Journal: Cells

Article Title: Generation of CD34 + CD43 + Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells

doi: 10.3390/cells11244046

Figure Lengend Snippet: Multipotent HPSCs were derived from hPSCs. ( A ) Schematic representation of the hematopoietic stem cell differentiation protocol. Between day 0 and day 9, EBs were treated to induce firstly mesoderm formation and then to induce specification towards the hematopoietic lineage. After 9 days, the EBs were dissociated and the cells were co-cultured on OP9-DLL1 or DLL4 cells in a specific medium to induce T-lymphoid commitment. ( B ) Evolution of the percentage of total cells expressing CD34 and CD43 between day 7 and day 9 and expression of CD45. ( C ) Flow cytometry analysis of differentiating cells at D7 and D9 in EB culture from hPSC. Representative dot plots of CD34 and CD43 co-staining on living cells are shown. The expressions of CD45 in CD34 + cells are shown. Red line represents cells stained with a fluorescent antibody and black line represents unstained cells. ( D ) Analysis of visual microscopic counting of CFU assay blood colonies for the indicated cell lines. Data are presented as a pool of experimental duplicates, n = 1.

Article Snippet: Briefly, on day 9, cells from EBs were dissociated with Accutase and CD34 + cells were MACS-purified with the human CD34 UltraPure MicroBead Kit (Miltenyi Biotech, Paris, France).

Techniques: Derivative Assay, Cell Differentiation, Cell Culture, Expressing, Flow Cytometry, Staining, Colony-forming Unit Assay

Journal: Cells

Article Title: Generation of CD34 + CD43 + Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells

doi: 10.3390/cells11244046

Figure Lengend Snippet: CD34 + CD43 + derived from hPSCs are closely related to cord blood HSCs. ( A ) Heatmap of Pearson correlation coefficients of HSCs and hPSCs differentiated from hPSCs. ( B ) Normalized enrichment score of biological pathways upregulated or downregulated in D9 CD34 + CD43 + EBs compared to HSCs. All represented pathways have an adjusted p -value < 0.05. ( C ) Gene expression heatmaps of selected markers characterizing hematopoietic differentiation are shown for digital gene expression sequencing (DGE-seq) for this study. The heatmap was generated by scaling and centering gene expression.

Article Snippet: Briefly, on day 9, cells from EBs were dissociated with Accutase and CD34 + cells were MACS-purified with the human CD34 UltraPure MicroBead Kit (Miltenyi Biotech, Paris, France).

Techniques: Derivative Assay, Gene Expression, Sequencing, Generated

Journal: Cells

Article Title: Generation of CD34 + CD43 + Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells

doi: 10.3390/cells11244046

Figure Lengend Snippet: hPSCs-Td25 are closed to CD8 + CD4 + thymocytes. ( A ) Representative histogram of CD7 expression in living cells (excluding OP9) on day 29. Red line represents cells stained with a fluorescent antibody and black line represents unstained cells. The expressions of CD8, CD4, CD3, and TCRαβ among CD7 + cells are shown in dot plot. ( B ) Heatmap of Pearson correlation coefficients of thymus cells and hPSCs differentiated from hPSCs. ( C ) Normalized enrichment score of biological pathways upregulated or downregulated in EB D9 CD34 + CD43 + compared to hPSC-Td25 (up) and in HSC compared to HSC-Td26 (down). All represented pathways have an adjusted p -value < 0.05.

Article Snippet: Briefly, on day 9, cells from EBs were dissociated with Accutase and CD34 + cells were MACS-purified with the human CD34 UltraPure MicroBead Kit (Miltenyi Biotech, Paris, France).

Techniques: Expressing, Staining

Journal: BMC Immunology

Article Title: P2 receptor mRNA expression profiles in human lymphocytes, monocytes and CD34+ stem and progenitor cells

doi: 10.1186/1471-2172-5-16

Figure Lengend Snippet: Relative P2 gene expression in CD34 + stem and progenitor cells. A, Bar graph shows relative P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 and P2Y 13 receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X 1 , P2X 4 and P2X 7 receptor gene expression normalized to GAPDH. P2Y 1 was chosen to be calibrator.

Article Snippet: CD34 + cells were isolated by 2passages through magnetic columns (MidiMacs;Miltenyi Biotec, Bergish Gladbach, Germany) by using a hapten-conjugated CD34 antibody (Qbend/10) and an antihapten antibody conjugated to magnetic beads (CD34 + isolation kit; Miltenyi Biotec).

Techniques: Gene Expression

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: Cancers

Article Title: Loss of MYBBP1A Induces Cancer Stem Cell Activity in Renal Cancer

doi: 10.3390/cancers11020235

Figure Lengend Snippet: MYBBP1A knock down increases c-MYB activity. ( A ) MYBBP1A knock down by the expression of a specific shRNA. Cell lines were transfected with MYBBP1A shRNA (sh) or a scramble vector (V). After selection, cells were grown to 80% confluence, and proteins were extracted. The figure shows the western blot results of MYBBP1A expression in all cell lines. Scale Bar: 8 µM. ( B ) Quantification of MYBBP1A expression in cells expressing MYBBP1A shRNA (sh) related to the scramble vector (V). ( C ) MYBBP1A knock down increases the expression of c-MYB target genes. mRNA levels of genes directly transcribed by c-MYB were measured by Q-RT-PCR. Graphs represent mRNA levels of cells expressing MYBBP1A shRNA normalized to mRNA levels of control cells. ( D ) Percentage of CD34 + cells measured by flow cytometry. ( E ) Percentage of CXCR4 + cells measured by flow cytometry. ( B – D ) Graphs show the mean ± SD of three independent experiments performed in triplicate. * p < 0.05; ** p < 0.01.

Article Snippet: Fluorochrome-conjugated monoclonal antibodies against human CD34 (FITC, MACS Miltenyi Biotec #130-098-142, Bergisch Gladbach, Germany) and CXCR4 (PE, MACS Miltenyi Biotec #130-098-354) were used.

Techniques: Knockdown, Activity Assay, Expressing, shRNA, Transfection, Plasmid Preparation, Selection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Flow Cytometry

Journal: Cancers

Article Title: Loss of MYBBP1A Induces Cancer Stem Cell Activity in Renal Cancer

doi: 10.3390/cancers11020235

Figure Lengend Snippet: MYBBP1A knock down in c-MYB + cell lines induces the expression of stem genes. ( A ) Measurement of NANOG, OCT4, SOX2 and TWIST1 expression by Q-RT-PCR. Graphs represent mRNA levels in MYBBP1A knock down cells normalized to the mRNA levels of control cells. ( B ) CaKi-1 and ACHN cell lines were transfected with c-MYB cDNA and the empty vector (pcDNA3). After selection, c-MYB, CXCR4, CD34, NANOG, SOX2 and TWIST1 expression were measured by Q-RT-PCR. Graphs represent mRNA levels in cells expressing c-MYB cDNA normalized to the mRNA levels of control cells. ( C ) CXCR4 mRNA levels from adherent cells and tumorspheres were measured by Q-RT-PCR. Graphs represent mRNA levels of 786-O and ACHN cells expressing sh2 normalized to mRNA levels of control cells (V2). ( A – C ) Graphs show the mean ± SD of three independent experiments performed in triplicate. * p < 0.05; ** p < 0.01.

Article Snippet: Fluorochrome-conjugated monoclonal antibodies against human CD34 (FITC, MACS Miltenyi Biotec #130-098-142, Bergisch Gladbach, Germany) and CXCR4 (PE, MACS Miltenyi Biotec #130-098-354) were used.

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Selection