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

Abstract

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.

Extracellular Vesicles After Allogeneic Hematopoietic Cell Transplantation: Emerging Role in Post-Transplant Complications

“…In agreement, a recent work showed that human BM-MSC-EVs can up-regulate the JAK/STAT pathway and increase the levels of phospho-STAT5 in in vitro-cultured CD34 + cells, enriched from leukapheresis (97). The involvement of the JAK/STAT signaling pathway in CD34 + cell proliferation is important in several hematologic neoplasms, including myelodysplastic syndromes and acute myeloid leukemia (AML).…”

“…Gene expression profile of CD34 + cells is also modulated by human MSC-EV-derived miRNAs through repression of the Wnt/β-catenin signaling pathway (42). Furthermore, both murine and human BM-MSC-EVs showed anti-apoptotic effect on CD34 + cells (97,99). When human CD34 + cells are co-cultured with human BM-MSC-EVs, there is an up-regulation of anti-apoptotic genes, such as BIRC2, BIRC3, and NFKB, a down-regulation of proapoptotic genes, including CASP3 and CASP6, and a decreased phosphorylation of H2AX.…”

“…Indeed, it has been observed that human BM-MSC-EVs can up-regulate CXCR4 expression in CD34 + HSCs, increasing their migration from the PB to the BM niche (96). Very recent findings supported this enhanced HSC migratory ability both in vitro and in vivo in the presence of human BM-MSC-EV stimulation, although the CXCR4 up-regulation was not confirmed (97).…”

“…In addition, it has been shown that this pathway plays a significant role in promoting cell survival (98). As shown in both humans and mice, MSC-EV treatment could also modify the gene expression profile of CD34 + cells and favor survival directly or indirectly, through microRNAs and Piwiinteracting RNAs (96,97,99). Gene expression profile of CD34 + cells is also modulated by human MSC-EV-derived miRNAs through repression of the Wnt/β-catenin signaling pathway (42).…”

Mesenchymal stromal cell‐derived extracellular vesicles as cell‐free biologics for the ex vivo expansion of hematopoietic stem cells

“…A hematopoietic xenotransplantation model evaluated the in vivo impacts of incorporating MSC‐derived EVs. They noted that when injected into the femur, the EVs improved the capacity of CD34 cells to lodge in the BM for 4‐week (Preciado et al, 2019).…”