ipsc-derived imscs  (Lonza)

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Substrate and BM‐MSC EV characterization. (a) Elastic moduli of substrates made with varying ratios of Sylgard 184 base to crosslinker reagents. All values expressed as mean ± SD ( n = 3). (b) Absorbance values indicating cell viability as determined by CCK8 assay over 5 days. All values expressed as mean ± SD ( n = 3). (c) Size distribution from nanoparticle tracking analysis of EVs isolated from BM‐MSCs seeded on Sylgard 184 PDMS substrates with differing base to crosslinker reagent ratios ( n = 3). (d) Representative Western blot of BM‐MSC EVs from each of the Sylgard 184 PDMS substrates and the corresponding cell lysates for EV‐positive markers ALIX, TSG101, and CD63 and cellular markers Calnexin and GAPDH (15 μg/lane). (e) Representative TEM images of BM‐MSC EVs from the softest Sylgard 184 PDMS substrates and collagen‐coated flasks. Statistical significance was determined by ANOVA; **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Human induced pluripotent stem cell‐derived MSCs (iMSCs) were also purchased from ATCC (ACS‐7010).

Techniques: CCK-8 Assay, Isolation, Western Blot

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Substrate stiffness influences BM‐MSC EV production and bioactivity. (a) EV production as quantified by EVs per cell from BM‐MSCs seeded on Sylgard 184 PDMS substrates with different base‐to‐crosslinker ratios. EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted ( n = 3). (b) After a scratch was induced, HUVECs were treated with BM‐MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy ( n = 3). (c) HUVECs were resuspended in EV treatments or growth or basal endothelial media, seeded in Matrigel‐coated wells, and tube formation after 3–6 h was quantified by the number of loops that had formed ( n = 3). All values expressed as mean ± SD. All data are representative of at least three independent experiments ( n = 3). Statistical significance was determined by ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Human induced pluripotent stem cell‐derived MSCs (iMSCs) were also purchased from ATCC (ACS‐7010).

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Softer 184:527 PDMS substrates improve the angiogenic bioactivity of BM‐MSC EVs. (a) EV production quantified as EV per cell from BM‐MSCs seeded on each substrate made with different ratios of Sylgard 184 and Sylgard 527 ( n = 2). EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted. (b) After a scratch was induced, HUVECs were treated with BM‐MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy ( n = 3). (c) HUVECs were resuspended in the different EV treatments or growth or basal endothelial basal media, and tube formation after 3–6 h was quantified by the number of loops that had formed ( n = 3). All values expressed as mean ± SD. Statistical significance was determined by ANOVA; * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Article Snippet: Human induced pluripotent stem cell‐derived MSCs (iMSCs) were also purchased from ATCC (ACS‐7010).

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: (a) EV production as quantified by EV per cell by EVs from iMSCs on different PDMS substrates. (b) EV size and concentration distribution from iMSCs cultured on different PDMS substrates as determined by nanoparticle tracking analysis. (c) iMSC proliferation/viability on PDMS substrates as measured by cell counting over 4 days. (d) Representative TEM images of F + C EVs and 527 EVs confirming morphology. (e) Western blot of EV markers CD63, ALIX, and TSG101, and EV‐negative marker, calnexin, on EVs from each PDMS substrate (12 μg/lane). (f) Western blot of MSC markers CD73, CD105, and CD90 and negative marker CD45 on iMSC lysate from each PDMS substrate. THP1 cell lysate was used as a positive control for CD45. (5 μg/lane). All values expressed as mean ± SD. Statistical significance was determined by ANOVA; ** p < 0.01.

Article Snippet: Human induced pluripotent stem cell‐derived MSCs (iMSCs) were also purchased from ATCC (ACS‐7010).

Techniques: Concentration Assay, Cell Culture, Cell Counting, Western Blot, Marker, Positive Control

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Substrate stiffness affects the pro‐angiogenic effect of iMSC EVs comparably to BM‐MSC EVs. (a) EVs isolated from iMSCs on different 184:527 PDMS substrates were used to treat HUVECs after a scratch had been induced, and percent gap closure after 20 h was evaluated via microscopy. (b) HUVECs were resuspended with the same EV groups and seeded, and tube formation after 3–6 h was quantified by the number of loops that had formed. All values expressed as mean ± SD. All data are representative of at least three independent experiments ( n = 3). Statistical significance was determined by ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Human induced pluripotent stem cell‐derived MSCs (iMSCs) were also purchased from ATCC (ACS‐7010).

