Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: The effect of statins on viability and growth of ( a ) stem ADMSC and non-cancerous HEK 293 cells and ( b ) cancer MiaPaCa-2 cells. ( a ) ADMSC—human adipose-derived mesenchymal stem cells, HEK 293—human embryonic kidney cells, exposure to statins—24 h, concentrations 0—100 µM, control—methanol, ( b ) previously published data , MiaPaCa-2—pancreatic cancer cells, exposure to statins—24 h, concentrations 0—40 µM, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Derivative Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of the effect of statins on the growth and viability of pancreatic cancer MiaPaCa-2 cells, non-cancerous HEK 293 cells, and ADMSC stem cells. Concentration of statins—20 µM, Time—exposure to statins—24, 48, and 72 h, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on size and compactness of spheroids. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr—methanol treated spheroids, P—pravastatin, R—rosuvastatin, L—lovastatin, F—fluvastatin, A—atorvastatin, Pi—pitavastatin, C—cerivastatin, S—simvastatin. Statins were added once, after spheroid formation, 10 weeks ( a ) or 3.5 weeks ( b ) after inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on the spheroid formation. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr methanol treated spheroids, P —pravastatin, R —rosuvastatin, L —lovastatin, F —fluvastatin, A —atorvastatin, Pi —pitavastatin, C —cerivastatin, S —simvastatin. Statins were added once, 24 h after cell inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of expression changes between statin treated and control MiaPaCa-2 and ADMSC cells. Displayed are only the genes that are differentially expressed upon at least one statin treatment in at least one cell type, requiring |log 2 FC|> 1 and FDR < 0.05. Statins were administered at a concentration of 12 µM for 24 h. ( FC fold change, FDR false discovery rate, horizontal and vertical axes—changes in ADMSC and MiaPaCa-2 cells, respectively, upon respective treatment). The red dashed lines indicate two-fold change increase or decrease in the gene expression. The genes with at least two-fold up-regulation (resp. down-regulation) in ADMSC stem cells are displayed to the right (resp. left) of the dashed lines. Similarly, genes with at least two-fold up-regulation (resp. down-regulation) in cancer cells are displayed above (resp. below) of the dashed lines. For details about differentially regulated transcripts see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579 .

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Expressing, Control, Concentration Assay, Gene Expression

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Cellular pathways most significantly affected by statins in cancer and stem cells. The gene set enrichment analysis (GSEA) revealed the KEGG pathways most affected by statin treatment in ADMSC and MiaPaCa-2 cells. Displayed is the union of the top five most enriched pathways among the comparisons. (Statin concentration—12 µM, treatment time—24 h, p-value—GSEA p-value, gene ratio—fraction of KEGG pathway genes among differentially expressed genes). For details about differentially regulated transcripts, see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Therapeutic role of aripiprazole in cartilage defects explored through a drug repurposing approach

doi: 10.1038/s41598-024-82177-1

Figure Lengend Snippet: Functional screening of drugs promoting chondrogenic differentiation ( A ) First Screening of Cartilage Differentiation-Promoting Drugs: Adipose-derived mesenchymal stem cells (ADMSCs) were treated with 10 µM of each drug for 72 h. The expression levels of COL2A1 (top) and SOX9 (bottom) mRNA were assessed using qRT-PCR. Dotted lines indicate drugs that significantly increased SOX9 and COL2A1 expression compared to DMSO control. ( B ) Second Screening: The Venn diagram shows the overlap of drugs that increased COL2A1 and SOX9 expression. Bar graphs present relative mRNA expression levels with statistical significance indicated. The cartilage-differentiation effect of seven drugs selected from the first screening was re-evaluated by measuring changes in mRNA levels of COL2A1, SOX9, and aggrecan (ACAN).: * P < 0.05, ** P < 0.01, *** P < 0.001 ( n = 3). ( C ) ADMSCs were cultured in pellet form and treated with DMSO, aripiprazole, or irinotecan for three weeks. Aripiprazole treatment showed increased cartilage differentiation compared to DMSO and irinotecan. ( D ) Hematoxylin and Eosin (H&E) staining and Alcian blue staining of pellet cultures. Aripiprazole treatment resulted in larger pellet size and more intense staining, indicating enhanced cartilage matrix production. Scale bars represent 100 μm.

