bone marrow primary human mscs (hmscs)  (Lonza)

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Functionality of lyophilized osteoinductive EVs: a mechanistic study

doi: 10.3389/fbioe.2024.1452428

Figure Lengend Snippet: Endocytosis of EVs by HMSCs (A) Representative confocal micrograph of fluorescently labeled fresh and lyophilized BMP2 EVs (green) endocytosed by naïve HMSCs at 37°C. Scale bar = 20 μm. (B) Dose-dependent endocytosis of fresh and lyophilized BMP2 EVs by HMSCs. Data points represent mean percentage fluorescence ± SD (n = 6). (C) Graph showing the inhibition of EV endocytosis after pre-treatment of the EVs with heparin to block interaction with the cell surface HSPGs. The reduction of EV endocytosis at 4°C was also measured compared to 37°C. Data represent mean percentage fluorescence with respect to control ± SD (n = 6). *: statistical significance with respect to No blocking control, #: statistical significance with respect to BMP2 EV group as measured by Student’s t-test (P< 0.05).

Article Snippet: Primary human bone marrow derived MSCs (HMSCs) were purchased from Lonza and cultured in αMEM basal media (Gibco) supplemented with 20% fetal bovine serum (FBS, Gibco), 1% (v/v) L-Glutamine (Gibco) and 1% (v/v) antibiotic-antimycotic solution (Gibco).

Techniques: Labeling, Fluorescence, Inhibition, Blocking Assay, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Functionality of lyophilized osteoinductive EVs: a mechanistic study

doi: 10.3389/fbioe.2024.1452428

Figure Lengend Snippet: Osteoinductive property of the lyophilized BMP2 EVs (A) Osteoinductive gene expression levels in fresh and lyophilized BMP2 EV HMSCs at day 3 and 5 with respect to control (no EV) group. Data are represented as mean fold changes ± SD (n = 4). *: statistical significance with respect to BMP2 EV group as measured by student’s t-test (P< 0.05). (B) Graph representing relative luciferase activity in HMSCs transfected with a BMP2 reporter luciferase plasmid (n = 4). Recombinant BMP2 protein (BMP2 GF) was used as positive control. Note the significant increase in luciferase activity in fresh and lyophilized BMP2 EV treated groups compared to control. *: statistical significance with respect to control, #: statistical significance with respect to BMP2 GF group as measured by Tukey’s test post ANOVA (P< 0.05). (C) Graph representing relative alkaline phosphatase (ALP) activity in HMSCs treated with osteogenic differentiation medium (OS) in the presence/absence of fresh and lyophilized BMP2 EVs at 3 and 7 days (n = 4). *: statistical significance with respect to control, #: statistical significance with respect to OS + BMP2 EV group as measured by Student’s t-test (P< 0.05). (D) Quantitative in-cell western for the presence of phosphorylated SMAD 1/5/8 and the corresponding tubulin expression in a 96 well plate assay (n = 6). The graph represents quantitation of the fluorescence from the wells using the LICOR Odyssey imager normalized to tubulin. *: statistical significance with respect to control group as measured by Tukey’s test post ANOVA (P< 0.05).

Article Snippet: Primary human bone marrow derived MSCs (HMSCs) were purchased from Lonza and cultured in αMEM basal media (Gibco) supplemented with 20% fetal bovine serum (FBS, Gibco), 1% (v/v) L-Glutamine (Gibco) and 1% (v/v) antibiotic-antimycotic solution (Gibco).

