Journal: Cells

Article Title: Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal

doi: 10.3390/cells13060484

Figure Lengend Snippet: ( A ) Gene construct of adenovirus vector containing the gene for CGRP 8–37 . The CGRP 8–37 fragment was inserted downstream of the signal sequence of peptidylglycine-amidating monooxygenase (ssPAM/pGEMT). This arrangement facilitates the amidation and secretion of CGRP 8–37 . ( B ) MSC were isolated from human infrapatellar fat pad (IFP), transduced with gene construct, and sorted by FACS cell sorting to generate aCGRP IFP-MSC. aCGRP IFP-MSC sEVs were isolated and characterized for their miRNA cargo. Functional assessment of aCGRP IFP-MSC sEVs was performed by macrophage polarization and cortical neurons neuroinflammation assays.

Article Snippet: Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia; ) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC, Waltham, MA, USA).

Techniques: Construct, Plasmid Preparation, Sequencing, Isolation, Transduction, FACS, Functional Assay

Journal: Cells

Article Title: Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal

doi: 10.3390/cells13060484

Figure Lengend Snippet: ( A ) 147 distinct miRNA were present in aCGRP IFP-MSC sEVs (n = 2). Nineteen highly present miRNAs are included in black-bordered box. ( B ) miRNAs present in aCGRP IFP-MSC sEVs were involved in the regulation of numerous genes and pathways. Predominantly, miRNAs present in sEVs are involved in the regulation of TGF-β/Wnt/FGFR pathways. Putative miRNA interactomes were generated using a miRNet centric network visual analytics platform. The miRNA target gene data were collected from well-annotated database miRTarBase v8.0 and miRNA-gene interactome network refining was performed with 2.0 betweenness cut-off. Values (with 34 cycles cut-off point) were represented in a topology miRNA-gene interactome network using force atlas layout and hypergeometric test algorithm.

Article Snippet: Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia; ) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC, Waltham, MA, USA).

Techniques: Generated, Refining

Journal: Cells

Article Title: Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal

doi: 10.3390/cells13060484

Figure Lengend Snippet: ( A ) In aCGRP IFP-MSC sEVs (n = 2), 19 miRNAs cargos were highly present. These distinct miRNAs regulate genes involved in the production of cytokines, the recruitment of monocytes, and cartilage homeostasis. From these miRNAs, 7 miRNAs were associated in previous studies with significant anti-inflammatory/immunomodulatory effects in vitro and in vivo. Putative miRNA interactomes were generated using a miRNet centric network visual analytics platform. The miRNA target gene data were collected from well-annotated database miRTarBase v8.0 and miRNA-gene interactome network refining was performed with 2.0 betweenness cut-off. Values (with 34 cycles cut-off point) were represented in a topology miRNA-gene interactome network using force atlas layout and hypergeometric test algorithm. ( B ) In silico analysis revealed a functional correlation of identified miRNAs in aCGRP IFP-MSC sEVs with genes involved in M2 macrophage polarization, immunomodulatory and pain signaling, and cartilage homeostasis. The miRDB online database for prediction of functional miRNA targets has been used to correlate highly expressed target genes in macrophages with specific miRNAs identified by aCGRP IFP-MSC sEV miRNA profiling. MirTarget prediction scores are in the range of 0–100% probability, and candidate transcripts with scores ≥ 50% are presented as predicted miRNA targets in miRDB.

Article Snippet: Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia; ) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC, Waltham, MA, USA).

Techniques: In Vitro, In Vivo, Generated, Refining, In Silico, Functional Assay

Journal: Cells

Article Title: Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal

doi: 10.3390/cells13060484

Figure Lengend Snippet: ( A , B ) Multiple immunomodulatory and reparative molecules secreted as a cargo of aCGRP IFP-MSC sEVs (n = 2). sEVs showed presence of key immunomodulatory molecules including TIMP-2, IL-8, MCP-1, IL-6, ICAM-1, sTNF-RI, MIP-1β, IL-10, and IP-10. In parallel, key reparative molecules including HGF, VEGF, EGFR, IGFBP-1, βFGF, and IGFBP-6 showed presence in aCGRP IFP-MSC sEVs. These proteins are listed in descending order based on their presence levels within sEVs. The miRDB online database for prediction of functional miRNA targets has been used to correlate highly expressed target genes in macrophages with specific miRNAs identified by aCGRP IFP-MSC sEV miRNA profiling. In terms of biological processes, various categories were highly affected and presented as % of proteins involved in a category to the total proteins detected. aCGRP IFP-MSC sEVs protein cargo have effects on PI3K-Akt signaling pathway (59%), MAPK signaling pathway (55%), Ras signaling pathway (52%), Rap1 signaling pathway (43%), cytokine-cytokine receptor interaction (24%), and Jak-STAT signaling pathway (19%). MirTarget prediction scores are in the range of 0–100% probability, and candidate transcripts with scores ≥ 50% are presented as predicted miRNA targets in miRDB.

