partially reflect gradual PSA degradation; however, further investigation is required to confirm this functional switch.
Furthermore, given PSA's capacity to modulate cell adhesion, future in vivo studies should explore its potential influence on NPC migration and recruitment within regenerative contexts. Clarifying these dynamics provides critical insights into designing PSA-functionalized MAP scaffolds optimized for neural regeneration and rehabilitation, particularly following ischemic stroke.

Acknowledgements and/or References (Optional): : 1. Feigin, V. L., Brainin, M., Norrving, B., Martins, S., Pandian, J. D., Lindsay, P., Fredin Grupper, M. M., & Rautalin, I. (2024). World Stroke Organization (WSO): Global Stroke Fact Sheet 2025. International Journal of Stroke, 20(2). https://doi.org/10.1177/17474930241308142
2. Rutishauser, U. (2008). Polysialic acid in the plasticity of the developing and adult vertebrate nervous system. Nature Reviews Neuroscience, 9(1), 26–35. https://doi.org/10.1038/nrn2285
3. Petridis, A. K., El Maarouf, A., & Rutishauser, U. (2004). Polysialic acid regulates cell contact‐dependent neuronal differentiation of progenitor cells from the subventricular zone. Developmental Dynamics, 230(4), 675–684. https://doi.org/10.1002/dvdy.20094
4. Denninger, J. K., Miller, L. N., Walters, A. E., Manal Hosawi, Sebring, G., Rieskamp, J. D., Ding, T., Rindani, R., Chen, K. S., Sakthi Senthilvelan, Volk, A., Zhao, F., Askwith, C., & Kirby, E. D. (2023). Neural stem and progenitor cells support and protect adult hippocampal function via vascular endothelial growth factor secretion. BioRxiv (Cold Spring Harbor Laboratory). https://doi.org/10.1101/2023.04.24.537801