Journal: Journal of Orthopaedic Translation

Article Title: Endocytosis-associated patterns in nerve regeneration after peripheral nerve injury

doi: 10.1016/j.jot.2021.09.004

Figure Lengend Snippet: The cellular uptake of exosomes into Schwann cells (A) The exosomes were labeled with CM-Dil (red), the cytoskeletal was labeled by F-actin/phalloidin (green) and the nuclei was labeled by Hoechst 33,342 (blue). All cells were detected after coculture with exosomes for 0, 1, 2, 3, 4 ​h, respectively. Scale bar: 20 ​μm ​(B and C) The diameter and particles concentration of exosomes were 141.7 ​nm and 2.97 ​× ​10 7 particles/mL, respectively (D) The size distribution of exosomes was 50–200 ​nm (E) The ASCs showed higher cellular uptake ability than NSCs from 1 to 4 ​h. ASCs, Activated Schwann cells. NSCs, Normal Schwann cells.

Article Snippet: For the cellular uptake of exosomes (hTERT MSC Exosomes, ATCC, USA), both ASCs and NSCs were seeded with 500 cells per well on the Cell Culture Slides (Corning®, USA).

Techniques: Labeling, Concentration Assay

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

doi: 10.1080/21691401.2019.1623232

Figure Lengend Snippet: Figure 1. Characterization of miR-185 enriched MSC-EVs. (A) Isolated miR-185 MSC-EVs were characterized by TEM. Representative image is shown (scale bar ¼ 50 nm). (B) NTA was performed on the EVs to determine their concentration and size. EV diameter was measured and represented as mean ± SD, n ¼ 3 independent experiments performed in triplicate. Western blotting (insets) for EV markers CD9, CD81 and flotillin-1. (C) qPCR analysis for expression of miR-185 in EVs. n ¼ 5 inde- pendent experiments performed in triplicate (p < .01 versus Ctrl).

Article Snippet: MSC-EVs were probed with primary antibodies against CD81 (1:400 dilution, ProSci, Collins, CO), CD9 (1:300 dilution, Lifespan Bioscience Inc., Seattle, WA) and flotillin-1 (1:500 dilution, Abcam, Cambridge, MA) by Western blotting.

Techniques: Isolation, Concentration Assay, Western Blot, Expressing

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

doi: 10.1080/21691401.2019.1623232

Figure Lengend Snippet: Figure 2. EVs binding abilities. (A) Binding of miR-185 MSC-EVs to buccal mucosa. miR-185 enriched MSC-EVs were prelabelled with PKH26 red dye and pasted on mucosa surface of lesion in hamsters. Images were captured at 12 h after the treatment and tissue distribution of PKH26 stained EVs was evaluated by fluorescent microscopy (blue indicates DAPI, red indicates PKH26-stained EVs, scale bar ¼ 50 lm). Most fluorescent red staining was localized in the epithelial layer of the mucosa (insets). (B) Intercellular internalization of miR-185 MSC-EVs to OSCC cells (Cal27). Cal27 cells were incubated with PKH26 labelled miR-185 MSC-EVs for 12 h and cellular localization of PKH26 fluorescence was evaluated by fluorescent microscopy. Cal27 cells were incubated with unstained EVs as a negative control (blue indicates DAPI, red indicates PKH26-stained EVs). Images shown are representative of five independent experiments (scale bar ¼ 20 lm).

Article Snippet: MSC-EVs were probed with primary antibodies against CD81 (1:400 dilution, ProSci, Collins, CO), CD9 (1:300 dilution, Lifespan Bioscience Inc., Seattle, WA) and flotillin-1 (1:500 dilution, Abcam, Cambridge, MA) by Western blotting.

Techniques: Binding Assay, Staining, Microscopy, Incubation, Fluorescence, Negative Control

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

doi: 10.1080/21691401.2019.1623232

Figure Lengend Snippet: Figure 3. MiR-185 MSC-EVs delay the progression of OPMD in DMBA-induced hamsters. (A) Schematic diagram of miR-185 MSC-EVs treatment in a DMBA induced OPMD hamster model. (B) Representative gross images of the hamster buccal mucosa on weeks 0, 3, and 6 of DMBA exposure (upper, white arrows indicate inflam- matory exudation (middle) and white patches (right)). Representative gross images of in vitro buccal tissue (lower). (C) Serum from blood collected at the time of sacrifice was assayed for the level of ALT, AST, Scr and BUN.