Techniques: Gene Expression, Control, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Recombinant, Positive Control, In-Cell ELISA, Expressing, Quantitation Assay, Fluorescence

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Functionality of lyophilized osteoinductive EVs: a mechanistic study

doi: 10.3389/fbioe.2024.1452428

Figure Lengend Snippet: Binding of lyophilized EVs to ECM proteins (A) Representative confocal image showing the binding of fluorescently labeled lyophilized EVs (green) to the decellularized ECM of HMSCs. Counterstain was performed for type I collagen (red, scale bar = 5 μm). (B) Dose-dependent binding of fluorescently labeled lyophilized EVs to type I collagen coated assay plates (data points represent mean ± SD, n = 6). (C) Representative confocal image showing the binding of fluorescently labeled lyophilized EVs (green) to the decellularized ECM of HMSCs. Counterstain was performed for fibronectin (red, scale bar = 20 μm). (D) Dose-dependent binding of fluorescently labeled lyophilized EVs to fibronectin coated assay plates (data points represent mean ± SD, n = 6).

Article Snippet: Primary human bone marrow derived MSCs (HMSCs) were purchased from Lonza and cultured in αMEM basal media (Gibco) supplemented with 20% fetal bovine serum (FBS, Gibco), 1% (v/v) L-Glutamine (Gibco) and 1% (v/v) antibiotic-antimycotic solution (Gibco).

Techniques: Binding Assay, Labeling

Journal: Regenerative therapy

Article Title: Generation of functional liver sinusoidal endothelial-like cells from human bone marrow-derived mesenchymal stem cells.

doi: 10.1016/j.reth.2023.07.006

Figure Lengend Snippet: Fig. 1. Screening of cytokines and small molecules for LSEC differentiation. (A) The schematic representation of BM-MSC differentiation. (B) qPCR analysis of the expression levels of LYVE1 and CD36 in BM-MSC-derived cells. On the y-axis, the expression levels are shown as a relative value to those of BM-MSCs. All data are presented as mean ± SD (n ¼ 3). Significant differences were evaluated using a one-way ANOVA followed by Dunnett's post-hoc test (*p < 0.05, ***p < 0.001 compared with BFTc [BMP4, FGF8b, TGF-b inhibitor, and cAMP]). Abbreviations: B; BMP4, F; FGF8b, T; TGF-b signal inhibitor, c; cAMP.

Article Snippet: Primary human bone marrow-derived MSCs (C-12974; PromoCell) were cultured in Mesenchymal Stem Cell Growth Medium 2 (C-28009; PromoCell).

Techniques: Expressing, Derivative Assay

Journal: Regenerative therapy

Article Title: Generation of functional liver sinusoidal endothelial-like cells from human bone marrow-derived mesenchymal stem cells.

doi: 10.1016/j.reth.2023.07.006

Figure Lengend Snippet: Fig. 2. Analysis of the expression of LSEC-related markers. (A) The schematic representation of BM-MSC differentiation to LSEC-like cells. (B) Brightfield images of the BM-MSCs and BM-MSC-derived cells. Scale bar ¼ 500 mm. (C) qPCR analysis of gene expression of LYVE1, CD36, CD32b, F8, PLVAP, CD31, and VEGFR2 in the BM-MSCs and BM-MSC-derived cells. On the y-axis, the expression levels are shown as a relative value to those of BM-MSCs. Data are presented as mean ± SD (n ¼ 6, sum of two independent experiments, n ¼ 3 for each experiment). (D) Immunocytochemistry analysis of LYVE1 (green) and CD36 (red) in the BM-MSCs and BM-MSC-derived cells. The nuclei were counterstained with DAPI (blue). Scale bar ¼ 50 mm. (E) The expression level of CD32 in the BM-MSCs and BM-MSC-derived cells was measured using flow cytometry analysis. The values of mean fluorescence intensity (MFI) of CD32 expression were normalized by the MFI of isotype control. Data are presented as mean ± SD (n ¼ 3). Significant differences were evaluated using an unpaired two-tailed Student's t-test (***p < 0.001). (F) The percentage of LYVE1- or CD36-positive cells in the BM-MSC-derived cells was measured using flow cytometry analysis.

Article Snippet: Primary human bone marrow-derived MSCs (C-12974; PromoCell) were cultured in Mesenchymal Stem Cell Growth Medium 2 (C-28009; PromoCell).

