Journal:

Article Title: Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

doi: 10.1182/blood-2006-05-023093

Figure Lengend Snippet: p38 Inhibitor SCIO-469 can reverse TNF-mediated myelosuppression. MDS1 cells were pretreated for 1 hour with vehicle (–) or 1.0 μM SCIO-469 (+) and then induced with either 1 ng/mL TNFα or 5 ng/mL TGFβ for 30 minutes. The p-p38 and total p38 levels were analyzed by Western blotting. Bar graph represents p-p38 levels relative to total p38 in each sample (A). Immunomagnetically selected bone marrow–derived CD34+ cells were differentiated into hematopoietic progenitors at the CFU-E stage of maturation as described before.30 These cells were treated with 20 ng/mL TNFα or 10 000U/mL IFN-γ in the presence and absence of 100 nM SCIO-469. Cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an antibody against the phosphorylated form of MapKapK-2 (threonine 334). The same blot was stripped and reprobed with an antibody against total MapKapK-2, to control for protein loading (B). Primary bone marrow–derived CD34+ cells were grown in cytokine-enriched liquid media in the presence and absence of 20 ng/mL TNFα and SCIO-469 (100 nM) for 24 hours. The percentages of apoptotic and dead cells were determined by staining with mixture of Annexin V–Alexa Fluor 488 and nucleic acid dye, Sytox green, respectively (Vybrant Apoptosis Kit; Molecular Probes) (C). Mean of 3 independent experiments showed significant decrease in TNFα-mediated apoptosis in the presence of SCIO-469 (P = .01, paired t test). BM CD34+ progenitors were cultured with TPO, Flt3L, and SCF with or without 20 ng/mL TNFα and in the presence and absence of 500 nM SCIO-469 for 6 days. BrDU incorporation was evaluated against the amount of 7-AAD by flow cytometry to determine the percentage of subpopulation at each cell-cycle stage in a gated population of CD34+ cells. Results from 3 experiments were used to compare the proportion of cells in G0/G1 and S phase of cell cycle by using 2-tailed t test. (D) Primary bone marrow–derived CD34+ cells were cultured in methylcellulose in the presence and absence of 20 ng/mL TNFα and SCIO-469. Colonies were scored on day 14. Results are expressed as mean ± SEM of 3 independent experiments (E). Treatment with SCIO-469 led to a significant reversal of TNF-mediated myelosuppression (P = .04, t test).

Article Snippet: The siRNA duplexes were labeled with FITC to show successful transfection in primary CD34 + cells (Label IT siRNA Tracker; Mirus, Madison, WI).

Techniques: Western Blot, Derivative Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Control, Staining, Cell Culture, BrdU Incorporation Assay, Flow Cytometry

Journal:

Article Title: Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

doi: 10.1182/blood-2006-05-023093

Figure Lengend Snippet: p38 inhibitor SCIO-469 can decrease apoptosis in MDS CD34+ progenitors. BM mononuclear cells from patients with MDS were cultured in the presence and absence of 500 nM SCIO-469 for 48 hours. Apoptosis in gated population of CD34+ cells was determined by Annexin V staining. Comparison of dot plots from 5 independent experiments shows a decrease in the percentage of Annexin V–positive CD34+ cells in samples treated with SCIO-469 (A). MDS CD34+ progenitors from 5 patients show significantly greater viability and decreased apoptosis after 48 hours of treatment with SCIO-469 (paired 2-tailed t test). Results are presented as means ± SEMs (B).

Article Snippet: The siRNA duplexes were labeled with FITC to show successful transfection in primary CD34 + cells (Label IT siRNA Tracker; Mirus, Madison, WI).

Techniques: Cell Culture, Staining, Comparison

Journal:

Article Title: Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

doi: 10.1182/blood-2006-05-023093

Figure Lengend Snippet: Down-regulation of p38α by siRNA can stimulate hematopoiesis in MDS CD34+ progenitors. A mixture of 4 siRNAs against p38α were transfected in primary CD34+ hematopoietic progenitors using Mirus TKO transfection reagent. Western blotting showed a specific and significant decrease in total p38 protein levels (A). High transfection efficiency was shown by using fluorescent-labeled siRNAs (B). MDS CD34+ cells were transfected with either anti-p38α or control-scrambled siRNAs and grown in vitro in methylcellulose with cytokines. Colonies were scored on day 14, and results were expressed as means ± SEMs of 3 independent experiments. Significantly higher number of both myeloid (CFU-GM) and erythroid (BFU-E) colonies were observed in cells transfected with anti-p38 siRNAs (C). MDS CD34+ cells transfected with anti-p38α and scrambled control siRNAs were evaluated after 48 hours by Annexin V staining. Flow cytometry revealed a significantly higher percentage of viable cells (P = .045, t test) and fewer number of apoptotic cells (P = .04, t test) when transfected with anti-p38α siRNA (D). Results are presented as means ± SEMs of 5 independent experiments.

