Roles of Exosomes in the Hematopoietic Stem Cell-Supporting Capacity of Stromal Cells

Hematopoietic stem cells (HSCs) are identified by their ability to self-renew and to differentiate into all blood cell lineages. In vivo, hematopoietic stem/progenitor cells (HSPCs) are in close association with stromal cells that constitute a supportive microenvironment also called niche. Recently, exosomes that are small microvesicles enclosed by a lipid bilayer and enriched in cytoplasmic proteins, mRNAs, microRNAs, have emerged as major communication mediators between cells. However, their implication in the cross-talk between HSCs and stromal cells is still largely unknown. This study aims to assess the existence and the functionality of stromal cell-derived exosomes in the HSPC support.

To address this issue, we used two murine stromal cell lines derived from the fetal liver and with differing capacity to maintain HSPCs ex vivo as revealed by repopulation assay and long-term cultures. AFT024 (AFT) harbors a potent HSPC supporting capacity in vitro whereas BFC012 (BFC) is non supportive. For each cell line, the exosome fractions were isolated from culture supernatant by ultra-centrifugation. Electron microscopy, western blot, and flow cytometry analyses revealed that both AFT and BFC stromal cells secrete exosomes. Interestingly, using PKH67 stained exosomes, we demonstrated that bone marrow Lin^-Sca-1⁺c-kit⁺ (LSK) cells preferentially uptake AFT-derived exosomes. This observation might be related to the different tetraspanin compositions of AFT and BFC derived exosomes as observed by flow cytometry. We then showed an increase in cell viability and clonogenic potential when LSK cells were exposed to AFT-derived exosomes for 96 hours in cytokine-free medium as compared to controls. Moreover, cultures with AFT-derived exosomes exhibited a 3.5 fold increase in the number of LSK cells as compared to untreated conditions. We then used high-throughput sequencing to explore the molecular signatures of AFT and BFC derived exosomes, as well as their cells of origin. We identified a list of 394 mRNAs and 6 microRNAs specifically expressed in exosomes and correlated to the HSPC support. Gene ontology analysis revealed that the apoptotic regulation, cell survival and proliferation pathways were significantly enriched in the AFT-derived exosomal signature. In addition, we showed the transfer of mRNAs involved in these pathways from the AFT-exosomes to the LSK recipient cells. Together with our observation of a decrease in the LSK apoptotic cells after co-culture with AFT-derived exosomes, these data suggest that exosomes released by AFT cells may protect HSPCs from apoptosis.

Collectively, our results revealed an important role for exosomes in the HSPC supporting capacity of stromal cells. This work provides new insights in our understanding of the molecular and cellular mechanisms involved in the cross-talk between HSPCs and their niches. It may also have interesting applications in regenerative medicine, regarding the ex vivo manipulation of HSCs in stromal-free conditions for cell therapy.