Bone Abstracts

Introduction: Umbilical cord blood (UCB) is increasingly used in hematopoietic stem cell (HSC) transplantations; however, the low cell numbers are still remaining as a limiting factor for proper engraftment. Osteoblasts are major constituents of HSC niche and 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. Therefore, in this study we focused on the characterization of human osteoblast EVs and investigated their potential in ex vivo expansion of human UCB–HSCs for clinical use.

Methods: We used human pre-osteoblasts (SV-HFO cells) to isolate EVs by a series of ultracentrifugation steps. We characterized osteoblast EVs by electron microscopy, proteomics, and miRNA sequencing, and investigated their effect on CD34⁺ UCB cells by single-platform counting and subset immunophenotyping using flow cytometry and qPCR.

Results: Treatment of CD34⁺ UCB cells with osteoblast EVs led to twofold expansion of the phenotypic HSCs three- and fivefold expansion of the CD34⁺ expressing progenitors. Microscopic analyses demonstrated that osteoblast EVs are very heterogenic in size and morphology. Mass spectrometry-based proteomic analyses identified an interesting range of novel osteoblast EV proteins primarily linked to ribosomal activity and RNA processing in addition to the well-known vesicle proteins. Moreover, EVs were enriched with small RNAs, and contained miRNAs known be involved in the regulation of early hematopoiesis. Interestingly, we discovered that EV treatment downregulated the expression of HMG-box transcription factor 1 (HBP1), which is one of the miR-29a targets, in CD34⁺ UCB cells.

Conclusion: In this study we demonstrated that osteoblasts secrete EVs that hold the potential to expand UCB–HSCs ex vivo. Elucidating the molecular mechanism of EV function is likely to provide us the means to increase the expansion efficiency and develop improved grafts for stem cell transplantations.