Article Snippet: The siRNA duplexes were labeled with FITC to show successful transfection in primary CD34 + cells (Label IT siRNA Tracker; Mirus, Madison, WI).

Techniques: Transfection, Western Blot, Labeling, Control, In Vitro, Staining, Flow Cytometry

Journal:

Article Title: Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

doi: 10.1182/blood-2006-05-023093

Figure Lengend Snippet: Pharmacologic p38 inhibitors stimulate hematopoiesis in MDS CD34+ progenitors. MDS bone marrow–derived CD34+ cells from 19 patients were plated in methylcellulose in the presence and absence of p38 inhibitors SB203580 (5 μM and 10 μM), SD-282, and SCIO-469; inactive structural analog SB202474 (10 μM); and Mek-1 inhibitor PD98059 (10 μM). Colonies were scored at day 14, and results were expressed as means ± SEMs of 19 independent experiments.

Article Snippet: The siRNA duplexes were labeled with FITC to show successful transfection in primary CD34 + cells (Label IT siRNA Tracker; Mirus, Madison, WI).

Techniques: Derivative Assay

Journal: Scientific Reports

Article Title: Ocimum flavone Orientin as a countermeasure for thrombocytopenia

doi: 10.1038/s41598-018-23419-x

Figure Lengend Snippet: Effect of Orientin on human hematopoietic stem cells. ( a – c ) Orientin induces human CD34 + cells differentiation towards MEP lineage. CD34 + cells purified from healthy donor’s PBMCs via density gradient ficol followed by magnetic bead separation were treated with or without Orientin in a serum free expansion medium (SFEM) supplemented with 100 ng/ml cytokine cocktail (CC100) for 7 days. Cells were harvested for lineage differentiation and stained with CD34-PE-Cy7, CD38-Pacblue, CD10-FITC, CD135-PE, CD45RA-APC antibodies and subjected to FACS analysis. (d) Orientin partially restores CD34 + cells differentiation potential after irradiation. CD34 + cells purified from healthy donor’s PBMCs (1 × 10 5 /ml in triplicates) were treated with or without Orientin (5 µM) and exposed to 4 Gy (fractionated dose of 2 × 2 Gy). These cells were then co-cultured on human mesenchymal stromal cell layer in SFEM medium supplemented with 100 ng/ml cytokine cocktail for normal differentiation in 7 day time period. The MEP cells were gated in lin − CD34 + CD38 + CD10 − CD135 − CD45RA − block in each sample. ( e ) Colony Assay in CD34 + cells (1 × 10 3 /ml in triplicates) post 0 or 2 Gy X-ray exposure. CFU-GM and GEMM colonies were scored on day 14 by applying standard morphologic criteria.

Article Snippet: CD34 + cells were purified from PBMCs using Diamond CD34 isolation kit for human (#130-094-531, MiltenyiBiotec) as described by manufacturer.

Techniques: Purification, Staining, Irradiation, Cell Culture, Blocking Assay, Colony Assay

Journal: Leukemia

Article Title: Spred1 deficit promotes treatment resistance and transformation of chronic phase CML

doi: 10.1038/s41375-021-01423-x

Figure Lengend Snippet: A Expression of SPRED1 in BM CD34+ cells from patients with BC CML and CP CML by Q-RT-PCR (n=8 samples for BC CML and n=12 samples for CP CML) and western blot and in BM by immunohistochemistry staining (one of three independent experiments with similar results was shown) (left), and expression of miR-126 in CD34+ and CD34+CD38− cells from BC CML (n=6 samples) and CP CML (n=10 samples) patients by Q-RT-PCR (right). B SPRED1 mRNA expression by Q-RT-PCR and protein expression by western blot, miR-126 levels by Q-RT-PCR, cell cycling by Ki-67 and DAPi staining (top) or by cell trace violet staining (bottom) followed by flow cytometry analysis in CML CD34+ cells transduced with SPRED1 siRNA to knock-down (KD) SPRED1 or with a non-targeting control siRNA (Ctrl). UND: undivided cells, G0; DIV: division. C Representative colonies and quantification of colony forming cells (CFC) in CML CD34+ (left) and CD34+CD38− (right) cells transduced with Spred1 siRNA to KD SPRED1 or with ctrl siRNA (n=3). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human CD34 + cells were selected using the indirect CD34 microbead kit (Miltenyi Biotec, San Diego, CA) and CD34 + CD38 − cells were sorted after staining with human antibodies against CD34 and CD38 ( Supplementary Table 1 ) or selected using CD34+CD38- cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Flow Cytometry, Transduction, Knockdown, Control

Journal: The Journal of International Medical Research

Article Title: Enumeration of viable CD34+ cells in cord blood using a novel stem cell enumeration kit

doi: 10.1177/03000605211055384

Figure Lengend Snippet: Examples of gating protocols. (a) BD SCE kit, acquisition/analysis using BD FACSCanto clinical software; (b) BCI Stem kit, acquisition/analysis using BD FACSDiva software. BD SCE, Beckton Dickinson stem cell enumeration; BCI, Beckman Coulter, Inc.

