Journal: PLoS ONE

Article Title: Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

doi: 10.1371/journal.pone.0108913

Figure Lengend Snippet: (a) Isolated HPC (CD34 + Lin − ) cells were cultured in IL-15-supplemented media, and the culture media was replaced every 48 h. The expression of CD56 as an NK cell surface marker was analyzed using FACS. (b) The kinetics of the mRNA expression of Id2, NKp30, NKG2D and Granzyme B were analyzed by real-time qPCR. (c) Stage 2 progenitors (CD34 + CD117 + CD94 − ) and stage 3 progenitors (CD34 − CD117 + CD94 − ) were isolated from UCB by flow sorting. The mRNA expression of IL2Rγ, NKp46 and NKG2D were analyzed by real-time qPCR. (d) A dendrogram of hierarchical clustering revealed genes that were altered more than 2-fold in 7d- and 14-d (mNK) cultured NK cell compared with 1 d-cultured cells. (E-F) The bar graphs represent the top seven functional categories of upregulated (e) or downregulated (f) genes according to the gene ontology analysis (as determined by DAVID, as described in the Methods). The data are representative of three independent experiments performed using three different UCB samples and represent the mean values ± S.E.M. of duplicates.

Article Snippet: Transfections of differentiating NK cell with miRNA mimic control, and miRNA mimics were performed by nucleofection using an Amaxa Human CD34 Cell Nucleofector kit (Lonza).

Techniques: Isolation, Cell Culture, Expressing, Marker, Functional Assay

Journal: PLoS ONE

Article Title: Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

doi: 10.1371/journal.pone.0108913

Figure Lengend Snippet: (a) Negatively correlated miRNA-mRNA interactions were visualized as a network using Magia (miRNAs and genes integrated analysis web-based tool). This network provides for the first time a theoretical outline of the concerted action of regulating miRNAs (red triangles) and their potential target mRNAs (green circles). (b) Isolated HPC (CD34 + Lin − ) cells were cultured as described in the Materials and Methods. After being cultured in IL-15-supplemented media, the cells were collected at the indicated time intervals. The expression of miR-143, miR-223, miR-150 and miR-583 was analyzed by real-time qPCR. The data are representative of five independent experiments performed using two different UCB samples and represent the mean values ± S.E.M. of duplicates.

Article Snippet: Transfections of differentiating NK cell with miRNA mimic control, and miRNA mimics were performed by nucleofection using an Amaxa Human CD34 Cell Nucleofector kit (Lonza).

Techniques: Isolation, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

doi: 10.1371/journal.pone.0108913

Figure Lengend Snippet: Isolated CB HPCs (CD34 + Lin − cells) were cultured as described in the Materials and Methods. After being cultured in IL-15-supplemented media, the cells were collected at 48 h intervals. (a) The expression of the IL2Rγ gene was analyzed by real-time quantitative RT-PCR. (b) The expression of IL2Rγ protein was analyzed by FACS. (c) Expression profiling of IL2Rγ, miR-583 and miR-143 after IL-15 treatment. The data are representative of three independent experiments performed using five different UCB samples and represent the mean values ± S.E.M. of duplicates.

Article Snippet: Transfections of differentiating NK cell with miRNA mimic control, and miRNA mimics were performed by nucleofection using an Amaxa Human CD34 Cell Nucleofector kit (Lonza).

Techniques: Isolation, Cell Culture, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

doi: 10.1371/journal.pone.0108913

Figure Lengend Snippet: (a) Differentiating cells were transfected with miR-143, a miR-583 mimic or negative control miRNA. NK cell populations (CD56 + CD3 − cells) were analyzed by FACS 10 days after transfection. *, p <0.1. (b) The expression level of miR-583 was decreased during NK cell development as shown by qRT-PCR. The results are shown as the mean expression values normalized against stage 2 (CD34 + CD117 + CD94 − ). *, p <0.1. (c) NK cell populations (CD56 + CD3 − cells) were analyzed by FACS after miR-583 transfection at regular intervals during NK cell differentiation. The absolute numbers of differentiated NK cells are shown in the parentheses (×10 5 ). *, p <0.01 **, p <0.001. The data are representative of three independent experiments performed using three different UCB samples and represent the mean values ± S.E.M. of duplicates.

Article Snippet: Transfections of differentiating NK cell with miRNA mimic control, and miRNA mimics were performed by nucleofection using an Amaxa Human CD34 Cell Nucleofector kit (Lonza).

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Cell Differentiation

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

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: Journal of medical genetics

Article Title: Germline mutations in the DNA damage response genes BRCA1, BRCA2, BARD1 and TP53 in patients with therapy related myeloid neoplasms.

doi: 10.1136/jmedgenet-2011-100674

Figure Lengend Snippet: Figure 1 (A) Pedigree and mutational analysis of index patient UPN 5869. The pedigree indicates a hereditary breast and ovarian cancer syndrome. Electropherograms illustrate the BRCA1 c.3112G/T mutation (arrow) as well as a nearby single nucleotide polymorphism (SNP) in DNA from fibroblasts and CD34+ leukaemic cells. Both mutation and SNP became homozygous in the leukaemic clone. The SNP array reveals a copy number state 1 and loss of heterozygosity (LOH) at the BRCA1 locus on chromosome 17q of CD34+ leukaemic blasts. (B) Pedigree and mutational analysis of index patient UPN 6371. The pedigree is not specific for a certain syndrome. Electropherograms illustrate the TP53 c.845_848dupGGCG mutation in germline as well as somatic DNA. SNP array shows copy number state 1 and LOH at the TP53 locus on chromosome 17p of CD34+ leukaemic cells. Filled symbols, subjects with malignancies; open symbols, asymptomatic subjects; the arrow indicates the index patient; number within symbol corresponds to the number of asymptomatic individuals. Red regions represent losses and violet regions LOH. BlaCa, bladder cancer; BrCa, breast cancer; Ca, cancer of unknown origin; GaCa, gastric cancer; Leuk, leukaemia of unknown type; Pheo, pheochromocytoma; Plas, plasmacytoma; ProCa, prostate cancer; UtCa, uterine cancer; y, age in years at diagnosis.

Article Snippet: CD34+ cells were separated from diagnostic blood or bone marrow specimens using the magnetic activated cell sorting CD34 MicroBead Kit together with MS Columns and MiniMACS Separator (all Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Mutagenesis, Biomarker Discovery