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: Molecular Therapy Oncology

Article Title: Bulk and single-cell transcriptomics identify gene signatures of stem cell-derived NK cell donors with superior cytolytic activity

doi: 10.1016/j.omton.2024.200870

Figure Lengend Snippet: Cytotoxicity analysis of 10 stem cell-derived NK batches against multiple tumor cell lines distinguishes donors with superior anti-tumor efficacy (A) Overview of the experimental setup. (B) NK cytotoxicity, expressed as percentage of tumor cell cytolysis, analyzed after 20 h of co-culture with different cell lines at multiple E:T ratios (from 1:3 to 10:1). Cytolysis was assessed via flow cytometry for K562, and on the impedance-based platform xCELLigence for A375, LoVo, and LN-18. Each dot represents a donor and is the average of a technical triplicate. (C) Flow cytometry-based overnight cytotoxicity assay at a 1:1 E:T ratio; cytolysis was assessed as percentage of 7AAD + cells. Data are shown as mean ± SD of technical triplicates. Donors are ordered from low to high cytotoxic capacity based on the mean cytotoxicity value across the four cell lines. (D–E) Heatmaps showing the hierarchical clustering of donors based on the mean cytotoxicity value across the four cell lines (D) or the expression of 30 surface antigens (E). Excellent and good donors are labeled with different colors. E:T, effector-to-target; 7AAD, 7-aminoactinomycin D; SD, standard deviation.

Article Snippet: Hematopoietic CD34 + stem cells were selected from the MNCs using the CD34 + magnetic microbead kit (Miltenyi Biotech) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Co-Culture Assay, Flow Cytometry, Cytotoxicity Assay, Expressing, Labeling, Standard Deviation

Journal: Immunotherapy

Article Title: Long-term survival and differentiation of human thymocytes in human thymus-grafted immunodeficient mice

doi: 10.2217/imt-2019-0030

Figure Lengend Snippet: (A) Schematic showing preparation of hu-mice. Human CD34+ FLC were transduced with GFP-lentiviruses and injected into sublethally irradiated NSG mice that were grafted with fresh (B–D) Or cryopreserved-thawed (E–G) Human thymic tissues (n = 5 per group). (B & E) Percentages of GFP+ cells in GFP-transduced CD34+ cells (i.e., GFP transduction efficiency). (C & F) Percentages of GFP+ human B and T cells in peripheral blood mononuclear cells at the indicated time points (mean ± standard error of the mean). (D & G) FACS profile showing the percentage of GFP+ cells in human CD45+ thymocytes from representative human thymic grafts at week 26 post-transplantation.

Article Snippet: Human CD34 + cells were purified from fetal liver cells (FLCs) by incubation with antihuman CD34 microbeads followed by positive selection using MACS (Miltenyi Biotech, CA, USA).

Techniques: Transduction, Injection, Irradiation, Transplantation Assay

Journal: Immunotherapy

Article Title: Long-term survival and differentiation of human thymocytes in human thymus-grafted immunodeficient mice

doi: 10.2217/imt-2019-0030

Figure Lengend Snippet: (A & B) Hu-mice were made by transplantation of human fetal thymic tissues together with allogeneic CD34+ cells (n = 3). (A) Percentages of human CD45+ cells in PBMCs (left) and of human CD3+ T cells in gated human CD45+ PBMCs (right). (B) Representative FACS profiles showing presence of both human CD3+ T cells and CD19+ B cells at the early time (6 weeks; left) and only CD3+ T cells at the later time (18 weeks; right) after transplantation. (C–E) FACS analysis of PBMCs, spleen cells and human thymic graft cells from a representative hu-mouse sacrificed 32 weeks after transplantation of HLA-A9+ fetal human thymic tissue and allogeneic (HLA-A9-) CD34+ cells. (C) Staining profiles of PBMCs and spleen cells: 1st row: human CD45+ cell chimerism; 2nd row: expression of human CD3 and HLA-A9 on gated human CD45+ cells; 3rd row: CD4 and CD8 expression on gated human CD3+ cells; and 4th row: human CD45RA and CD31 expression on gated human CD4+ cells. (D) Macroscopic image of a thymic graft (on the top of a kidney). (E) Human thymic graft cells stained with antimouse CD45 versus antihuman CD45 (left panel), and the expression of human CD4 versus CD8 and CD3 versus HLA-A9 in gated human CD45+ thymocytes (right panel).