Article Snippet: MSC-EVs were probed with primary antibodies against CD81 (1:400 dilution, ProSci, Collins, CO), CD9 (1:300 dilution, Lifespan Bioscience Inc., Seattle, WA) and flotillin-1 (1:500 dilution, Abcam, Cambridge, MA) by Western blotting.

Techniques: In Vitro

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

doi: 10.1080/21691401.2019.1623232

Figure Lengend Snippet: Figure 4. MiR-185 MSC-EVs suppress inflammation. (A) Inflammatory cells in buccal sections were stained with H&E (left, dotted squares indicate the area with dense inflammatory cells of buccal mucosa painted with DMBA, 10 sections per animal, n ¼ 5 in each group, scale bar ¼ 50 lm) and then quantified (right, miR-185 EVs versus DMBA-scramble, p < .05). (B) Protein array analysis showing altered expression of cytokines and chemokines in buccal tissue (left) and quantification (right). Numbers above the X axis indicate partial pro-inflammatory cytokines and chemokines, and numbers below the X axis indicate partial anti-inflammatory cytokines and chemokines. (C) Serum concentrations of IL-6, IL-1b and IL-10 were assessed by ELISA (n ¼ 5 in each group, miR-185 EVs versus DMBA-scramble, p < .05, p < .01). (D) The protein levels of IL-6 and IL-1b, and pathway-related protein of Akt, p-Akt, NF-jB and p-NF-jB in buccal tissue were determined by Western blotting (left) and quantified (right, n ¼ 5 in each group, miR-185 EVs versus DMBA-scramble, p < .05, p < .01).

Article Snippet: MSC-EVs were probed with primary antibodies against CD81 (1:400 dilution, ProSci, Collins, CO), CD9 (1:300 dilution, Lifespan Bioscience Inc., Seattle, WA) and flotillin-1 (1:500 dilution, Abcam, Cambridge, MA) by Western blotting.

Techniques: Staining, Protein Array, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

doi: 10.1080/21691401.2019.1623232

Figure Lengend Snippet: Figure 5. MiR-185 MSC-EVs alleviate pathological features and inhibit proliferation and angiogenesis. (A) Representative images of histopathological grading in buc- cal sections by H&E staining (arrows indicate the location of hyperplasia, dotted squares indicate the location of typical dysplasia with partially magnified images (right), 10 sections per animal, n ¼ 5 in each group, scale bar ¼ 50 lm (100 lm for Ctrl)). (B) Incidence of dysplasia on weeks 3, 4, 5 and 6 of DMBA exposure (n ¼ 5 in each group, p < .01). (C) Hyperplasia and dysplasia were quantified and visualized by scatter-plot diagram (n ¼ 5 in each group, p < .05). (D) IHC analysis of PCNA and CD31 protein expression levels in buccal sections. Homogenous brown nuclear and vascular endothelial staining indicate respectively the positive expres- sion for PCNA and CD31 (upper, arrows indicate positive expression, scale bar ¼ 50 lm). Staining was then quantified (lower, MOD: mean optical density, MVD: microvessel density determined by CD31 staining, n ¼ 5 in each group, miR-185 EVs versus DMBA-scramble, p < .05, p < .01). (E) The protein levels of PCNA and VEGFA in buccal tissue were determined by Western blotting (upper) and then quantified (lower, n ¼ 5 in each group, p < .01).

Article Snippet: MSC-EVs were probed with primary antibodies against CD81 (1:400 dilution, ProSci, Collins, CO), CD9 (1:300 dilution, Lifespan Bioscience Inc., Seattle, WA) and flotillin-1 (1:500 dilution, Abcam, Cambridge, MA) by Western blotting.

Techniques: Staining, Expressing, Western Blot

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

doi: 10.1080/21691401.2019.1623232

Figure Lengend Snippet: Figure 6. MiR-185 MSC-EVs induce apoptosis. (A) Apoptotic cells in buccal sections were determined by TUNEL assay (10 sections per animal, green indicates apop- totic bodies, blue indicates DAPI-stained nuclei, scale bar ¼ 20 lm). Positive (apoptotic) cells were quantified (right, n ¼ 5 in each group, p < .05). (B) Pro-caspase- 9, cleaved caspase-9, pro-caspase-3 and cleaved caspase-3 protein levels in buccal tissue were assessed by Western blotting (left) and were quantified (right, n ¼ 5 in each group, p < .05, p < .01).