Article Snippet: Human adipose tissue-derived mesenchymal stem cells (ADMSCs) were obtained from PromoCell GmbH (Heidelberg, Germany), ADMSCs were cultured in standard culture and chondrogenic differentiation media (Gibco).

Techniques: Functional Assay, Derivative Assay, Expressing, Quantitative RT-PCR, Control, Cell Culture, Staining

Journal: Cell Death Discovery

Article Title: Proinflammatory cytokines sensitise mesenchymal stromal cells to apoptosis

doi: 10.1038/s41420-025-02412-0

Figure Lengend Snippet: A Quantification of Annexin V + cells in Jurkat T lymphoma cells treated with increasing concentrations of anti-FAS antibody (aFAS) for 24 h ( n = 3) and representative Annexin V/PI staining following stimulation with 1 μg/ml of anti-Fas antibody for 24 h. B Quantification of Annexin V + cells in human BM-MSCs treated with increasing concentrations of anti-FAS antibody for 24 h ( n = 3) and representative Annexin V/PI staining following stimulation with 10 μg/ml of anti-Fas antibody for 24 h. C Representative flow cytometric analysis of FAS expression in one of three human BM-MSCs in comparison to peripheral blood mononuclear cells (PBMC). D Quantification of Annexin V + cells in Jurkat cells treated with increasing concentrations of FcFASL for 24 h ( n = 3). E – G Quantification of Annexin V + cells in human BM-MSCs ( E ), mouse BM-MSCs ( F ), and primary MEFs and SV40-immortalised MEFs ( G ) treated with increasing concentrations of FcFASL for 24 h with or without pre-treatment with 50 μM zVAD-FMK for 30 min ( n = 3). H Quantification of Annexin V + cells in human BM-MSCs treated with 10 μg/ml anti-FAS antibody for 24 h in the presence or absence of 500 nM SMAC mimetic (Compound A) with or without pre-treatment with 50 μM zVAD-FMK for 30 min ( n = 3). I Representative Annexin V/PI staining in human BM-MSCs treated with 100 ng/ml TNF for 24 h, and quantification of Annexin V + cells in human BM-MSCs treated with increasing concentrations of TNF ( n = 3). J Quantification of Annexin V + cells in human BM-MSCs (left panel) and mouse BMDMs (right panel) treated with 100 ng/ml TNF, or 25 μg/ml poly(I:C), or 25 μg/ml LPS for 24 h in the presence or absence of 500 nM SMAC mimetic, with or without pre-treatment with the pan-caspase inhibitors Q-VD-OPh or zVAD-FMK for 30 min ( n = 3). K Quantification of Annexin V + cells in human BM-MSCs treated with 100 ng/ml TNF, or 100 μg/ml poly(I:C), or 50 μg/ml LPS for 24 h in the presence or absence of 500 nM SMAC mimetic with or without pre-treatment with increasing concentrations of Q-VD-OPh or zVAD-FMK for 30 min ( n = 3). Data expressed as the mean ± S.E.M. and representative of at least two independent experiments, p values by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Human adipose-derived MSCs were either purchased from ScienCell or isolated from subcutaneous adipose tissue obtained from outpatient liposuction procedures (Monash human ethics approval #2007/1798; performed with informed patient consent).

