Background & Aims

Time series experimental designs and approaches have been widely used to study the dynamic regulations of transcripts and explore the time-dependent changes of gene expression in biological systems. In this study, we used a multifaceted approach to analyze and interpret the temporal dynamics of gene expression data at both bulk and single cell levels, with a specific focus on neuronal genes derived from human spinal cord organotypic slice culture. This involved profiling gene expression patterns, identification of the cell types and their relative abundance, and the biological pathways that change over time.

Methods

Spinal cord organotypic slices were obtained from 3 donors at five distinct time points (0, 5, 10, 15, 20 days). Samples from day 0 were snap frozen, whereas samples from days 5, 10, 15 and 20 were in organotypic culture. At each timepoint, the slices were harvested for genomic analysis including single cell nuclear RNAseq (10X Genomics) and total bulk RNAseq (Illumina).

Results

Pairwise comparison of gene expression between each of the 5 time points showed the effect of culturing human spinal cord slices in organotypic culture on cellular populations, as evidenced by significant cell proliferation between these time points. Functional enrichment analysis of the significant differentially expressed genes showed a predominant association with biological pathways related to immune and inflammatory responses. Cell type enrichment of the biological pathways corroborated the results demonstrating enrichment in astrocytes, microglia and macrophages. A propensity for state transitions over time was observed within a neuronal cluster in which a trajectory analysis revealed an early and late overexpression correlation of immediate early genes (IEGs) like EGR1, ATF3, GEM and voltage-gated ion channel genes like SCN9A and KCND3.

Conclusions

The findings of this study represent a significant advancement in the comprehensive characterization of neuronal activity within spinal cord slices in an organotypic culture, achieved through the analysis of gene expression and cell type profiling over time. The results will contribute to a refined understanding of a pivotal platform for investigating specific targets involved in the intricacies of pain biology.

References

Korsunsky, I., Millard, N., Fan, J., Slowikowski, K., Zhang, F., Wei, K., Baglaenko, Y., Brenner, M., Loh, P., & Raychaudhuri, S. (2019). Fast, sensitive and accurate integration of single-cell data with harmony. Nature Methods, 16(12), 1289-1296. https://doi.org/10.1038/s41592-019-0619-0

Trapnell, C., Cacchiarelli, D., Grimsby, J., Pokharel, P., Li, S., Morse, M., Lennon, N. J., Livak, K. J., Mikkelsen, T. S., & Rinn, J. L. (2014). The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nature Biotechnology, 32(4), 381-386. https://doi.org/10.1038/nbt.2859

Yadav, A., Matson, K. J. E., Li, L., Hua, I., Petrescu, J., Kang, K., Alkaslasi, M. R., Lee, D. I., Hasan, S., Galuta, A., Dedek, A., Ameri, S., Parnell, J., Alshardan, M. M., Qumqumji, F. A., Alhamad, S. M., Wang, A. P., Poulen, G., Lonjon, N., … Levine, A. J. (2023). A cellular taxonomy of the adult human spinal cord. Neuron, 111(3). https://doi.org/10.1016/j.neuron.2023.01.007

Presenting Author

Anne Andere

Poster Authors

Anne Andere

PhD

Eli Lilly and Company

Lead Author

E-mail

Sarubini Kananathan

MSc

Eli Lilly and Co.

Lead Author

E-mail

Mahmoud Haroun

BSc

Eli Lilly and Co.

Lead Author

E-mail

Jeff Krajewski

Eli Lilly and Company

Lead Author

E-mail

Jeff McDermott

Eli Lilly and Co.

Lead Author

E-mail

Gopuraja Dharmalingam

Eli Lilly and Co.

Lead Author

E-mail

Ryan Smith

Eli Lilly

Lead Author

E-mail

Chris Mathes

PhD

AnaBios Corporation

Lead Author

E-mail

Andre Ghetti

PhD

AnaBios Corporation

Lead Author

E-mail

Richard Kondo

AnaBios Corporation

Lead Author

E-mail

Mari Kennedy

AnaBios Corporation

Lead Author

E-mail

Kevin Carlin

AnaBios Corporation

Lead Author

E-mail

Topics

  • Models: Chronic Pain - Inflammatory