Kulkarni Rohan S, Bajaj Manmohan S, Kale Vaijayanti P
Stem Cell Lab, National Centre for Cell Science, Pune, India.
Symbiosis Center for Stem Cell Research, Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Pune, India.
Methods Mol Biol. 2019;1854:21-34. doi: 10.1007/7651_2018_166.
Autophagy is an important cellular process for maintenance of quality and functionality of cells. This happens through repair and renewal of cellular components like proteins and mitochondria. Reduction in autophagy process in aged hematopoietic stem cells (HSCs) leads to their compromised stemness and self-renewal capacity, and consequently, their applicability in various regenerative therapies also reduces. HSC functions are regulated by their microenvironment, known as "HSC niche," which comprises of mesenchymal stromal cells (MSCs), osteoblasts, endothelial cells, etc. In this niche, the MSCs are known to closely interact with the HSCs, and therefore, they can directly influence the stem cell fate. In our earlier studies, we have demonstrated that young MSCs or aged MSCs rejuvenated by treating them with LY294002, a PI3K inhibitor (rescued aged MSCs), rejuvenate aged HSCs via intercellular transfer of microvesicles (MVs) harboring autophagy-inducing mRNAs.Here, we describe the protocol for induction of autophagy in aged HSCs by incubating them with microvesicles (MVs) collected from young MSCs or rescued aged MSCs. We also describe the protocols for determination of autophagy levels in these HSCs.
自噬是维持细胞质量和功能的重要细胞过程。这通过修复和更新蛋白质和线粒体等细胞成分来实现。老年造血干细胞（HSC）自噬过程的减少会导致其干细胞特性和自我更新能力受损，因此，它们在各种再生疗法中的适用性也会降低。HSC的功能受其微环境（称为“HSC生态位”）调节，该微环境由间充质基质细胞（MSC）、成骨细胞、内皮细胞等组成。在这个生态位中，已知MSC与HSC密切相互作用，因此，它们可以直接影响干细胞的命运。在我们早期的研究中，我们已经证明，用PI3K抑制剂LY294002处理使其恢复活力的年轻MSC或老年MSC（挽救的老年MSC），通过携带自噬诱导mRNA的微泡（MV）的细胞间转移使老年HSC恢复活力。在这里，我们描述了通过将老年HSC与从年轻MSC或挽救的老年MSC收集的微泡（MV）孵育来诱导老年HSC自噬的方案。我们还描述了测定这些HSC中自噬水平的方案。