Techniques: Staining, Expressing, Comparison

Journal: Cell Death Discovery

Article Title: Proinflammatory cytokines sensitise mesenchymal stromal cells to apoptosis

doi: 10.1038/s41420-025-02412-0

Figure Lengend Snippet: A , B Quantification of Annexin V + cells in Jurkat cells ( A ) and human BM-MSCs ( B ) treated with increasing concentrations of the BH3 mimetic drugs ABT199 (BCL-2 inhibitor, iBCL2), A1331852 (BCL-XL inhibitor, iBCLxL) for 3 h. C Representative Annexin V/PI staining in human BM-MSCs treated with increasing concentrations of BH3 mimetic drugs, and BAK/BAX-deficient (BKX) MSCs treated with 1.25 μM BH3 mimetic drugs for 3 h. D Quantification of Annexin V + cells in human MSCs ( D ) and mouse MSCs ( E ) treated with increasing concentrations of BH3 mimetic drugs for 2 h ( n = 3). F , G Quantification of Annexin V + cells at various time points following treatment of human MSCs with 1.25 μM BH3 mimetic drugs ( F ) and mouse MSCs, MEFs and BAK/BAX deficient MEFs treated with 10 μM BH3 mimetic drugs ( G ) ( n = 3). H Quantification of Annexin V + cells in human MSCs treated with increasing concentrations of various BH3 mimetic drugs combinations for 24 h ( n = 3). I Quantification of Annexin V + cells in mouse MSCs treated with various BH3 mimetic drugs combinations at 10 μM for 24 h ( n = 3). Data expressed as the mean ± S.E.M. and representative of at least two independent experiments.

Article Snippet: Human adipose-derived MSCs were either purchased from ScienCell or isolated from subcutaneous adipose tissue obtained from outpatient liposuction procedures (Monash human ethics approval #2007/1798; performed with informed patient consent).

Techniques: Staining

Journal: Cell Death Discovery

Article Title: Proinflammatory cytokines sensitise mesenchymal stromal cells to apoptosis

doi: 10.1038/s41420-025-02412-0

Figure Lengend Snippet: A Quantification of live cells (Annexin V − ) in human AD-MSCs, UC-MSCs, and BM-MSCs treated with increasing concentrations of BH3 mimetic drugs for 2 h ( n = 3 donors per tissue type). B Relative expression level of the anti-apoptotic genes BCL-XL , BCL-2 , and MCL-1 in cultured AD-MSCs, UC-MSCs, and BM-MSCs ( n = 3 donors per tissue type). Data expressed as the mean ± S.E.M. pooled from two independent experiments, p values by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Human adipose-derived MSCs were either purchased from ScienCell or isolated from subcutaneous adipose tissue obtained from outpatient liposuction procedures (Monash human ethics approval #2007/1798; performed with informed patient consent).

Techniques: Expressing, Cell Culture

Journal: Cell Death Discovery

Article Title: Proinflammatory cytokines sensitise mesenchymal stromal cells to apoptosis

doi: 10.1038/s41420-025-02412-0

Figure Lengend Snippet: A , B Representative Annexin V/PI staining in unprimed BM-MSCs and BM-MSCs primed with single (TNF or IFN-γ) or dual (TNF and IFN-γ) cytokines at the indicated concentrations for 24 h prior to treatment with vehicle control (DMSO) ( A ) or 1.25 μM BH3 mimetic drugs ( B ) for 2.5 h. C Representative Annexin V/PI staining of two additional BM-MSC donors primed with 10 ng/ml TNF and 100 ng/ml IFN-γ for 24 h prior to treatment with BH3 mimetic drugs for 2.5 h. D Quantification of the proportion of live Annexin V − PI − cells (left panel), early Annexin V + PI − (middle panel) and late Annexin V + PI + apoptotic cells (right panel) in unprimed and primed BM-MSCs from three donors treated with increasing concentrations of BH3 mimetic drugs for 2.5 h ( n = 3). E Representative Annexin V/PI staining in unprimed and primed BM-MSCs treated with 0.125 μM BH3 mimetic drugs for 2.5 h. F Quantification of live (Annexin V − PI − ), early apoptotic (Annexin V + PI - ) and late apoptotic (Annexin V + PI + ) cells from three BM-MSC donors treated with 0.125 μM BH3 mimetic drugs (as shown in E ) for 2.5 h ( n = 3). G Representative Annexin V/PI staining in unprimed and primed MSCs treated with 1.25 μM BH3 mimetic drugs for 30 min. H Representative Annexin V/TO-PRO-3 staining and gating of Annexin V + TO-PRO-3 hi late apoptotic cells in unprimed and primed BM-MSCs treated with BH3 mimetic drugs for 30 min. I , J Representative histograms showing the proportion of TO-PRO-3 int cells after exclusion of Annexin V + TO-PRO-3 hi late apoptotic (as shown in H ) in unprimed and primed BM-MSCs treated with BH3 mimetic drugs (blue histograms) or vehicle (grey histograms) for 30 min. Data expressed as the mean ± S.E.M. and representative of two independent experiments, p values by two-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Human adipose-derived MSCs were either purchased from ScienCell or isolated from subcutaneous adipose tissue obtained from outpatient liposuction procedures (Monash human ethics approval #2007/1798; performed with informed patient consent).

