Bose Bipasha, Nihad Muhammad, P Sudheer Shenoy
Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Karnataka, India.
Cytometry A. 2023 May;103(5):368-377. doi: 10.1002/cyto.a.24726. Epub 2023 Mar 14.
Pluripotent stem cell research has revolutionized the modern era for the past 14 years with the advent of induced pluripotent stem cells. Before this time, scientists had access to human and mouse embryonic stem cells primarily for basic research and an attempt towards lineage-specific differentiations for cell therapy applications. Regarding pluripotent stem cells, expression of bonafide marker proteins such as Oct4, Nanog, Sox2, Klf4, c-Myc, and Lin28 have been considered giving a perfect readout for pluripotent stem cells and assessed using an analytical flow cytometer. In addition to the intracellular markers, surface markers such as stage-specific embryonic antigen-1 for mouse cells and SSEA-4 for human cells are needed to sort pure populations of stem cells for further downstream applications for cell therapy. The surface marker SSEA-4 is the most appropriate for obtaining pure populations of human pluripotent stem cells. When differentiated in a controlled manner using growth factors or small molecules, it is mandatory to assess the downregulation of pluripotency markers (Oct4, Nanog, Sox2, and Klf4) with subsequent up-regulation of stage-specific differentiation markers. Such assessments are done using flow cytometry. Pluripotent stem cells have a high teratoma-forming potential in vivo. Small amounts of undifferentiated PSCs might lead to dangerous teratomas upon transplantation if leftover in the pool of differentiated cells. Hence, flow cytometry is essential for sorting out PSC populations with teratoma-forming potential. The pure populations of differentiated progenitors need to be flow-sorted before differentiating them further for cell therapy applications. For example, Glycoprotein 2 is a specific cell-surface marker for pancreatic progenitors that enables one to sort the pancreatic progenitors differentiated from human PSCs. Taken together, analytical flow cytometry, and cell sorting provide indispensable tools in PSC research and cell therapy.
在过去的14年里，随着诱导多能干细胞的出现，多能干细胞研究彻底改变了现代医学。在此之前，科学家主要将人类和小鼠胚胎干细胞用于基础研究，并尝试将其定向分化用于细胞治疗。对于多能干细胞，Oct4、Nanog、Sox2、Klf4、c-Myc和Lin28等真正的标记蛋白的表达被认为是多能干细胞的完美读数，并使用分析流式细胞仪进行评估。除了细胞内标记物外，还需要表面标记物，如小鼠细胞的阶段特异性胚胎抗原-1和人类细胞的SSEA-4，以分选纯净的干细胞群体，用于细胞治疗的进一步下游应用。表面标记物SSEA-4最适合用于获得纯净的人类多能干细胞群体。当使用生长因子或小分子以可控方式进行分化时，必须评估多能性标记物（Oct4、Nanog、Sox2和Klf4）的下调以及阶段特异性分化标记物的随后上调。这种评估通过流式细胞术进行。多能干细胞在体内具有很高的形成畸胎瘤的潜力。如果分化细胞池中残留少量未分化的多能干细胞，移植后可能会导致危险的畸胎瘤。因此，流式细胞术对于分选具有形成畸胎瘤潜力的多能干细胞群体至关重要。在将分化的祖细胞进一步用于细胞治疗应用之前，需要通过流式分选获得纯净的群体。例如，糖蛋白2是胰腺祖细胞的一种特异性细胞表面标记物，可用于分选从人类多能干细胞分化而来的胰腺祖细胞。综上所述，分析流式细胞术和细胞分选在多能干细胞研究和细胞治疗中提供了不可或缺的工具。
