Single cell transcriptomic analysis of human mesenchymal stem cells reveals limited heterogeneity

Abstract

Mesenchymal stem cells (MSCs) are a population of multipotent cells with a superior ability to promote tissue repair by regulating regeneration and inflammation. Effective application of MSCs in disease treatment relies on the production of relatively homogeneous cell population. However, the cellular heterogeneity and the differentiation trajectories of in vitro expanded MSCs remain largely unclear. We profiled the transcriptomes of 361 single MSCs derived from two umbilical cords (UC-MSCs). These UC-MSCs were harvested at different passages and stimulated with or without inflammatory cytokines. Weighted gene correlation network analysis revealed that UC-MSCs surprisingly possess only limited heterogeneity, regardless of donors, and passages. We also found that upon pretreatment with inflammatory cytokines (IFNγ and TNFα), a classical strategy that can improve the efficiency of MSC-based therapy, MSCs exhibited uniformed changes in gene expression. Cell cycle-based principal component analysis showed that the limited heterogeneity identified in these UC-MSCs was strongly associated with their entrance into the G2/M phase. This was further proven by the observation that one featured gene, CD168, was expressed in a cell cycle-dependent manner. When CD168 high UC-MSCs were sorted and cultured in vitro, they again showed similar CD168 expression patterns. Our results demonstrated that in vitro expanded UC-MSCs are a well-organized population with limited heterogeneity dominated by cell cycle status. Thus, our studies provided information for standardization of MSCs for disease treatment.

Revealing Cellular Heterogeneity andIn VitroDifferentiation Trajectory of Cultured Human Endometrial Mesenchymal-like Stem Cells Using Single-cell RNA Sequencing

“…At sample level, one menstrual phase sample (M1) and one secretory phase sample (S3) manifested quiescent/senescence characteristic with a high proportion of cells at the G1 phase, whereas the remaining samples demonstrated a high proportion of cells in the proliferative phase ( Figure 1B). From overview of all single cells, PCA results based on normalized gene expression matrix showed that variance of the first 3 principal components were mainly caused by cell cycle effect ( Figure 1C) and batch/donor effect (Figure 1D), which were consistently observed in other single cell studies on MSCs [13][14][15]. The results indicated that unwanted major source of variation should be removed before investigation on the molecular heterogeneity of eMSCs.…”

Single-cell RNA-seq highlights heterogeneity in human primary Wharton’s jelly mesenchymal stem/stromal cells cultured in vitro

“…Recently, several studies have been performed to investigate into the heterogeneity of cultured MSCs by single-cell transcriptomic analysis [35,[81][82][83]. Huang et al profiled the transcriptomes of 361 single MSCs derived from two umbilical cords (UC-MSCs) that were harvested at different passages and stimulated with or without inflammatory cytokines.…”

“…Huang et al profiled the transcriptomes of 361 single MSCs derived from two umbilical cords (UC-MSCs) that were harvested at different passages and stimulated with or without inflammatory cytokines. Following the analysis, they concluded that in vitro expanded UC-MSCs are a well-organized population with limited heterogeneity, which is mainly caused by distinct distribution in cell cycle phases [81]. However, the number of cells sequenced for each condition is small (~50 cells per condition), and they did not remove the cell cycle effects for the subpopulations identification.…”

Single-cell RNA-seq highlights heterogeneity in human primary Wharton’s Jelly mesenchymal stem/stromal cells cultured in vitro

“…Recently, several studies have been performed to investigated into the heterogeneity of cultured MSCs by single cell transcriptomic analysis (Huang et al, 2019; Khong et al, 2019; Liu et al, 2019; Wang et al, 2019). Huang et al profiled the transcriptomes of 361 single MSCs derived from two umbilical cords (UC-MSCs) that were harvested at different passages and stimulated with or without inflammatory cytokines.…”

“…Huang et al profiled the transcriptomes of 361 single MSCs derived from two umbilical cords (UC-MSCs) that were harvested at different passages and stimulated with or without inflammatory cytokines. Following analysis, they concluded that in vitro expanded UC-MSCs are a well-organized population with limited heterogeneity, which is mainly caused by distinct distribution in cell cycle phases (Huang et al, 2019). However, the number of cells sequenced for each condition is small (~50 cells per condition), and they did not remove the cell cycle effects for the subpopulations identification.…”