Techniques: Staining, Control, Comparison

Journal: Cell Death Discovery

Article Title: Proinflammatory cytokines sensitise mesenchymal stromal cells to apoptosis

doi: 10.1038/s41420-025-02412-0

Figure Lengend Snippet: A Live cell imaging of parental MSCs (top panels; Video ) and apoptosis-deficient BKX-MSCs (bottom panels; Video ) following apoptosis induction with BH3 mimetic drugs. B Live cell imaging of untreated human bone marrow MSCs (top panels; Video ) and BH3 mimetic drug-treated MSCs (bottom panels; Video ) stained with Annexin V, showing formation of apoptotic bodies (arrows). C Representative Annexin V staining (left panel) and quantification of the proportion of apoptotic bodies (right panel) in human MSCs treated with BH3 mimetic drugs ( n = 3). Data are expressed as the mean ± S.E.M and representative of three independent experiments. D Schematic for detection of MSCs and apoptotic bodies within the lungs at various time points after intravenous injection into mice. E Representative staining for CTV and CD45 to detect MSCs in digested lung tissue harvested 10 min post intravenous MSC injection F Representative staining for active caspase 3 within the CTV + cell population, used to identify apoptotic MSCs and apoptotic bodies within digested lung tissue following intravenous injection of CTV-labelled parental MSCs (top panel) or BKX-MSCs (bottom panel). G Quantification of apoptotic bodies detected in the lungs of mice (as shown in E ). Data represent the mean ± S.E.M. of n = 3 mice, p values by one-way ANOVA with Tukey’s post hoc test. Panel D was created with BioRender.com.

Article Snippet: Human adipose-derived MSCs were either purchased from ScienCell or isolated from subcutaneous adipose tissue obtained from outpatient liposuction procedures (Monash human ethics approval #2007/1798; performed with informed patient consent).

Techniques: Live Cell Imaging, Staining, Injection

Journal: Cell Death Discovery

Article Title: Proinflammatory cytokines sensitise mesenchymal stromal cells to apoptosis

doi: 10.1038/s41420-025-02412-0

Figure Lengend Snippet: A Schematic of how the survival of unprimed and primed MSCs was analysed within mouse lung tissue. B Representative staining for CTV and hCD73 to detect MSCs in digested lung tissue harvested 30 min post intravenous MSC injection. C Gating strategy used to identify live MSCs, apoptotic MSCs and apoptotic bodies, based on pooled samples of viable MSCs and BH3-mimetic drug treated MSCs stained with FLICA to detect active caspase 3/7. D Representative FLICA staining within the CTV + cell population used to detect live MSCs, apoptotic MSCs and apoptotic bodies in digested lung tissue. E Quantification of the number of live MSCs, apoptotic MSCs and apoptotic bodies within the lungs (as shown in D ). F Quantification of the proportion of CD45 − (left panel) and CD45 + cells (right panel) within the CTV + FLICA + apoptotic MSC gate. G , H Detection of human CD73 + MSCs within ex vivo cultured lung cells. Digested lung cells from untreated mice, or mice that received intravenous unprimed MSCs or primed MSCs were cultured for six days and the number of human MSCs was quantified. G Representative staining of human CD73 + MSCs within the CD45 − population of cultured lung cells. H Quantification of the number (left panel) and proportion (right panel) of human CD73 + MSCs in cultured lung cells six days after plating. Data represent the mean ± S.E.M. of n = 3 mice, unpaired T -test; ** p ≤ 0.01. Panel A was created with BioRender.com.

