Poster U17 - Metabolic Glycoengineering induces Neural Stem Cell Differentiation in a Thiol Analog-Dependent Manner

Introduction: Ischemic brain injuries often result in neuronal apoptosis, worsening neurological dysfunction. Our team explored improving outcomes for brain injuries from cardiac infarcts by transplanting exogenous neural stem cells (NSCs) at damage sites, which improved recovery and neuronal function in rat models. However, challenges like poor post-injection cell viability and slow differentiation persist. To address these, we utilized metabolic glycoengineering (MGE) with thiol-modified ManNAc analogs to enhance differentiation, adhesion, and recovery outcomes. Though results were promising, the biochemical basis of these effects remains unclear. We are now expanding our analysis to better understand the effects of chemical structure, aiming to optimize their therapeutic potential and facilitate clinical translation. This comprehensive approach will provide deeper insights into the mechanisms driving the observed benefits of MGE NSC-based therapies.

Materials and

Methods: hNSCs (ReNcell VM Human Neural Progenitor, Millipore) were cultured and passaged according to the manufacturer’s instructions. We synthesized, purified, and characterized thiol-modified analogs TGc (Ac5ManNTGc) and TProp (Ac5ManNTProp) and their size-controlled alkane counterparts Prop (Ac4ManNProp) and But (Ac4ManNBut). These compounds were added to the cells after solvation in ethanol (controls received equivalent volumes of EtOH). Cell physiology assays were conducted by CyQUANTTM Cell Proliferation Assay and CCK8 assay. The characterization of sialic acid levels was performed by periodate-resorcinol assay. Metabolomics analysis provided by Gigantest Inc and signaling pathway profiling through RT2 Profiler PCR Arrays utilized standardized protocols. Lastly, Wnt pathway analysis included western blotting of key proteins (e.g., beta-catenin, LRP6, Akt).

Results, Conclusions, and Discussions:

Results:
Comparing metabolism-based assays (e.g., MTT, CyQUANT, and CCK8) for cell quantification revealed inconsistent results between thiol-modified and non-thiolated ManNAc analogs. These inconsistencies were due to the metabolic basis of the assays (e.g., MTT measures mitochondrial activity, while CCK8 is broader based). Metabolomics analysis of hNSCs showed differences between control, But-, and TProp-treated cells. TProp treatment upregulated β-catenin and LRP6 in a manner consistent with previous results, indicating Wnt pathway activation. Meanwhile, But down-regulated these proteins, despite its known neuro-modulatory activity.

Discussion:
Our team has recently published on the front-cover of Advanced Functional Materials that metabolically glycoengineered human neural stem cells pre-treated with thiol-modified hNSCs provide substantial regenerative benefits in a rat model of brain damage after cardiac infarction. The current presentation delves into the underlying biology and biochemistry of the beneficial responses elicited by “TProp” and provides additional corroborating support that this analog, as compared to earlier reported non thiol-modified MGE analogs, is a prime candidate for clinical translation. In addition, the current work expands the scope of our investigation of the impact of MGE beyond our previous focus on the Wnt pathway to other signaling pathways involved in neural cell differentiation. In so doing, it finds that non thiol-modified MGE analogs interrogate pathways and cellular processes involved in neuronal differentiation beyond Wnt signaling, yielding unwanted impacts in neuro-modulatory activity.

Conclusions:
The results confirm that thiol-modified ManNAc analogs in MGE are critical for promoting Wnt pathway responses, essential for hNSCs in neural regeneration. The data highlights the complex interplay between analog structure and biological impact, supporting "TProp" as the preferred candidate for clinical translation.

Acknowledgements (Optional): Acknowledgements: Partially supported by R01NS125232 and R01NS110387 from NINDS, and 2024-MSCRFD-6401 from Maryland Stem Cell Research Fund (all to X. Jia).