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Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model

ABSTRACT: The objective of this study was to explore the characteristics and roles of extracellular vesicles (EVs) obtained from human mesenchymal stem cells (MSCs) under two culture conditions: undifferentiated MSCs (Naïve-EVs) and MSCs undergoing early-stage osteogenesis (Osteo-EVs). Additionally, the study aimed to evaluate the potential of Osteo-EVs to induce bone formation for the purpose of regenerating bone defects in rat calvaria. To accomplish these goals, EVs from both groups were isolated using a technique called size-exclusion chromatography. The isolated EVs were then subjected to various analyses to gain insights into their properties. This included examining their size distribution, morphology, flow cytometry analysis, and proteome profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with Label Free Quantification.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell, Bone Marrow

SUBMITTER: Niyaz Alsharabi

LAB HEAD: Kamal Mustafa

PROVIDER: PXD043335 | Pride | 2024-01-10

REPOSITORIES: Pride

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Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. To analyse whether the BM-MSC and Fat-MSC (ASC) differentiation stage may define the sarcoma phenotype, RbloxP/loxPp53loxP/loxP BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage and both Rb and p53 were excised with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre) at different stages (day 0 and 10) along osteogenic differentiation. NSG mice were inoculated subcutaneously with 5M-CM-^W10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using: WT BM-MSCs and ASCs, Rb-/-p53-/- BM-MSCs and ASCs previously differentiated to the osteogenic lineage for 10 days and a tumor cell line derived from p53-/-Rb-/- BM-MSC differentiated to the osteogenic lineage for 10 days.

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Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model

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