Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modulate Hematopoietic Stem and Progenitor Cell Viability and the Expression of Cell Cycle Regulators in an Age-dependent Manner

Abstract

Aging of the hematopoietic system is characterized by an expansion of hematopoietic stem and progenitor cells (HSPCs) with reduced capacity for engraftment, self-renewal, and lymphoid differentiation, resulting in myeloid-biased hematopoiesis. This process is mediated by both HSPC intrinsic and extrinsic factors, e.g., the stromal environment. A relevant cellular component of the bone marrow (BM) microenvironment are mesenchymal stromal cells (MSCs) which regulate fate and differentiation of HSPCs. The bi-directional communication with HSPCs is mediated either by direct cell-cell contacts or by extracellular vesicles (EVs) which carry bioactive substances such as small RNA, DNA, lipids and proteins. So far, the impact of MSC-derived EVs on human hematopoietic aging is poorly investigated. BM MSCs were isolated from young (n = 3, median age: 22 years) and aged (n = 3, median age: 70 years) donors and the EVs were isolated after culturing the confluent cell layer in serum-free medium for 48 h. CD34+ HSPCs were purified from peripheral blood of healthy donors (n = 3, median age: 65 years) by magnetic sorting. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot detection of EV markers CD63, CD81 and Flotillin-1 revealed no significant differences between young and aged MSC-EVs. Interestingly, young MSCs secreted a significantly higher miRNA concentration than aged cells. However, the amount of distinct miRNAs such as miR-29a and miR-34a was significantly higher in aged MSC-EVs. HSPCs incubated with young EVs showed a significant increase in cell number and a higher viability. The expression of the tumor suppressors PTEN, a known target of mir-29a, and CDKN2A was increased in HSPCs incubated with young EVs. The clonogenic assay demonstrated a decreased colony number of CFU-GM after treatment with young EVs and an increased number of BFU-E/CFU-E after incubation with aged MSC-EVs. Xenogenic transplantation experiments showed no significant differences concerning the engraftment of lymphoid or myeloid cell compartments, but the overall human chimerism 8–16 weeks after transplantation was higher after EV treatment. In conclusion, our data suggest that HSPC characteristics such as cell cycle activity and clonogenicity can be modulated by MSC-derived EVs. Further studies have to elucidate the potential therapeutic relevance of our findings.

Characterization of a Stemness-Optimized Purification Method for Human Dental-Pulp Stem Cells: An Approach to Standardization

“…Clonogenicity describes the feature of cells to form cell clones, i.e., colonies of cells originating from a single cell. Since whole tissues and organs are derivates of SCs, clonogenicity can be seen as a basic feature of these types of cells [ 105 , 106 ]. Therefore, limiting dilution was applied to create single cell isolates, which were then checked for their ability to form colonies [ 107 ].…”

Non-Classical Intercellular Communications: Basic Mechanisms and Roles in Biology and Medicine

“…As a result, EVs from donors older than 40 years stimulated HSCs, in contrast to the EVs from younger persons. A new study [ 103 ] examined the modulation of cell cycle activity and clonogenicity of HSCs by extracellular vesicles. EVs from young MSCs were able to support the expansion of HSCs in vitro, while vesicles from old MSCs did not have a positive effect on cell survival and proliferation.…”

The potential therapeutic role of extracellular vesicles in osteoarthritis

“…The mechanism underlying the MSCs therapeutic effect might be secretion of bioactive molecules (Eleuteri and Fierabracci, 2019), additionally, application of EVs secreted from MSCs possess inherent advantages compared to direct MSC therapy (lower immunogenicity, tumorigenicity, etc.) (Ankrum et al, 2014;Fichtel et al, 2022). Thus, EVs therapy has gained more and more attention in disease treatment and tissue reconstruction.…”