Article Snippet: Human adipose-derived MSCs were either purchased from ScienCell or isolated from subcutaneous adipose tissue obtained from outpatient liposuction procedures (Monash human ethics approval #2007/1798; performed with informed patient consent).

Techniques: Staining, Injection, Ex Vivo, Cell Culture

Journal: Clinical Epigenetics

Article Title: MEST mediates the impact of prenatal bisphenol A exposure on long-term body weight development

doi: 10.1186/s13148-018-0478-z

Figure Lengend Snippet: In vitro adipocyte differentiation from human MSCs: exposure to BPA (10 μM, 50 μM) compared to solvent control (EtOH 0.05%). a Real-time monitoring of cell differentiation (xCELLigence: normalized cell index) over a 17-day period (mean ± SD, n = 4). b Quantification of Oil Red O stained area (mean ± SD, n ≥ 20 from one experiment). c Exemplary histological Oil Red O staining of adipocytes (black bar = 100 μm). d qPCR data of genes involved in adipogenesis ( n ≥ 3) normalized to EtOH control ( Lep = leptin, LPL = lipoprotein lipase, PPAR γ = peroxisome proliferator activated receptor gamma, IRS2 = insulin receptor substrate 2, FASN = fatty acid synthase, SREBF 1 = sterol receptor element binding factor 1, ESR1 = estrogen receptor alpha). e Targeted MassARRAY analysis of MEST promoter methylation, shown are the measurement of the single CpG cg17580798 covered by the amplicon (gray bars, n = 3) and the mean of the MassARRAY amplicon (black bars). f qPCR data of MEST ( n ≥ 3, relative to EtOH control); * p < 0.05, ** p < 0.01, *** p < 0.001 from Student’s t test/ANOVA

Article Snippet: For adipocyte differentiation MSCs at passage 1–3 were seeded at 9600 cells/cm 2 and were cultured with Adipocyte Differentiation Initiation Medium (ADIM; ATCC Adipocyte Differentiation Toolkit PCS-500-050) for 4 days.

Techniques: In Vitro, Solvent, Control, Cell Differentiation, Staining, Binding Assay, Methylation, Amplification