Article Snippet: MSC-EVs were probed with primary antibodies against CD81 (1:400 dilution, ProSci, Collins, CO), CD9 (1:300 dilution, Lifespan Bioscience Inc., Seattle, WA) and flotillin-1 (1:500 dilution, Abcam, Cambridge, MA) by Western blotting.

Techniques: TUNEL Assay, Staining, Western Blot

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Delivery of mesenchymal stem cells-derived extracellular vesicles with enriched miR-185 inhibits progression of OPMD.

doi: 10.1080/21691401.2019.1623232

Figure Lengend Snippet: Figure 7. Schematic representation of the mechanism by which miR-185 encapsulated by MSC-EVs ameliorates OPMD by inhibition of inflammation and induction of apoptosis via Akt/NF-jB and Akt/caspase-9 related pathways.

Article Snippet: MSC-EVs were probed with primary antibodies against CD81 (1:400 dilution, ProSci, Collins, CO), CD9 (1:300 dilution, Lifespan Bioscience Inc., Seattle, WA) and flotillin-1 (1:500 dilution, Abcam, Cambridge, MA) by Western blotting.

Techniques: Inhibition

Journal: PeerJ

Article Title: Quality assessment of a serum and xenofree medium for the expansion of human GMP-grade mesenchymal stromal cells

doi: 10.7717/peerj.13391

Figure Lengend Snippet: Supernatant of THP-1 cells co-cultured with MSC at P1 and P2 was assayed for TNF-α and IL-1ra secretion. The cytokine concentration is normalized by the one measured in the LPS-treated THP-1 monoculture . Data are presented as median ± interquartile range with n = 7 for TNF- α secretion by BM-MSC, n = 6 for IL-1ra secretion by BM-MSC, n = 5 for TNF- α secretion by Ad-MSC and n = 4 for IL-1ra secretion by Ad-MSC. Friedman test was used to compare the effect of MSC expanded in the 2 media (hCPL and SXFM) to THP-1 stimulated by LPS, ns = non-significant, ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: As highly encouraged by the regulators to have a backup media in all Good Manufacturing Practices (GMP) processes (Guideline on human cell-based medicinal products, EMEA/CHMP/410869/2006), the aim of this study was to validate the use of a GMP standardized SXFM medium: the MSC-Brew GMP Medium (Miltenyi Biotec ® , Bergisch Gladbach, Germany).

Techniques: Cell Culture, Concentration Assay

Journal: Human Reproduction (Oxford, England)

Article Title: Putative human myometrial and fibroid stem-like cells have mesenchymal stem cell and endometrial stromal cell properties

doi: 10.1093/humrep/dez247

Figure Lengend Snippet: Mesenchymal lineage differentiation of SUSD2 + cells. (A) αSMA immunofluorescence in SUSD2+ myometrial cells after differentiation and (B) αSMA mRNA fold induction with differentiation. Day 0 represents the SUSD2+ cells before plating. Columns represent the average of n = 3 patient myometrial samples. Error bars equal SEM, and ratio paired t test was used to determine significance. Representative images of SUSD2+ myometrial (C, D, G, H) and fibroid (E, F, I, J) cells grown in control growth media (C, E, G, I) and adipogenic (D, F) or osteogenic (H, J) differentiation media. Adipogenic and control cultures were stained with Oil Red O (red colour, black arrows), and osteogenic and control cultures were stained for alkaline phosphatase activity (purple colour, black arrows). 3/3 myometrial and 2/3 fibroid samples analysed showed adipogenic differentiation. All myometrial and fibroid samples showed osteogenic differentiation. Adipogenic and control cultures were counter stained with hematoxylin. Scale bars are 0.1 mm for αSMA, adipogenic and control cultures and 2 mm for osteogenic and control cultures.

Article Snippet: Briefly, cells were incubated with anti-SUSD2-PE antibody (Miltenyi Biotec #130-106-325), diluted in staining buffer (PBS, BSA, EDTA), washed and then incubated with anti-PE MicroBeads in staining buffer.