Article Snippet: Measurement of CD34+ cells in cord blood using the BD™ stem cell enumeration kit was highly consistent at all three hospitals (all ICCs >0.95; .

Techniques: Software

Journal: The Journal of International Medical Research

Article Title: Enumeration of viable CD34+ cells in cord blood using a novel stem cell enumeration kit

doi: 10.1177/03000605211055384

Figure Lengend Snippet: Linear regression between absolute counts of CD34+ cells in cord blood determined using the BD SCE kit and the BCI Stem kit. Measurements of absolute counts of CD34-positive cells using the two methods were highly linear and nearly equivalent. BD SCE, Beckton Dickinson stem cell enumeration; BCI, Beckman Coulter, Inc.

Article Snippet: Measurement of CD34+ cells in cord blood using the BD™ stem cell enumeration kit was highly consistent at all three hospitals (all ICCs >0.95; .

Techniques:

Journal: The Journal of International Medical Research

Article Title: Enumeration of viable CD34+ cells in cord blood using a novel stem cell enumeration kit

doi: 10.1177/03000605211055384

Figure Lengend Snippet: Linear regression between relative counts of CD34+ cells in cord blood determined using the BD SCE kit and those from the BCI Stem kit. Measurements of the relative counts of CD34-positive cells between the two methods were highly linear and nearly equivalent. BD SCE, Beckton Dickinson stem cell enumeration; BCI, Beckman Coulter, Inc.

Article Snippet: Measurement of CD34+ cells in cord blood using the BD™ stem cell enumeration kit was highly consistent at all three hospitals (all ICCs >0.95; .

Techniques:

Journal: The Journal of International Medical Research

Article Title: Enumeration of viable CD34+ cells in cord blood using a novel stem cell enumeration kit

doi: 10.1177/03000605211055384

Figure Lengend Snippet: Bland–Altman plot of absolute counts of CD34+ cells in cord blood showing agreement between the BD SCE kit and the BCI Stem kit. The two measurement methods had high agreement for absolute counts of CD34-positive cells. BD SCE, Beckton Dickinson stem cell enumeration; BCI, Beckman Coulter, Inc.

Article Snippet: Measurement of CD34+ cells in cord blood using the BD™ stem cell enumeration kit was highly consistent at all three hospitals (all ICCs >0.95; .

Techniques:

Journal: The Journal of International Medical Research

Article Title: Enumeration of viable CD34+ cells in cord blood using a novel stem cell enumeration kit

doi: 10.1177/03000605211055384

Figure Lengend Snippet: Bland–Altman plot of relative counts of CD34+ cells in cord blood showing agreement between the BD SCE kit and the BCI Stem kit. The two measurements methods had high agreement for relative counts of CD34-positive cells. BD SCE, Beckton Dickinson stem cell enumeration; BCI, Beckman Coulter, Inc.

Article Snippet: Measurement of CD34+ cells in cord blood using the BD™ stem cell enumeration kit was highly consistent at all three hospitals (all ICCs >0.95; .

Techniques:

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Stem cell transcriptome of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst MSC, hESC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Dendrogram showing hierarchical clustering of MSC, hESC and MSC-hiPSC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing MSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC (d) and MSC-hiPSC (e) is reported in the indicated tables [Fisher's exact test].

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Mesenchymal potential of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst different F-hiPSC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Principal Component Analysis (PCA) showing 3D visualization of Principal Component (PC) 1, PC2 and PC3 of differentially expressed genes for different F-hiPSC, MSC-hiPSC and hESC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing F-hiPSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC-hiPSC (d) and F-hiPSC (e) is reported in the indicated tables [Fisher's exact test]. f) Schematic of the differentiation protocol toward MSC-like cells. g) Representative images of adipogenic (A, scale bar is 50 µm), osteogenic (O, scale bar is 50 µm) and chondrogenic (C, scale bar is 400 µm) mesenchymal derivatives. h) Left panel: representative density plot showing CD45 + hematopoietic cells (P3) gated from total cells of the cobblestone area-forming cell assay; FSC-A, forward scatter area, a.u., arbitrary units. Right panel: representative histograms showing CD34 + hematopoietic progenitor subpopulation (purple) of CD45 + cells compared to unstained control (grey); the vertical axis represents event percentage count (Count%).

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test, Control