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: MiRNA qPCR array data of differentially expressed MSC EV‐associated miRNAs in flask vs. soft substrate‐generated EVs. (a) Fold change of miRNAs within EVs from iMSCs seeded on 527 PDMS normalized to the miRNA levels within EVs from iMSCs seeded on collagen‐coated flasks. (b) The same data represented as the log2 of the fold change, again normalized to the miRNA levels within EVs from iMSCs seeded on collagen‐coated flasks. All data are representative of at least three independent experiments ( n = 3).

Article Snippet: Human induced pluripotent stem cell‐derived MSCs (iMSCs) were also purchased from ATCC (ACS‐7010).

Techniques: Generated

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: FIGURE 1 Substrate and BM-MSC EV characterization. (a) Elastic moduli of substrates made with varying ratios of Sylgard 184 base to crosslinker reagents. All values expressed as mean ± SD (n = 3). (b) Absorbance values indicating cell viability as determined by CCK8 assay over 5 days. All values expressed as mean ± SD (n = 3). (c) Size distribution from nanoparticle tracking analysis of EVs isolated from BM-MSCs seeded on Sylgard 184 PDMS substrates with differing base to crosslinker reagent ratios (n = 3). (d) Representative Western blot of BM-MSC EVs from each of the Sylgard 184 PDMS substrates and the corresponding cell lysates for EV-positive markers ALIX, TSG101, and CD63 and cellular markers Calnexin and GAPDH (15 μg/lane). (e) Representative TEM images of BM-MSC EVs from the softest Sylgard 184 PDMS substrates and collagen-coated flasks. Statistical significance was determined by ANOVA; **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Human induced pluripotent stem cell-derived MSCs (iMSCs) were also purchased from ATCC (ACS-7010).

Techniques: CCK-8 Assay, Isolation, Western Blot

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: FIGURE 2 Substrate stiffness influences BM-MSC EV production and bioactivity. (a) EV production as quantified by EVs per cell from BM- MSCs seeded on Sylgard 184 PDMS substrates with different base-to-crosslinker ratios. EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted (n = 3). (b) After a scratch was induced, HUVECs were treated with BM-MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy (n = 3). (c) HUVECs were resuspended in EV treatments or growth or basal endothelial media, seeded in Matrigel-coated wells, and tube formation after 3–6 h was quantified by the number of loops that had formed (n = 3). All values expressed as mean ± SD. All data are representative of at least three independent experiments (n = 3). Statistical significance was determined by ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Human induced pluripotent stem cell-derived MSCs (iMSCs) were also purchased from ATCC (ACS-7010).

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: FIGURE 3 Softer 184:527 PDMS substrates improve the angiogenic bioactivity of BM-MSC EVs. (a) EV production quantified as EV per cell from BM-MSCs seeded on each substrate made with different ratios of Sylgard 184 and Sylgard 527 (n = 2). EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted. (b) After a scratch was induced, HUVECs were treated with BM-MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy (n = 3). (c) HUVECs were resuspended in the different EV treatments or growth or basal endothelial basal media, and tube formation after 3–6 h was quantified by the number of loops that had formed (n = 3). All values expressed as mean ± SD. Statistical significance was determined by ANOVA; *p < 0.05, **p < 0.01, and ****p < 0.0001.

Article Snippet: Human induced pluripotent stem cell-derived MSCs (iMSCs) were also purchased from ATCC (ACS-7010).

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: FIGURE 4 (a) EV production as quantified by EV per cell by EVs from iMSCs on different PDMS substrates. (b) EV size and concentration distribution from iMSCs cultured on different PDMS substrates as determined by nanoparticle tracking analysis. (c) iMSC proliferation/viability on PDMS substrates as measured by cell counting over 4 days. (d) Representative TEM images of F + C EVs and 527 EVs confirming morphology. (e) Western blot of EV markers CD63, ALIX, and TSG101, and EV-negative marker, calnexin, on EVs from each PDMS substrate (12 μg/lane). (f) Western blot of MSC markers CD73, CD105, and CD90 and negative marker CD45 on iMSC lysate from each PDMS substrate. THP1 cell lysate was used as a positive control for CD45. (5 μg/lane). All values expressed as mean ± SD. Statistical significance was determined by ANOVA; **p < 0.01.