Article Snippet: Pre-designed human MSC exosome 166 miRNA qPCR arrays (GeneCopoeia; ) were performed using 1000 ng cDNA per IFP-MSC sample (n = 2), and processed using StepOne Real-time thermocycler (Applied Biosystems, LLC, Waltham, MA, USA).

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Perfusion Bioreactor Culture Incorporating Mechanical Confinement Enhances Mesenchymal Stem Cell Extracellular Vesicle Production and Wound Healing Potential

doi: 10.1101/2025.08.12.669872

Figure Lengend Snippet: A) Timeline of experiment setup and injection schedule. Mice received injections of 1.1×10 10 EVs or an equivalent volume of PBS on days 0, 3, 6, and 9. B) Representative photos of splinted wounds receiving each treatment over 14 days (n=4-6). C) Percent wound closure based on wound area over 14 days (n=4-6). D) Fold change of M1 markers CD86, iNOS, and TNF11 and E) M2 markers IL-10, CD206, and Arg1 in harvested tissue from mice receiving flask and bioreactor EV injections relative to tissue from mice receiving PBS, as quantified by qPCR. Data represents 4 biological replicates analyzed with 3 technical replicates each per treatment group (n=4). F) Length of regenerating epithelium from H&E staining as a percentage of the length of the wound bed (n=4-6). G) Collagen deposition from Masson’s Trichrome staining quantified as a percentage of the total tissue volume (n=4-6). G) Granulation tissue area from H&E staining (n=4-6). I) CD31 fluorescence intensity normalized to DAPI over multiple fields of view and representative confocal microscopy images taken at 10X magnification (n=4-5). All values expressed as mean +/- standard error of the mean (ns – no significance; * p<0.05; ** p<0.01).

Article Snippet: Resulting cDNA was analyzed in a human MSC exosome miRNA qPCR array (GeneCopoeia; QM0460B6) per the manufacturer’s protocol.

Techniques: Injection, Staining, Fluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: Perfusion Bioreactor Culture Incorporating Mechanical Confinement Enhances Mesenchymal Stem Cell Extracellular Vesicle Production and Wound Healing Potential

doi: 10.1101/2025.08.12.669872

Figure Lengend Snippet: A) Heat map of the miRNA content of 5 µm static and 5 µm bioreactor EVs represented as the log2(fold change) relative to flask EVs (n≥2). B) Volcano plot comparing the miRNA within static and flask EVs, with the horizontal line at 1.3 (p = 0.05) representing statistical significance (n≥2). C) Volcano plot comparing the miRNA within bioreactor and flask EVs, with the horizontal line at 1.3 (p = 0.05) representing statistical significance. The labeled points indicate those in common between both static and bioreactor EVs (n≥2). All data represents the average of at least 2 independent experiments.

Article Snippet: Resulting cDNA was analyzed in a human MSC exosome miRNA qPCR array (GeneCopoeia; QM0460B6) per the manufacturer’s protocol.

Techniques: Labeling

Journal: Biomedicines

Article Title: Neuroprotective Effects of Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cell Extracellular Vesicles in Ischemic Stroke Models

doi: 10.3390/biomedicines11092550

Figure Lengend Snippet: Characterization of hiPS-MSCs derived extracellular vesicles (hiPS-MSCs-EV) ( A ). Flowchart of hiPS-MSC-EVs isolation from the culture medium. ( B ). Morphology of hiPS-MSC-EVs under transmission electron microscopy. ( C ). Representative bioanalyzer profile of the RNA contained in hiPS-MSC-EVs. ( D ). Western blotting analysis of exosomal positive markers CD63, CD9, and CD81 and negative marker Calnexin in MSC lysate and hiPS-MSC-EVs. Tubulin was used as the loading control. ( E ). Flow cytometry analysis of positive marker CD63 expression after binding to CD9 and CD81 antibody-coated beads. The solid gray line represents cells that were unstained. while solid pink line represents the experimental samples. ( F ). Particle size distribution of iPSC-MSC-EVs measured using a flow nanoanalyzer.

Article Snippet: The cells were incubated with primary antibodies from the human MSC analysis kit (Cat#562245, BD Biosciences, San Jose, CA, USA) to identify the characteristic human MSC surface markers.

Techniques: Derivative Assay, Isolation, Transmission Assay, Electron Microscopy, Western Blot, Marker, Flow Cytometry, Expressing, Binding Assay