Article Snippet: Human CD34 + cells were purified from fetal liver cells (FLCs) by incubation with antihuman CD34 microbeads followed by positive selection using MACS (Miltenyi Biotech, CA, USA).

Techniques: Transplantation Assay, Staining, Expressing

Journal: Immunotherapy

Article Title: Long-term survival and differentiation of human thymocytes in human thymus-grafted immunodeficient mice

doi: 10.2217/imt-2019-0030

Figure Lengend Snippet: (A) Percentages of human CD45+ cells in PBMCs at the indicated time points (n = 4; each symbol represents an individual animal). (B) Representative FACS profiles showing human CD45+, CD3+ and CD19+ cell chimerism in PBMCs 18 weeks after human fetal thymus/CD34+ fetal liver cell (FLC) transplantation. Human CD34+ FLCs from the same fetus were used in the experiment presented in Figure 2.

Article Snippet: Human CD34 + cells were purified from fetal liver cells (FLCs) by incubation with antihuman CD34 microbeads followed by positive selection using MACS (Miltenyi Biotech, CA, USA).

Techniques: Transplantation Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: In vitro differentiated plasmacytoid dendritic cells as a tool to induce anti-leukemia activity of natural killer cells

doi: 10.1007/s00262-017-2022-y

Figure Lengend Snippet: AHR antagonists increase the yield of in vitro differentiated pDC. Cord blood CD34 + progenitors were cultured for 2 weeks in the absence (CTL—control) or in the presence of AHR antagonists (CH223191 or StemRegenin-1, SR1). Absolute numbers of differentiated pDC were determined by using immunostaining and flow cytometry analysis. a Differentiated pDC were identified as HLA-DR + CD123 hi by flow cytometry. Representative contour plots are presented with percentage of pDC for each culture condition. b Absolute numbers of pDC obtained from 10 5 CD34 + cells are presented with median ( n = 7 independent experiments). Statistical analyses were performed by using one-way analysis of variance. ** p < 0.01, *** p < 0.001

Article Snippet: Mononuclear cells were isolated by gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Bio-Science AB, Uppsala, Sweden), and CD34 + cells were positively selected using magnetic beads (Miltenyi Biotec, San Diego, CA, USA).

Techniques: In Vitro, Cell Culture, Control, Immunostaining, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy

Article Title: In vitro differentiated plasmacytoid dendritic cells as a tool to induce anti-leukemia activity of natural killer cells

doi: 10.1007/s00262-017-2022-y

Figure Lengend Snippet: Activated ivD-pDC infusions cure human ALL in humanized mice. Irradiated newborn NSG mice were transplanted with 10 4 human cord blood-derived CD34 + cells. 10 5 REH ALL cells were intravenously injected in humanized mice 8 weeks after transplantation. Forty-eight hours after leukemia injection, mice were injected with activated pDC (a-pDC; n = 12) or unstimulated pDC (unst-pDC, n = 5) (once a week, for 5 weeks), 20,000 IU of IL-2 (daily injections; n = 7) or saline solution for control mice (control; n = 18). a In vivo bioluminescence imaging of ALL-bearing mice was performed weekly. Images of representative mice are shown; units in rainbow color scales are proportional to the numbers of photons per second. b Survival curves of ALL-bearing humanized mice treated with unstimulated or TLR9-activated ivD-pDC, IL-2 or saline solution injections. Mice were euthanized after overt leukemia onset. Flow cytometry analysis of bone marrow samples confirmed complete leukemia involvement. Log-rank test was used to compare survival

Article Snippet: Mononuclear cells were isolated by gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Bio-Science AB, Uppsala, Sweden), and CD34 + cells were positively selected using magnetic beads (Miltenyi Biotec, San Diego, CA, USA).

Techniques: Irradiation, Derivative Assay, Injection, Transplantation Assay, Saline, Control, In Vivo, Imaging, Flow Cytometry