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: a, Standard scRNA-seq equipment including Sony cell sorter, 10X Chromium scRNA-seq device, and Illumina sequencers are used in the different steps of the SEC-seq workflow (top to bottom). Checkmarks indicate the steps that were validated with corresponding equipment. b, Standard curve of VEGF-A on nanovials using recombinant VEGF-A, immobilized via the VEGF-A capture antibody, and detected with AF647-tagged anti-VEGF-A detection antibody shows a dynamic range across 3 orders of magnitude. Horizontal line represents the detection threshold (see ). c, (left) Schematic showing the steps of the VEGF-A secretion assay in single MSC-loaded nanovials. (middle) Flow cytometry histograms of VEGF-A secretion from single MSCs on nanovials after 0, 6, 12 hours of incubation. (right bottom) VEGF-A secretion assay on single MSC-loaded nanovials with and without VEGF-A capture antibody (Ab). More than 90% of cells in nanovials with capture antibody had fluorescence signal above the threshold (dotted line). (right top) The fluorescence microscopy image shows single MSCs on nanovials with secreted VEGF-A detected with a fluorescently (AF647)-tagged VEGF-A detection antibody (magenta) and cells stained with calcein AM (green). Scale bar is 50 µm. d, Bar plot shows cell viability measured by image analysis of live/dead stain (see ) following flow sorting of cells in suspension or loaded in nanovials. e, (top) Schematic of nanovial loading into droplets with 10X training gel beads in the absence of detergent (to prevent cell lysis). (bottom) Brightfield and fluorescence images of nanovials (red) with single MSCs (green) together with a gel bead in a droplet following emulsification. Scale bar is 50 µm. f , Graph showing the proportion of all nanovial-containing droplets with the indicated number of nanovials. g, After droplet formation and in the presence of lysis buffer, lysis of calcein (green)-labeled cells on nanovials was observed by diffusion of the green fluorescent signal throughout the droplets containing single-MSC loaded nanovials. Overlaid fluorescence and brightfield images of droplets generated with a 10X Genomics NextGEM kit. Scale bar is 50 µm. h, Distribution of species-specific reads from a scRNA-seq experiment with nanovials containing human MSCs or mouse fibroblasts pooled in a 1:1 ratio. Species identity was called by mapping to a joined genome contig and determining the ratio of reads from each species’ genome. i, Comparison of transcripts per cell for either suspended (unsorted) MSCs, suspended and sorted (sorted) MSCs, or MSCs loaded on nanovials and sorted (nanovial). j, Scheme explaining the experiment where MSCs cultured under normoxic and hypoxic conditions, respectively, were loaded on nanovials labeled with different oligo-barcoded streptavidin molecules (‘normoxic’ and ‘hypoxic’ barcodes) and analyzed in a 1:1 ratio in a single 10X channel. The scatter plot below depicts the assignment of cells based on the normoxic or hypoxic oligo-barcode attached to nanovials via streptavidin. Mixed cells have a signal for both barcodes. k , UMAP plots of the scRNA-seq data derived from the experiments in (j), where each cell is labeled by their oligo-barcode assignment. Mixed cells are excluded in UMAPs. l, The UMAP from (k) labeled by the hypoxic gene expression signature to identify MSCs cultured in hypoxic conditions.

Article Snippet: Immortalized adipose-derived MSCs , ATCC , SCRC-4000.

Techniques: Recombinant, Flow Cytometry, Incubation, Fluorescence, Microscopy, Staining, Suspension, Lysis, Emulsification, Labeling, Diffusion-based Assay, Generated, Comparison, Cell Culture, Derivative Assay, Gene Expression

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: a, Schematic of the detection of secreted VEGF-A protein and corresponding global gene expression for individual MSCs using the SEC-seq method. b, UMAP dimensionality reduction based on transcriptomes from SEC-seq experiments on normoxic and hypoxic MSCs in nanovials. Cells are labeled according to the culture condition. c, UMAP displaying VEGF-A secretion level, shown as log transformation of the UMI collapsed anti-VEGF-A oligo-barcode reads per cell. d, UMAP displaying VEGFA transcript levels, shown as normalized transcripts per cell. e, Distribution of VEGF-A secretion for cell-loaded nanovials in normoxic and hypoxic conditions, detected by FACS using a fluorescent anti-VEGF-A antibody (top) or by the SEC-seq experiment in (b) using the oligo-barcoded anti-VEGF-A antibody (middle). The last plot shows distribution of VEGFA transcript levels from the SEC-seq experiment cells in (b) (bottom). f, UMAP displaying cluster assignment. g, Violin plots by cluster showing VEGFA transcripts and VEGF-A secretion levels for all cells in the normoxic clusters (N1-N5, red shades), hypoxic clusters (H1–4, blue shades), and mixed clusters (M1–3, green shades) from (f). For mixed clusters, the levels are shown separately for normoxic and hypoxic cells. The dashed line represents the mean across all cells for each plot. Data below this threshold are lightened to highlight differences. h, Scatter plots showing the relationship between VEGFA transcript and VEGF-A secretion levels for individual normoxic (left) and hypoxic (right) cells from the experiment in (b). Best fit regression lines and Pearson correlation coefficients are shown. i, Plot showing the ranking of all detected genes based on the correlation of their transcript levels to the VEGF-A secretion level per cell for normoxic (top) and hypoxic (bottom) MSCs. The rank of the VEGFA gene is highlighted, and the top three genes per sample are also noted.

Article Snippet: Immortalized adipose-derived MSCs , ATCC , SCRC-4000.

