Single-cell RNA sequencing of equine mesenchymal stromal cells from primary donor-matched tissue sources reveals functional heterogeneity in immune modulation and cell motility

Abstract

Background The efficacy of mesenchymal stromal cell (MSC) therapy is thought to depend on the intrinsic heterogeneity of MSC cultures isolated from different tissue sources as well as individual MSCs isolated from the same tissue source, neither of which is well understood. To study this, we used MSC cultures isolated from horses. The horse is recognized as a physiologically relevant large animal model appropriate for translational MSC studies. Moreover, due to its large size the horse allows for the simultaneous collection of adequate samples from multiple tissues of the same animal, and thus, for the unique collection of donor matched MSC cultures from different sources. The latter is much more challenging in mice and humans due to body size and ethical constraints, respectively. Methods In the present study, we performed single-cell RNA sequencing (scRNA-seq) on primary equine MSCs that were collected from three donor-matched tissue sources; adipose tissue (AT), bone marrow (BM), and peripheral blood (PB). Based on transcriptional differences detected with scRNA-seq, we performed functional experiments to examine motility and immune regulatory function in distinct MSC populations. Results We observed both inter- and intra-source heterogeneity across the three sources of equine MSCs. Functional experiments demonstrated that transcriptional differences correspond with phenotypic variance in cellular motility and immune regulatory function. Specifically, we found that (i) differential expression of junctional adhesion molecule 2 (JAM2) between MSC cultures from the three donor-matched tissue sources translated into altered cell motility of BM-derived MSCs when RNA interference was used to knock down this gene, and (ii) differences in C-X-C motif chemokine ligand 6 (CXCL6) expression in clonal MSC lines derived from the same tissue source correlated with the chemoattractive capacity of PB-derived MSCs. Conclusions Ultimately, these findings will enhance our understanding of MSC heterogeneity and will lead to improvements in the therapeutic potential of MSCs, accelerating the transition from bench to bedside.

The Unique Immunomodulatory Properties of MSC-Derived Exosomes in Organ Transplantation

“…For MSCs from different sources, although their proliferation and differentiation abilities are similar, they may show different specific phenotypes and immunomodulatory properties, resulting in differences in efficacy. Harman et al ( 25 ) performed single-cell RNA sequencing (scRNA-seq) on primary equine MSCs that were collected from adipose tissue, bone marrow, and peripheral blood. They observed that transcriptional differences corresponded with phenotypic variance in cellular motility and the immune regulatory function ( 25 ).…”

Translational Animal Models Provide Insight Into Mesenchymal Stromal Cell (MSC) Secretome Therapy

“…Additional studies from our group identified anti-microbial peptides in the horse PB-MSC secretome that inhibit the growth of contaminating bacteria commonly found in skin wounds, as well as cysteine proteases that destabilize bacterial biofilms in vitro ( Harman et al, 2017b ; Marx et al, 2020 ). Moreover, we confirmed that the chemokine C-X-C motif ligand 6 (CXCL6) secreted by horse PB-MSC acts as a chemoattractant for neutrophils in vitro ( Harman et al, 2020 ). Collectively, our research has identified specific factors secreted by PB-MSC that promote various aspects of skin wound healing, supporting the notion that the complete MSC secretome provides therapeutic benefits by targeting various aspects of specific disease processes.…”

“…Single-cell RNA sequencing of mouse BM-derived MSCs revealed multiple profiles as well, some associated with distinct differentiation potential ( Freeman et al, 2015 ). Our group used single-cell transcriptomics to analyze donor-matched equine MSCs isolated from three different tissue sources and we found inter- and intra-source genetic heterogeneity that resulted in functional heterogeneity in immune function and cell motility ( Harman et al, 2020 ). The emerging technology of high-resolution precision proteomics is currently only being used to evaluate cancer cellular heterogeneity ( Waas and Kislinger, 2020 ), but will certainly be transferrable to MSCs, where this technique can provide additional insights into the heterogeneity of MSC populations to allow for the purification of MSC subpopulations with high secretory potential.…”

“…This is illustrated by previously discussed data from our own group. We have identified proteins with regenerative properties and proteins with antimicrobial properties in the secretome of horse PB-MSCs ( Bussche and Van de Walle, 2014 ; Bussche et al, 2015 ; Harman et al, 2017a , b , 2018 , 2020 , Marx et al, 2020 ). Using these identified factors individually could be therapeutically useful to promote wound healing or to fight bacterial infections.…”

“…Although it is clear that MSCs have great therapeutic potential and are being explored in multiple medical fields, results are often inconsistent and (pre-)clinical studies show only minor effects or do not lead to the desired outcomes at all ( Lukomska et al, 2019 ; Wilson et al, 2019 ). A potential reason for the observed inconsistencies is the heterogeneity of cultured MSCs, which is influenced by the individual donor and tissue of origin, isolation technique, culture environment, and cell passage number ( Wilson et al, 2019 ; Harman et al, 2020 ). Purposefully manipulating MSCs to improve therapeutic benefits, which by default could result in a more standardized and/or customized secretome, is not a novel idea, but one that has not yet been maximally explored ( Liesveld et al, 2020 ; Ocansey et al, 2020 ).…”