EXOSOMES SECRETED BY STROMAL CELLS CONTRIBUTE TO THE HEMATOPOIETIC STEM CELL NICHE
(Abstract release date: 05/19/16)
EHA Library. Stik G. 06/09/16; 135367; LB2256
Dr. Gregoire Stik
Contributions
Contributions
Abstract
Abstract: LB2256
Type: Eposter Presentation
Background
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.
Aims
This study aims to assess the existence and the functionality of stromal cell-derived exosomes in the HSPC support.
Methods
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 were performed to characterize the exosomal fraction isolated. Using PKH67 staining we analyze the uptake of exosomes after co-culture with Lin- Sca1+ cKit+ (LSK) cells, total bone marrow or after in vivo injection. Clonogenic assay and FACS analysis were performed after co-culture of LSK cells with exosomes to assay their biological effect. High-throughput sequencing was realized to explore the molecular signature followed by several bioinformatic analyses. Finally, RNA transfer from exosomes to recipient cells was analyzed by qPCR.
Results
Electron microscopy performed on stromal cells showed multivesicular bodies containing exosomes. Additional electron microscopy, FACS and western blot analyses performed on the fraction isolated by ultracentrifugation revealed that both AFT and BFC stromal cells secrete exosomes. We then investigate if these exosomes could be taken up by HSPCs. 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. Furthermore, in vitro and in vivo assays showed that AFT exosomes specifically target hematopoietic CD45+ cells. 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 324 mRNAs and 23 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.
Conclusion
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.
Session topic: E-poster
Keyword(s): Hematopoietic stem and progenitor cells, Microenvironment, Microvesicles, Stromal cell
Type: Eposter Presentation
Background
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.
Aims
This study aims to assess the existence and the functionality of stromal cell-derived exosomes in the HSPC support.
Methods
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 were performed to characterize the exosomal fraction isolated. Using PKH67 staining we analyze the uptake of exosomes after co-culture with Lin- Sca1+ cKit+ (LSK) cells, total bone marrow or after in vivo injection. Clonogenic assay and FACS analysis were performed after co-culture of LSK cells with exosomes to assay their biological effect. High-throughput sequencing was realized to explore the molecular signature followed by several bioinformatic analyses. Finally, RNA transfer from exosomes to recipient cells was analyzed by qPCR.
Results
Electron microscopy performed on stromal cells showed multivesicular bodies containing exosomes. Additional electron microscopy, FACS and western blot analyses performed on the fraction isolated by ultracentrifugation revealed that both AFT and BFC stromal cells secrete exosomes. We then investigate if these exosomes could be taken up by HSPCs. 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. Furthermore, in vitro and in vivo assays showed that AFT exosomes specifically target hematopoietic CD45+ cells. 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 324 mRNAs and 23 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.
Conclusion
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.
Session topic: E-poster
Keyword(s): Hematopoietic stem and progenitor cells, Microenvironment, Microvesicles, Stromal cell
Abstract: LB2256
Type: Eposter Presentation
Background
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.
Aims
This study aims to assess the existence and the functionality of stromal cell-derived exosomes in the HSPC support.
Methods
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 were performed to characterize the exosomal fraction isolated. Using PKH67 staining we analyze the uptake of exosomes after co-culture with Lin- Sca1+ cKit+ (LSK) cells, total bone marrow or after in vivo injection. Clonogenic assay and FACS analysis were performed after co-culture of LSK cells with exosomes to assay their biological effect. High-throughput sequencing was realized to explore the molecular signature followed by several bioinformatic analyses. Finally, RNA transfer from exosomes to recipient cells was analyzed by qPCR.
Results
Electron microscopy performed on stromal cells showed multivesicular bodies containing exosomes. Additional electron microscopy, FACS and western blot analyses performed on the fraction isolated by ultracentrifugation revealed that both AFT and BFC stromal cells secrete exosomes. We then investigate if these exosomes could be taken up by HSPCs. 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. Furthermore, in vitro and in vivo assays showed that AFT exosomes specifically target hematopoietic CD45+ cells. 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 324 mRNAs and 23 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.
Conclusion
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.
Session topic: E-poster
Keyword(s): Hematopoietic stem and progenitor cells, Microenvironment, Microvesicles, Stromal cell
Type: Eposter Presentation
Background
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.
Aims
This study aims to assess the existence and the functionality of stromal cell-derived exosomes in the HSPC support.
Methods
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 were performed to characterize the exosomal fraction isolated. Using PKH67 staining we analyze the uptake of exosomes after co-culture with Lin- Sca1+ cKit+ (LSK) cells, total bone marrow or after in vivo injection. Clonogenic assay and FACS analysis were performed after co-culture of LSK cells with exosomes to assay their biological effect. High-throughput sequencing was realized to explore the molecular signature followed by several bioinformatic analyses. Finally, RNA transfer from exosomes to recipient cells was analyzed by qPCR.
Results
Electron microscopy performed on stromal cells showed multivesicular bodies containing exosomes. Additional electron microscopy, FACS and western blot analyses performed on the fraction isolated by ultracentrifugation revealed that both AFT and BFC stromal cells secrete exosomes. We then investigate if these exosomes could be taken up by HSPCs. 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. Furthermore, in vitro and in vivo assays showed that AFT exosomes specifically target hematopoietic CD45+ cells. 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 324 mRNAs and 23 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.
Conclusion
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.
Session topic: E-poster
Keyword(s): Hematopoietic stem and progenitor cells, Microenvironment, Microvesicles, Stromal cell
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