Do endothelial cells belong to the primitive stem leukemic clone in CML? Role of extracellular vesicles

Ramos, Teresa Lopes; Sánchez‐Abarca, Luis Ignacio; López-Ruano, Guillermo; Muntión, Sandra; Preciado, Silvia; Hernández-Ruano, Montserrat; Rosado, Belén; Heras, Natalia de las; Chillón, M. Carmen; Hernández‐Hernández, Ángel; González, Marcos; Sánchez‐Guijo, Fermín; Cañizo, Consuelo del

doi:10.1016/j.leukres.2015.05.014

Cited by 14 publications

(13 citation statements)

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“…We have also shown that MSC‐EV carry bioactive molecules and can transfer mRNA and proteins to the target cells . The bioactive cargo enclosed in MSC‐EV may play a role in favoring hematopoietic engraftment.…”

Section: Discussionmentioning

confidence: 87%

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The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability

et al. 2019

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Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34 + cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34 + cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34 + cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34 + cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34 + cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34 + cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34 + cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34 + cells, increasing their clonogenic capacity and their bone marrow lodging ability. STEM CELLS 2019;37:1357-1368 SIGNIFICANCE STATEMENTIn the current study, the authors validate for the first time that preincubating human CD34 + cells with extracellular vesicles derived from human mesenchymal stromal cells not only modifies the gene expression of the recipient cells (inducing a downregulation of proapoptotic genes and overexpression of genes involved in colony formation and JAK-STAT pathway) but also significantly increases their in vitro clonogenic ability and, most importantly, increases their 4-week bone marrow lodging ability in vivo in a standard xenotransplantation model. This strategy could potentially be exploited to increase the hematopoietic engraftment in the clinical setting.

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Section: Discussionmentioning

confidence: 87%

“…To allow their tracking before coculture, EV were stained with Vybrant Dil cell labeling solution (Life Technology), following manufacturer's instructions . As a control, an ultracentrifugation tube was included containing only phosphate‐buffered saline and Vybrant Dil.…”

Section: Methodsmentioning

confidence: 99%

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability

et al. 2019

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“…Interestingly, a recent study provided preliminary evidence that EVs released by CML cells can mediate the transfer of BCR-ABL mRNA to endothelial cells (42).…”

Section: Chronic Myeloid Leukemia (Cml)mentioning

confidence: 99%

Endothelial progenitor cells in hematologic malignancies

Testa

Saulle

Castelli

et al. 2016

Stem Cell Investig.

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“…Previous studies reported by our group showed that BCR-ABL cell release an EV containing the oncogene (mRNA), that can be incorporated into endothelial cells leading to the expression of the oncoprotein in negative BCR-ABL cells (Ramos, et al 2015). In the context of MPN, other groups also showed that vesicles released by CML cell lines induce the reduction of VCAM-1 and CXCL12 expression in endothelial cells, demonstrating the capacity of EV of reprogramming the microenvironment (Taverna, et al 2014).…”

Section: Extracellular Vesiclesmentioning

confidence: 69%

“…In myelodysplastic syndromes we have shown that microvesicles from MSC of these patients change HPC properties, modifying the expression of important genes (MDM2 and TP53), and increasing their clonogenic capacity (Muntion, et al 2016). Recently we also have shown that CML cells derived EV, which carry the transcript BCR-ABL that can be incorporated in endothelial cells, leading to the expression of the oncogene and oncoprotein in these cells (Ramos, et al 2015). Other studies showed that the transference of the BCR-ABL gene from CML-derived EV to neutrophils may provide in vivo transformation of normal cells (Cai, et al 2014).…”

Section: Extracellular Vesicles From Bm-mscmentioning

confidence: 99%

Role of Mesenchymal Stromal Cells and their Extracellular Vesicles microRNAs in JAK2 Myeloproliferative Neoplasms

Ramos¹,

Conceiçao²

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Compared to HD, BM-MSC from MPN patients showed similar morphology and differentiation capacity, with an increased proliferation rate with less apoptotic cells. BM-MSC from MPN expressed comparable levels of CD73, CD44, CD90 and CD166, whereas they were negative for haematopoietic markers. The median expression of CD105 was lower in BM-MSC from MPN patients (p <0.05) when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD. RT-PCR was performed in two genes to confirm these results, demonstrating that HDAC8 and MYADM genes were upregulated. Next, we observed a significant increase in the number of CFU-GM when MPN-HPC were co-cultured with MPN-MSC compared to HD-MSC. MPN-MSC also showed the ability to support healthy HPC in LTBMC. However, MPN-MSC showed alterations in the expression of genes associated to the maintenance of haematopoiesis. The inhibition of HDAC8 in BM-MSC from MPN was confirmed by RT-PCR and WB assays, when the cells were treated with PCI34051. HDAC8-selective inhibition also induced a cell cycle arrest in the MPN BM-MSC, with an increase of the percentage of apoptotic cells. Co-cultures of BM-MNC from MPN patients with neoplastic stroma previously treated with HDAC8 inhibitor induced a decrease in MNC cell viability (p=0.028), CFU-GM (p=0.018) and an increase of TP53 expression. Regarding the EV studies, we showed that the characterisation by TEM and NanoSight revealed that EV-MSC from both groups exhibited a size and morphology characteristic of EV, and were positive for CD63 (WB), the characteristic marker of EV. By MFC, EV released from BM-MSC of both groups (HD and JAK2) were defined as particles less than 1µM of diameter, positive for CD90, CD44 and CD73 and for EV markers. At the same time they were negative for CD34 and CD45, demonstrating the specificity of monoclonal labelling. When the content of microRNA was analysed (8 HD-MSC and 11 MPN-MSC), we observed an overall increase in the microRNA expression in the EV from patients, yet without reaching statistical significance. Using RT-PCR, we observed a significant overexpression (p=0.032) of miR-155 in the EV derived from MPN-MSC. We also observed an increase of CD34+ cell viability, after the incorporation of EV from both groups (HD and JAK2). A significant increase (p=0.04) of miR-155 expression was observed in the HD CD34+ cells, after incorporating MPN-MSC-derived EV. In addition, an increase of CFU-GM was observed when neoplastic CD34+ cells incorporated the EV derived from MSC from MPN patients (p=0.056). Conclusions These results suggest that MPN-MSC display different proliferative rate, immunophenotypic markers, gene expression profile and HDAC8 overexpression compared to HD-MSC. The inhibition of HDAC8 expression by its specific inhibitor decreases the capacity of the stroma to support haematopoietic cells from MPN patients, suggesting that HDAC8 may be a potential therapeutic target. Furthermore, we suggest that EV rel...

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Do endothelial cells belong to the primitive stem leukemic clone in CML? Role of extracellular vesicles

Cited by 14 publications

References 20 publications

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability

Endothelial progenitor cells in hematologic malignancies

Role of Mesenchymal Stromal Cells and their Extracellular Vesicles microRNAs in JAK2 Myeloproliferative Neoplasms

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