Differentiation of Mesenchymal Stem/Stromal Cells into CD45+ Macrophage-like Cells: Expanding Insights into MSC Plasticity

Single-Cell RNA-Seq supports transition of PαS cells into macrophage and fibrocyte-like
lineages.
To further investigate these results, single-cell RNA-Seq (scRNA-seq) was performed with freshly
isolated PαS cells, which were cultured for 1 week in DMEM or RM. First, the t-distributed stochastic
neighbor embedding was investigated for the positive isolation markers PDGFRα (denoted as
PDGFRα) and Ly6a (Sca-1) Ptprc, Cd45 (PTPRCATXN1) (Figure 4A). Although both samples
displayed a similar distribution, only the RM-cultured sample clearly contained CdD45 (PTPRC)-
expressing cells.
Next, the cells were clustered into 10 clusters using the Louvain Clustering of BBrowser (Figure 4B).
However, owing to the distinct location and different levels of key marker expression, cluster 6 was
manually subdivided into clusters 6 and cluster 6a (Figure 4B, and 4C). The isolation markers Sca-1
(denoted as LY6A) (LY6A), PDGFRα (PDGFRA), CdD73 (NT5E), CdD45 (PTPRC), as well as CdD68
(CD68), CdD11b (ITGAM), CsfSF1r (CSF1R)R, CdD34 (CD34), and Ccollagen 1 (COL1A1) were
blotted in a bubble heatmap (Figure 4C). Clusters 4, 5, and 6a–10 all highly expressed Sca-1 and
PDGFRα, whereas clusters 1, 2, and 3 expressed low levels of Sca-1 and PDGFRα, while cluster 6
did not express PDGFRα and expressed low levels of Sca-1. CdD73 was only notably expressed in
clusters 3 and 4. Cluster 3 was the only cluster with high CdD34 expression. While devoid in the
other clusters, clusters 6 and 6a both expressed high levels of Csf1rCSF1R, CdD11b, CdD68, and
CdD45. Notably, Ccollagen 1 expression was high in all clusters except for cluster 6, which was
approximately half of that in the others (Figure 4C).
Based on these markers and co-expression within clusters, cells were divided into MSCs (Figure 4E,
blue, clusters 5, 7–10) based on Sca-1 and PDGFRα expression; adipocyte progenitors (yellow,
cluster 4) based on CdD73 expression; fibroblast progenitors (green, cluster 3) based on CdD73 and
CdD34 expression; macrophages (red, cluster 6) based on CdD45, CdD68, CdD11b, and Csf1CSF1R
expression; and fibrocyte-like macrophages (violet, cluster 6a) based on the low expression of
macrophage markers and high expression of collagen I. Both cluster (macrophages and fibrocyte-like

macrophages) clearly express macrophage relevant genes such as CdD14 (CD14), CdD48 (CD48),
CdD53 (CD53), Stab1 (STAB1), Ccl9 (CCL9), and CdD206 (MRC1), and lose the characteristics of
MSCs (Figure 4F). These data strongly suggest that freshly isolated PαS cells contain heterogeneous
subpopulations, including macrophages and fibrocyte-like macrophages, that emerge in response to
M-CSF stimulation.