Using Single Cell RNA-Sequencing to Define the Heterogeneity of Equine MSC

Presented by: Rebecca Harman


Authors: Rebecca Harman1, Roosheel Patel2, Jenna Park1, Brad Rosenberg2 and Gerlinde Van de Walle1.

Affiliations: 1 Baker Institute for Animal Health, Cornell University, Ithaca, NY. 2 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY.

Introduction: In the broad field of regenerative medicine, the efficacy of mesenchymal stromal cell (MSC) treatments is highly dependent on the intrinsic heterogeneity of MSC, both between preparations of cells isolated from different tissue sources and within individual preparations from the same tissue source. In equine regenerative medicine, the most common sources of MSC isolated for clinical use are bone marrow (BM), adipose tissue (AT), and peripheral blood (PB). Our lab and others have used candidate-based approaches to compare specific features of MSC derived from different sources, however, the global heterogeneity of MSC, and how this contributes to treatment outcomes, is not well appreciated.

Hypothesis / Objectives: Our objective is to explore the global heterogeneity of equine MSC isolated from different tissue sources using an unbiased approach. Transcriptomic data from this study will provide the first comprehensive overview of equine MSC heterogeneity. If observed gene expression differences translate into differences in functional properties of MSC, this knowledge will contribute to improvements in MSC therapy in equine medicine.

Materials and Methods: Single cell RNA-sequencing is a recently developed technology designed for whole transcriptomic analyses of individual cells in a mixed population. We used this technology to determine the global levels of heterogeneity of equine MSC isolated from the BM, AT and PB of an adult mare. In addition to comparing the heterogeneity of MSC cultures isolated from one tissue source to those isolated from another in an unbiased manner, these data allow us to also compare MSC heterogeneity within isolations from the same tissue source. To verify the RNA-sequencing data, we will methodically knock down the expression of specific genes characteristic of MSC derived from one tissue source, to determine what genes are critical to distinguishing that population of MSC from MSC derived from the other tissue sources. In addition, we have used limiting dilution cloning to enrich for sub-populations of MSC derived from a single tissue source. We will carry out functional experiments, such as differentiation assays, to characterize heterogeneity from sub-populations within isolations from the same tissue source.

Results: Our single cell RNA-sequencing analysis revealed that (i) MSC cultures isolated from different tissue sources exhibit distinct transcriptional profiles and (ii) MSC prepared from one tissue source are heterogeneous and can be separated into discreet sub-populations based on gene expression patterns. For example, we found that BM-MSC express higher levels of the chemoattractant CCL2 compared to AT- and PB-MSC. We also found that ALPL and COL6A3, two genes involved in osteoblast differentiation, are more highly expressed in one specific AT-MSC cluster (aka sub-population) compared to other AT-MSC clusters. Based on these data, we expect to uncover functional differences in MSC isolated from different sources, as well as in MSC sub-populations isolated from single tissue sources.

Conclusions: The heterogenity of equine MSC is likely to contribute to the efficacy of these cells as a therapy. Understanding (i) the global differences in MSC isolated from different sources and (ii) the heterogeneity of MSC isolated from single tissue sources, will allow clinicians to make informed decisions about which MSC are most appropriate to administer to equine patients.

Acknowledgements, Funding, and Conflicts of Interest: This work is funded by an unrestricted fund from the Harry M. Zweig Memorial Fund to G. Van de Walle. The authors declare no conflict of interest.