Evaluating the Endocytosis and Lineage-Specification Properties of Mesenchymal Stem Cell Derived Extracellular Vesicles for Targeted Therapeutic Applications

Abstract

Mesenchymal stem cells (MSCs) are multipotent cells with regenerative and immunomodulatory properties. Several aspects of MSC function have been attributed to the paracrine effects of MSC derived extracellular vesicles (EVs). Although MSC EVs show great promise for regenerative medicine applications, insights into their uptake mechanisms by different target cells and the ability to control MSC EV properties for defined function in vivo have remained elusive knowledge gaps. The primary goal of this study is to elucidate how the basic properties of MSC derived EVs can be exploited for function-specific activity in regenerative medicine. Our first important observation is that, MSC EVs possess a common mechanism of endocytosis across multiple cell types. Second, altering the MSC state by inducing differentiation into multiple lineages did not affect the exosomal properties or endocytosis but triggered the expression of lineagespecific genes and proteins in vitro and in vivo respectively. Overall, the results presented in this study show a common mechanism of endocytosis for MSC EVs across different cell types and the feasibility to generate functionally enhanced EVs by modifications to parental MSCs.

Mesenchymal stem cells secretome: The cornerstone of cell-free regenerative medicine

“…In fact, inhibitors of these surface molecules have been shown to decrease EVs uptake[ 86 ]. EVs uptake requires energy and is also cholesterol-dependent[ 88 , 89 ]. Endocytic uptake after EVs recognition can be as diverse as the diversity of EVs and acceptor cells.…”

“…Phagocytosis of EVs by monocytes is described to decrease in preeclampsia due to higher expression of CD47, which inhibits EVs uptake[ 90 ]. However, the main pathways described for EVs uptake are caveolar[ 88 , 89 , 91 ] and clathrin endocytic pathways, being the last pathways mediated by integrins[ 86 ] and by the dynamin GTPase[ 92 ]. In addition, other investigations show that EVs cargo can also be delivered by lipid raft endocytosis[ 82 , 89 ] and by membrane fusion[ 82 , 93 ].…”

“…The therapeutic effect of EVs secreted by MSCs has also been described in dental pulp stem cells (DPSCs)[ 91 ] where Exos derived from DPSCs undergoing osteogenic differentiation are able to interact with fibronectin and collagen type I fibres of the ECM and promote teeth regeneration[ 91 ]. Importantly, there seems to be marked differences, probably due to changes on EVs cargo, in the effect over acceptor cells between Exos secreted by naïve and differentiated cells[ 89 ]. According to Narayanan et al [ 99 , 100 ], Exos from MSCs growing in osteogenic differentiation media were the most effective in inducing osteogenic differentiation to other MSCs[ 99 , 100 ].…”

The importance of cellular and exosomal miRNAs in mesenchymal stem cell osteoblastic differentiation

“…MSC conditioned media can substitute for the MSC in directing bone regeneration 28 . More recent studies have extended our understanding of exosomes as key signaling components of the MSC secretome 5 , 29 , 30 and the treatment of experimental osseous defects with MSC exosomes enhances bone regeneration 31 – 33 . This use of MSC exosomes in bone regeneration has potential advantages versus MSCs themselves.…”

“…miRNAs are small non-coding RNAs (21–23 nucleotides) that function to regulate the expression of target genes by repression of gene transcription or by targeted degradation of specific mRNAs or by inhibiting protein translation. The miRNA cargo of MSC exosomes is representative of the parental cell status and is specific to the cell type and cellular state 5 . Individual miRNAs function in regulation of gene expression and cell physiology 6 and have used knockout strategies, synthetic miRNA mimics or their antagomirs to identify the functions of individual miRNAs.…”