Article Snippet: Human induced pluripotent stem cell-derived MSCs (iMSCs) were also purchased from ATCC (ACS-7010).

Techniques: Concentration Assay, Cell Culture, Cell Counting, Western Blot, Marker, Positive Control

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: FIGURE 5 Substrate stiffness affects the pro-angiogenic effect of iMSC EVs comparably to BM-MSC EVs. (a) EVs isolated from iMSCs on different 184:527 PDMS substrates were used to treat HUVECs after a scratch had been induced, and percent gap closure after 20 h was evaluated via microscopy. (b) HUVECs were resuspended with the same EV groups and seeded, and tube formation after 3–6 h was quantified by the number of loops that had formed. All values expressed as mean ± SD. All data are representative of at least three independent experiments (n = 3). Statistical significance was determined by ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Human induced pluripotent stem cell-derived MSCs (iMSCs) were also purchased from ATCC (ACS-7010).

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: FIGURE 7 MiRNA qPCR array data of differentially expressed MSC EV-associated miRNAs in flask vs. soft substrate-generated EVs. (a) Fold change of miRNAs within EVs from iMSCs seeded on 527 PDMS normalized to the miRNA levels within EVs from iMSCs seeded on collagen- coated flasks. (b) The same data represented as the log2 of the fold change, again normalized to the miRNA levels within EVs from iMSCs seeded on collagen-coated flasks. All data are representative of at least three independent experiments (n = 3).

Article Snippet: Human induced pluripotent stem cell-derived MSCs (iMSCs) were also purchased from ATCC (ACS-7010).

Techniques: Generated

Journal: ACS nano

Article Title: Co-Delivery of Bioengineered Exosomes and Oxygen for Treating Critical Limb Ischemia in Diabetic Mice

doi: 10.1021/acsnano.3c08088

Figure Lengend Snippet: Characterization of iMSC-derived exosomes without modification (EXO) and modified with ischemic-limb-targeting capability (ILTEXO). (A) Schematic illustration of EXO isolation and ILTEXO preparation. (B) Biomarkers on EXO analyzed by using an Exo-Check Exosome Antibody Array. (C–E) Quantitative analysis of angiogenesis-related growth factors, including PDGF-BB (C), VEGF (D), and HGF (E), in exosomes isolated from iMSCs cultured in different oxygen environments (n = 4). (F–H) Comparison of miRNA contents, including miR126–3p (F), miR21–5p (G), and miR182–5p (H), in exosomes isolated from iMSCs cultured under hypoxia and normoxia (n = 6). (I, J) TEM images of EXO (I) and ILTEXO (J). Scale bar = 100 nm. (K) Fluorescent images (middle) corresponding to differential interference contrast (DIC) images (left) and merged images (right) for both EXO (upper) and ILTEXO (lower); scale bar = 5 μm. The images were taken for aggregated exosomes since confocal microscopes have limited resolution for clearly imaging a single exosome. (L, M) Size distribution of EXO (L) and ILTEXO (M) using nanoparticle tracking analysis (NTA). (N) Cellular internalization of ILTEXO by C2C12 cells (left), HUVECs (center), and THP-1 derived macrophages (right) after incubation for 2 h under hypoxia (1% O2). Co-localization of ILTEXO within the cells is denoted in yellow and emphasized by white arrows. Included are scale bars 50 μm for the upper images and 20 μm for the enlarged lower field of views. (O) Quantification of cell uptake efficiency of ILTEXO by three types of cells (n = 7). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) were obtained from FUJIFILM Cellular Dynamics, Inc. and were maintained in serum-free maintenance medium mainly containing Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco), Ham’s F-12 medium (Corning), 50 ng/mL bFGF (Peprotech), 50 ng/mL PDGF-BB (Peprotech), 50 μ g/mL ascorbic acid (Sigma-Aldrich), and 1% penicillin/streptomycin (Gibco).

Techniques: Derivative Assay, Modification, Isolation, Ab Array, Cell Culture, Comparison, Imaging, Incubation