Mesenchymal stromal cells (MSCs) are potential treatments for a variety of veterinary medical conditions. However, clinical trials have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. Identification and characterization of subpopulations within MSC cultures may improve those outcomes. Therefore, the functional heterogeneity of different-sized subpopulations of MSCs was evaluated. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization, as well as to evaluate the impact of culture conditions on their functional heterogeneity. We found that culture-expanded canine Ad-MSCs comprise distinct subpopulations: larger MSCs (mean diameter of 18.6 ± 0.2 μm), smaller MSCs (mean diameter of 15.3 ± 0.2 μm), and intermediate MSCs (mean diameter of 16.9 ± 0.1 μm). In addition, proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. We observed that larger MSCs proliferate more slowly, senesce at earlier passages, and are inclined to differentiate into adipocytes compared with smaller MSCs. Most importantly, these size-dependent functions are also affected by the presence of serum in the culture medium, as well as time in culture. Cell surface staining for MSC-specific CD44 and CD90 antigens showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs. Finally, transcriptome analysis showed differential gene expression between larger and smaller subpopulations of MSCs. Larger MSCs expressed genes involved in cellular senescence such as cyclin-dependent kinase inhibitor 1A and smaller MSCs expressed genes that promote cell growth [mechanistic target of rapamycin 1 (mTORC1) pathway] and cell proliferation [myelocytomatosis (myc), e2f targets]. These results suggest that different subpopulations of MSCs have specific properties.

中文翻译：

---

犬脂肪来源的间充质基质细胞的微流体分离

间充质基质细胞 (MSC) 是治疗各种兽医疾病的潜在疗法。然而，临床试验往往达不到预期，部分原因是 MSC 的异质性和缺乏表征。MSC 培养物中亚群的鉴定和表征可能会改善这些结果。因此，评估了不同大小的 MSCs 亚群的功能异质性。使用高通量、生物物理、无标记微流体分选方法根据大小分离犬脂肪来源的 MSC (Ad-MSC) 亚群，以进行后续表征，并评估培养条件对其功能异质性的影响. 我们发现培养扩增的犬 Ad-MSC 包含不同的亚群：较大的 MSC（平均直径为 18.6 ± 0.2 μm），较小的 MSC（平均直径为 15.3 ± 0.2 μm）和中间 MSC（平均直径为 16.9 ± 0.1 μm）。此外，犬 Ad-MSCs 的增殖特性、衰老和分化潜能也取决于细胞大小。我们观察到，与较小的 MSCs 相比，较大的 MSCs 增殖更慢，在较早的传代中衰老，并且倾向于分化为脂肪细胞。最重要的是，这些大小依赖的功能还受到培养基中血清的存在以及培养时间的影响。MSC 特异性 CD44 和 CD90 抗原的细胞表面染色表明，所有 MSC 亚群都无法区分，这表明该标准与定义 MSC 亚群无关。最后，转录组分析显示较大和较小的 MSC 亚群之间的差异基因表达。较大的 MSC 表达参与细胞衰老的基因，例如细胞周期蛋白依赖性激酶抑制剂 1A，较小的 MSC 表达促进细胞生长 [雷帕霉素 1 (mTORC1) 通路的机制靶点] 和细胞增殖 [骨髓细胞瘤病 (myelocytomatosis) 的基因。myc ), e2f目标]。这些结果表明 MSC 的不同亚群具有特定的特性。