Single-cell RNA-seq of bone marrow-derived mesenchymal stem cells reveals unique profiles of lineage priming

The plasticity and immunomodulatory capacity of mesenchymal stem cells (MSCs) have spurred clinical use in recent years. However, clinical outcomes vary and many ascribe inconsistency to the tissue source of MSCs. Yet unconsidered is the extent of heterogeneity of individual MSCs from a given tissue source with respect to differentiation potential and immune regulatory function. Here we use single-cell RNA-seq to assess the transcriptional diversity of murine mesenchymal stem cells derived from bone marrow. We found genes associated with MSC multipotency were expressed at a high level and with consistency between individual cells. However, genes associated with osteogenic, chondrogenic, adipogenic, neurogenic and vascular smooth muscle differentiation were expressed at widely varying levels between individual cells. Further, certain genes associated with immunomodulation were also inconsistent between individual cells. Differences could not be ascribed to cycles of proliferation, culture bias or other cellular process, which might alter transcript expression in a regular or cyclic pattern. These results support and extend the concept of lineage priming of MSCs and emphasize caution for in vivo or clinical use of MSCs, even when immunomodulation is the goal, since multiple mesodermal (and even perhaps ectodermal) outcomes are a possibility. Purification might enable shifting of the probability of a certain outcome, but is unlikely to remove multilineage potential altogether. Examination was performed using single-cell RNA-seq of sixteen mouse MSCs (mMSC1-mMSC16), non MSC single cell controls (HL1cm1-HL1cm5), and the population controls (mMSC-PC and HL1cm-PC).

间充质干细胞（mesenchymal stem cells, MSCs）的可塑性与免疫调节能力，近年来推动了其临床转化应用。然而临床应用结局存在显著异质性，诸多研究将这种不一致性归因于MSCs的组织来源差异。但目前尚未有研究系统探讨同一组织来源的单个MSCs在分化潜能与免疫调节功能层面的异质性程度。本研究采用单细胞RNA测序（single-cell RNA-seq）技术，对源自骨髓的小鼠间充质干细胞的转录组多样性进行评估。研究结果显示，与MSCs多能性相关的基因呈高表达状态，且在单个细胞间表达一致性较好；但与成骨、成软骨、成脂、成神经及血管平滑肌分化相关的基因，在单个细胞间的表达水平存在显著差异。此外，部分与免疫调节功能相关的基因，在单个细胞间的表达同样存在不一致性。上述表达差异并非由增殖周期、培养偏倚或其他以规律或周期性模式改变转录本表达的细胞过程所导致。本研究结果支持并拓展了MSCs谱系预置的学术概念，同时提醒研究者在MSCs的体内实验或临床应用中需保持谨慎：即便以免疫调节为研究或治疗目标，也存在向多个中胚层（甚至可能为外胚层）谱系分化的可能性。细胞纯化手段或许能够调整特定分化结局的发生概率，但无法完全消除MSCs的多系分化潜能。本研究通过对16株小鼠MSCs（mMSC1~mMSC16）、非MSCs单细胞对照（HL1cm1~HL1cm5）以及群体对照（mMSC-PC与HL1cm-PC）进行单细胞RNA测序，完成了上述实验分析。