Umbilical cord blood (UCB) is increasingly used in haematopoietic stem cell (HSC) transplantations; however, the low cell numbers are still remaining as a limiting factor for proper engraftment. Osteoblasts play important roles in regulating HSC self-renewal and differentiation. Recently, extracellular vesicles (EVs) have been implicated in stem cell fate regulation via horizontal transfer of proteins and nucleic acids between cells. In this study, we focused on the characterisation of osteoblast EVs and investigated their potential in ex vivo expansion of CD34+ UCB cells for clinical use. We used human pre-osteoblasts (SVHFO cells) to isolate EVs, and characterised EVs by electron microscopy, proteomics, and RNA sequencing, and investigated their functional effect on human CD34+ UCB cells by qPCR and flow cytometry. Characterisation analyses demonstrated that osteoblast EVs are heterogenic in size, contain novel osteoblast EV proteins primarily linked to ribosomal activity and RNA processing, and are enriched with small RNAs. Treatment of CD34+ UCB cells with osteoblast EVs led to donor-dependent two to threefold expansion (P<0.01) of the CD34+ expressing progenitors in 10 days. MicroRNA profiling demonstrated that osteoblast EVs contain abundant amounts of miR-29a, one of the key regulators of early haematopoiesis. Interestingly, EVs treatment led to the twofold down-regulation (P<0.01) of HBP1, a miR-29a target that has been shown to be a cell cycle inhibitor, in CD34+ UCB cells. Consequently, cell cycle analysis showed that EVs stimulated progression from G0/G1 to S/G2 phase (P<0.05), which may explain the mechanism by which EVs stimulate UCB cell expansion. Finally, EV-expanded CD34+ UCB cells showed good clonogenicity and differentiation potential in vitro and successful engraftment in a NOD/SCID-IL2Rγ (NSG mice) xenograft model in vivo. In this study, we demonstrated that osteoblasts secrete EVs that expand UCB cells ex vivo, and uncovered the first clues that contributed to the understanding of EV function.
Disclosure: The authors declared no competing interests. Erasmus MC Stem Cell and Regenerative Medicine Institute.