Cellular and Tissue Therapies Branch, Division of Cellular and Gene Therapies, Office of Cellular, Tissue, and Gene Therapies, FDA, Center for Biologics Evaluation and Research, Bethesda, MD, USA.
Tissue Eng Part C Methods. 2012 Nov;18(11):877-89. doi: 10.1089/ten.TEC.2011.0736. Epub 2012 Jun 25.
Bone marrow-derived multipotent stromal cells (MSCs), also known as mesenchymal stem cells, have great promise due to their capacity for tri-lineage differentiation and immunosuppressive properties, which allows for their allogeneic use and ultimately may allow for treatment of many diseases. MSCs will require extensive expansion and passaging to obtain cells in sufficient numbers necessary for cell therapies. MSCs from many donors could potentially be used. Because of this, there is a need to understand the role of passaging and donor differences on differentiation capacity using quantitative approaches. Here, we evaluated MSCs from two donors (noted as PCBM1632 and PCBM1641 by the manufacturer) at tissue culture passages 3, 5, and 7. We used a colony forming unit (CFU) assay and limiting dilution to quantify clonogenicity and precursor frequency during adipogenesis, and quantitative real-time-polymerase chain reaction for adipogenic markers to evaluate changes on a gene expression level. Further, we observed changes in cell size, and we sorted small and large populations to evaluate size-related adipogenic potential. While the adipogenic precursor frequency of ∼1 in 76 cells remained similar through passages for cells from PCBM1641, we found a large decrease in the adipogenic potential of MSCs from PCBM1632, with 1 in 2035 cells being capable of differentiating into an adipocyte at passage 7. MSCs from both donors showed an increase in cell diameter with increasing passage, which correlates with a decrease in clonogenicity by CFU analysis. We also measured adipose lineage gene expression following induction of adipocyte differentiation. Expression of these genes decreased with passage number for MSCs from PCBM1632 and correlated with the decrease in adipogenic potential by passage 7. In contrast, MSCs from PCBM1641 showed increased expression of these genes with increasing passage. We have shown that several quantitative assays can detect differences in MSC differentiation capacity, clonogenicity, and cell size between donors and passages. These quantitative methods are useful to assess the quality of MSCs.
骨髓来源的多能基质细胞（MSCs），也称为间充质干细胞，由于其具有三系分化能力和免疫抑制特性，因此具有很大的应用前景，允许其同种异体使用，最终可能用于治疗许多疾病。MSCs 需要进行广泛的扩增和传代，以获得足够数量的细胞用于细胞治疗。可能会使用来自许多供体的 MSCs。因此，需要使用定量方法来了解传代和供体差异对分化能力的影响。在这里，我们评估了制造商标记为 PCBM1632 和 PCBM1641 的两名供体的 MSCs，在组织培养传代 3、5 和 7 时。我们使用集落形成单位（CFU）测定和有限稀释法来量化脂肪生成过程中的克隆形成能力和前体频率，并使用定量实时聚合酶链反应来评估脂肪生成标志物的基因表达水平上的变化。此外，我们观察到细胞大小的变化，并对小细胞和大细胞群体进行分选，以评估与大小相关的脂肪生成潜能。虽然 PCBM1641 来源的细胞的脂肪生成前体频率在传代过程中保持在 1/76 左右相似，但我们发现 PCBM1632 来源的 MSC 的脂肪生成潜力有很大的下降，在第 7 代时，1/2035 个细胞能够分化为脂肪细胞。来自两个供体的 MSC 随着传代的增加而增加细胞直径，这与 CFU 分析中克隆形成能力的下降相关。我们还在诱导脂肪细胞分化后测量脂肪谱系基因的表达。对于 PCBM1632 的 MSC，这些基因的表达随着传代次数的增加而减少，并且与第 7 代时的脂肪生成潜力下降相关。相比之下，PCBM1641 的 MSC 随着传代次数的增加，这些基因的表达增加。我们已经表明，几种定量测定方法可以检测供体和传代之间 MSC 分化能力、克隆形成能力和细胞大小的差异。这些定量方法可用于评估 MSC 的质量。