Techniques: Immunofluorescence, Staining, Activity Assay

Journal: Human Reproduction (Oxford, England)

Article Title: Putative human myometrial and fibroid stem-like cells have mesenchymal stem cell and endometrial stromal cell properties

doi: 10.1093/humrep/dez247

Figure Lengend Snippet: In vitro decidualisation of SUSD2 + myometrial cells. To assess the ability of SUSD2+ and SUSD2− cells to decidualise, cells were cultured in untreated low serum media or with decidualisation cocktail (E2, MPA and db-cAMP). Representative images of SUSD2− (A, B) and SUSD2+ (C, D) cells grown in untreated (A, C) or with decidualisation cocktail (B, D) in low serum media after 5 days. Black arrows point to epithelial-like cells in (B) and large epithelial-like nests in (D). Immunocytochemistry for IGFBP1 is shown in (E, F) and for CD10 in (G, H). Nuclei are stained with DAPI. Fold induction of mRNA levels for PRL (I)IGFBP1 (J) and MME (CD10) (K) of SUSD2− and SUSD2+ myometrial cells, and endometrial stromal (Endo, control) cells (n = 3 each) grown in decidual induction media relative to RPL17, and untreated control cells. (L) mRNA levels of MME in SUSD2+ myometrial cells before plating (Day 0) and 5 days after treatment (Day 5) with the decidualisation cocktail. Statistical analysis was performed using the ratio paired t test; *P < 0.05. Columns represent mean fold change, and error bars represent SEM. Endo is shown for visual comparison and was not included in the statistical analyses.

Article Snippet: Briefly, cells were incubated with anti-SUSD2-PE antibody (Miltenyi Biotec #130-106-325), diluted in staining buffer (PBS, BSA, EDTA), washed and then incubated with anti-PE MicroBeads in staining buffer.

Techniques: In Vitro, Cell Culture, Immunocytochemistry, Staining

Journal: Human Reproduction (Oxford, England)

Article Title: Putative human myometrial and fibroid stem-like cells have mesenchymal stem cell and endometrial stromal cell properties

doi: 10.1093/humrep/dez247

Figure Lengend Snippet: Xenotransplantation and tumour formation of SUSD2 + cells. Xenotransplantation was performed to assess in vivo stem cell activity. (A) Trichrome staining of the parental fibroid for the xenotransplant images shown in this figure. Red colour shows staining of cytoplasm of muscle cells, and blue shows staining of collagen. Bar = 100 μm. (B) Graph of tumour size 8 weeks after transplantation. Statistical analysis performed was ratio paired t test for tumour area (mm2) from SUSD2− and SUSD2+ cells. *P = 0.038. (C) Representative image (n = 4) of fibroid-like tumours that formed from SUSD2− and SUSD2+ cell pellets transplanted under the renal capsule of immunocompromised mice. Representative low magnification images (D, E; bar = 1 mm) and high-magnification images (F, G; bar = 100 μm) of trichrome staining of tumours that formed in C. Dotted lines demarcate fibroid-like tumours from kidney tissue. Representative αSMA (H) and CD10 (I) immunofluorescence images of the fibroid xenotransplants. Green fluorescence observed in the kidney is background fluorescence. Merged αSMA and CD10 image with added DAPI channel shown in (J). Bar = 100 μm

Article Snippet: Briefly, cells were incubated with anti-SUSD2-PE antibody (Miltenyi Biotec #130-106-325), diluted in staining buffer (PBS, BSA, EDTA), washed and then incubated with anti-PE MicroBeads in staining buffer.

Techniques: In Vivo, Activity Assay, Staining, Transplantation Assay, Immunofluorescence, Fluorescence

Journal: Human Reproduction (Oxford, England)

Article Title: Putative human myometrial and fibroid stem-like cells have mesenchymal stem cell and endometrial stromal cell properties

doi: 10.1093/humrep/dez247

Figure Lengend Snippet: CFU assay of SUSD2 + myometrial and fibroid cells under normoxic and hypoxic conditions. Colony formation assays were performed to assess stem cell activity in normal (20%, Normox) and hypoxic (2%, Hypox) oxygen levels. Representative images are of colonies formed by myometrial (A–D) and fibroid (F–I) SUSD2− (A, B, F, G) and SUSD2+ (C, D, H, I) cells grown under Normox (A, C, F, H) or Hypox (B, D, G, I) conditions. Graphs show the rate of colony forming efficiency represented as %CFUs/days for myometrial (E) and fibroid (J) cells. Statistical analysis performed by beta mixed effects with n = 4% transformed matched myometrial and fibroid sample values. Different letters indicate significant differences (P < 0.5). Error bars are +/− SEM.

Article Snippet: Briefly, cells were incubated with anti-SUSD2-PE antibody (Miltenyi Biotec #130-106-325), diluted in staining buffer (PBS, BSA, EDTA), washed and then incubated with anti-PE MicroBeads in staining buffer.

Techniques: Colony-forming Unit Assay, Activity Assay, Transformation Assay