Techniques: Gene Expression, Labeling, Transformation Assay

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: a, Scatter plot of the transcript to VEGF-A secretion correlation for all genes from SEC-seq experiments for normoxic and hypoxic MSCs from . The 10 most highly correlating genes based on both experiments are labeled. b, Table giving the ranking (based on average correlation), gene name, correlation to secretion in normoxic and hypoxic cells, and the average of those two values for the ten top genes from (a). c, UMAPs showing VEGF-A secretion levels and expression of 5 select correlated genes from (b) in normoxic and hypoxic MSCs from . The VEGF-A secretion UMAP is given from for comparison. d, As in (c), for a separate SEC-seq experiment performed on MSCs in the normoxic culture condition. e, Cell clusters projected onto the UMAP of the replicate SEC-seq experiment f, Heatmap of the top 10 differentially expressed genes from each cluster (indicated on top) of the SEC-seq experiment in (c,d) (rows=genes, columns=individual cells). Displayed at the top are the log transformed VEGF-A secretion and VEGFA transcript levels. Right: top 3 genes differentially expressed gene for each cluster. g, Heatmap of the top GO terms found for all of the differentially expressed genes from the clusters in (e). The (–logP) value indicates if the term was enriched for a given cluster. h , Venn diagram showing the overlap of differentially expressed genes from the highly secreting cluster in 3 SEC-seq experiments (top left: normoxic MSCs from (3b), top right: hypoxic MSCs from (3b), bottom: normoxic MSCs from (4e)). Overlapping genes form the Vascular Regenerative Signal (VRS). i, Gene ontology analysis for VRS genes from (h). Similar terms were collapsed. j , Average of the normalized transcripts level of VRS genes per cell, displayed for the SEC-seq experiment in (e) and (3b). k, As in (j), for MSCs loaded in oligo-barcoded nanovials (see - ). l, As in (j), for a standard scRNA-seq experiment on unsorted, suspended MSCs. m, Comparison of gene type classification for VRS genes and genes differentially expressed in all clusters in (e) except for those from cluster c5. n, Enrichment of possible TF regulators of the VRS genes based on the TRRUST database. o, Consensus rank of VEGF-A secretion to gene correlation based the SEC-seq experiments used in (h), with red dots displaying all VRS genes. p, Schematic depicting the heterogeneity of VEGF-A secretion in MSCs under normoxic and hypoxic conditions, highlighting the importance of the VRS genes for marking high VEGF-A secretion.

Article Snippet: Immortalized adipose-derived MSCs , ATCC , SCRC-4000.

Techniques: Labeling, Expressing, Comparison, Transformation Assay

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: List of reagents and resources

Article Snippet: Immortalized adipose-derived MSCs , ATCC , SCRC-4000.

Techniques: Conjugation Assay, Recombinant, Modification, Cell Culture, Enzyme-linked Immunosorbent Assay, Sequencing, Software

Journal: Korean Journal of Neurotrauma

Article Title: Evaluation of Bone Marrow-derived Stem Cells and Adipose-derived Stem Cells Co-cultured on Human Nucleus Pulposus Cells: A Pilot Study

doi: 10.13004/kjnt.2020.16.e36

Figure Lengend Snippet: SA-β-gal: senescence associated-β-gal, NP: nucleus pulposus, NPC: nucleus pulposus cell, MSC: mesenchymal stem cell, ADMSC: adipose-derived mesenchymal stem cell, BDMSC: bone marrow-derived mesenchymal stem cell.

Article Snippet: BDMSC complete culture medium (ATCC ® PCS-500-030) plus one MSC growth kit (ATCC ® PCS-500-041) and ADMSC complete culture medium (ATCC ® PCS-500-030) plus one MSC growth kit (ATCC ® PCS-500-040) were used to cultivate both MSCs; this medium consists of 485 mL Human MSC Basal Medium, 35 mL Human MSC-Qualified FBS, 0.5 mL penicillin/streptomycin-amphotericin and 6 mL L-alanyl-L-glutamine.

Techniques: Derivative Assay