Spurlock Brian, Hanumanthu Vidya Sagar, Mitra Kasturi
Department of Genetics, University of Alabama at Birmingham, AL 35294, USA.
Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, AL 35294, USA.
Bio Protoc. 2021 Mar 5;11(5):e3945. doi: 10.21769/BioProtoc.3945.
Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are "primed" by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these "primed" cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol can be directly used or modified to be used in various cell types. Thus, this protocol is anticipated to be invaluable for the basic understanding of mitochondrial and energetic heterogeneity within stem cell population, and may also prove valuable in translational studies in regenerative medicine and cancer biology.
已发现多种干细胞依赖线粒体能量代谢。线粒体在调节正常干细胞和肿瘤起始干细胞（TICs）的自我更新中的作用日益受到重视。我们提出，TIC群体中有一部分细胞被线粒体“预激发”以进行自我更新。利用卵巢癌模型，我们开发了一种方案，通过荧光辅助细胞分选（FACS）来鉴定和分离这些“预激发”细胞。我们将用于TIC功能标记物和线粒体跨膜电位的活细胞染色相结合，以富集具有较高线粒体电位的TIC，这些TIC形成球体的能力是线粒体电位较低的其他TIC的10倍。该方案可直接使用或经修改后用于各种细胞类型。因此，预计该方案对于深入理解干细胞群体中线粒体和能量异质性具有重要价值，并且在再生医学和癌症生物学的转化研究中也可能具有重要价值。