Techniques: Expressing, Derivative Assay, Gene Expression, Immunocytochemistry, Cytometry, Control, Two Tailed Test

Journal: Regenerative therapy

Article Title: Generation of functional liver sinusoidal endothelial-like cells from human bone marrow-derived mesenchymal stem cells.

doi: 10.1016/j.reth.2023.07.006

Figure Lengend Snippet: Fig. 3. Evaluation of endothelial cell-related functions. (A) Tube-formation assay of the BM-MSCs, BM-MSC-derived cells, and HUVECs. Scale bar ¼ 200 mm. (B) acLDL-uptake assay. BM-MSCs, BM-MSC-derived cells, and HUVECs were cultured in a medium containing Alexa Fluor 488-conjugated acLDL to measure their ability to take up acLDL. Scale bar ¼ 20 mm. Abbreviation: LDL, low-density lipoprotein.

Article Snippet: Primary human bone marrow-derived MSCs (C-12974; PromoCell) were cultured in Mesenchymal Stem Cell Growth Medium 2 (C-28009; PromoCell).

Techniques: Tube Formation Assay, Derivative Assay, Cell Culture

Journal: Regenerative therapy

Article Title: Generation of functional liver sinusoidal endothelial-like cells from human bone marrow-derived mesenchymal stem cells.

doi: 10.1016/j.reth.2023.07.006

Figure Lengend Snippet: Fig. 4. Evaluation of LSEC-related functions. (A) HA-uptake assay. BM-MSCs, BM-MSC-derived cells, and HUVECs were cultured in a medium containing fluoresceinamine- conjugated HA to measure their ability to take up HA. (B) IgG-uptake assay. BM-MSCs, BM-MSC-derived cells, and HUVECs were cultured in a medium containing Alexa Fluor 488-conjugated IgG to measure their ability to endocytose IgG. Scale bar ¼ 20 mm. Abbreviation: HA, hyaluronic acid.

Article Snippet: Primary human bone marrow-derived MSCs (C-12974; PromoCell) were cultured in Mesenchymal Stem Cell Growth Medium 2 (C-28009; PromoCell).

Techniques: Derivative Assay, Cell Culture

Journal: Regenerative therapy

Article Title: Generation of functional liver sinusoidal endothelial-like cells from human bone marrow-derived mesenchymal stem cells.

doi: 10.1016/j.reth.2023.07.006

Figure Lengend Snippet: Fig. 5. Evaluation of the differentiation capacity of primary human BM-MSCs into LSEC-like cells (A) The schematic representation of the differentiation of primary human BM- MSCs into LSEC-like cells. (B) qPCR analysis of gene expression of LYVE1, CD36, CD32b, and F8 in primary human BM-MSCs and primary human BM-MSC-derived cells. On the y-axis, the expression levels are shown as a relative value to those of primary human BM-MSCs. Data are presented as mean ± SD (n 8, sum of two independent experiments, n 3 for each experiment.). Significant differences were evaluated using an unpaired two-tailed Student's t-test (*p < 0.05, ***p < 0.001). (C) The percentage of LYVE1- or CD36- positive cells in the primary human BM-MSC-derived cells was measured using flow cytometry analysis. Data are presented as mean ± SD (n ¼ 3). (D) acLDL-uptake assay. BM-MSCs, BM-MSC-derived cells, and HUVECs were cultured in a medium containing Alexa Fluor 488-conjugated acLDL to measure their ability to take up acLDL. Scale bar ¼ 20 mm. (E) HA- uptake assay. Primary human BM-MSCs and primary human BM-MSC-derived cells were cultured in a medium containing fluoresceinamine-conjugated HA to measure their ability to take up HA. Scale bar ¼ 20 mm. Abbreviation: LDL, low-density lipoprotein; HA, hyaluronic acid.

Article Snippet: Primary human bone marrow-derived MSCs (C-12974; PromoCell) were cultured in Mesenchymal Stem Cell Growth Medium 2 (C-28009; PromoCell).

Techniques: Gene Expression, Derivative Assay, Expressing, Two Tailed Test, Cytometry, Cell Culture