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Induction and Detection of Autophagy in Aged Hematopoietic Stem Cells by Exposing Them to Microvesicles Secreted by HSC-Supportive Mesenchymal Stromal Cells

  • Rohan S. Kulkarni
  • Manmohan S. Bajaj
  • Vaijayanti P. Kale
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1854)

Abstract

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.

Keywords

Autophagy Hematopoietic stem cells HSC niche LC3 Mesenchymal stromal cells Microvesicles 

References

  1. 1.
    Lymperi S, Ferraro F, Scadden DT (2010) The HSC niche concept has turned 31. Ann N Y Acad Sci 1192(1):12–18CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Méndez-Ferrer S, Michurina TV, Ferraro F et al (2010) Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466(7308):829CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ratajczak J, Miekus K, Kucia M et al (2006) Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia 20(5):847CrossRefPubMedGoogle Scholar
  4. 4.
    Stik G, Crequit S, Petit L et al (2017) Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells. J Cell Biol.  https://doi.org/10.1083/jcb.201601109
  5. 5.
    Geiger H, Rudolph KL (2009) Aging in the lympho-hematopoietic stem cell compartment. Trends Immunol 30(7):360–365CrossRefPubMedGoogle Scholar
  6. 6.
    Ho TT, Warr MR, Adelman ER et al (2017) Autophagy maintains the metabolism and function of young and old stem cells. Nature 543(7644):205CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kulkarni R, Bajaj M, Ghode S et al (2018) Intercellular transfer of microvesicles from young mesenchymal stromal cells rejuvenates aged murine hematopoietic stem cells. Stem Cells 36(3):420–433CrossRefPubMedGoogle Scholar
  8. 8.
    Klionsky D, Abeliovich H, Agostinis P et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175Google Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  • Rohan S. Kulkarni
    • 1
  • Manmohan S. Bajaj
    • 1
  • Vaijayanti P. Kale
    • 1
    • 2
  1. 1.Stem Cell LabNational Centre for Cell SciencePuneIndia
  2. 2.Symbiosis Center for Stem Cell Research, Symbiosis School of Biological SciencesSymbiosis International (Deemed University)PuneIndia

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