Journal: Nature

Article Title: MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment

doi: 10.1038/s41586-019-1790-2

Figure Lengend Snippet: a, Venn diagram of microarray gene expression data, identifying genes enriched in self-renewing human FL-HSPCs. Number of genes downregulated after differentiation (pink) of fetal liver CD34+CD38−/loCD90+GPI80+ HSCs to CD34+CD38−/loCD90+GPI80− progenitors3; number of genes downregulated in FL-HSPCs during 5-week culture on OP9M2 stroma (green)4; and number of genes suppressed in human embryonic stem (ES)-cell-derived HSPCs (purple)5 are shown. b, FACS analysis 30 days after transduction of CD34+CD38−/loCD90+ HSPCs with MLLT3 shRNA (MLLT3-KD) or empty vector control (CTR) (representative of three plots). c, Quantification of cells as in b after 5, 15 and 30 days in culture (n = 3). d, FACS analysis of bone marrow from NSG mice 12 weeks after transplantation of FL- HSPCs transduced with MLLT3-KD or empty vector control (representative of 10 mice). e, Quantification of human (h) CD45+ cells in bone marrow (BM) from NSG mice treated as in d (n = 10 mice, two independent experiments). f, FACS analysis of CD34+CD38−/loCD90+ FL-HSPCs transduced with control or MLLT3- OE lentiviral vector (representative of six experiments). g, Expansion of HSPCs as in f (n = 6 independent experiments). h-j, FACS analysis showing human haematopoietic reconstitution (hCD45 expression) (h) and quantification of total hCD45 cells (i) or human HSPCs (hCD45+CD34+CD38−/lo) (j) in bone marrow from NSG mice. In i andj, the number of mice showing multi-lineage reconstitution versus the number of total transplanted mice is shown (n = 13 or 14 mice, 4 independent experiments). APCcy7, PEcy7, BV421 and BV711 denote fluorochrome dyes. Data in c denote mean values; data in e, i and j denote mean and individual values; data in g are mean ± s.e.m. All P values determined by two-sided t-test.

Article Snippet: CD34+ cells were magnetically isolated from mononuclear cell fraction using anti-CD34 microbeads (Miltenyi Biotech).

Techniques: Microarray, Expressing, Derivative Assay, Transduction, shRNA, Plasmid Preparation, Transplantation Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

doi: 10.1002/stem.3259

Figure Lengend Snippet: Differentiation of CB-CD34+ cells in a feeder- and serum-free culture system. A, Schematic of culture system. B, Conceptual schema of human T-cell development. CD7, CD5, and CD1a are sequentially expressed on CD34+ cells entering into the thymus (DN cells), and develop into CD4- or CD8-expressing single positive (SP) cells through immature SP (iSP) and immature double-positive (iDP). Finally, mature SP T cells leave thymus and spread to periphery. C, Representative histograms and flow cytometry plots on day 14. Mean ± SEM values are presented for percent of CD7+ and CD7+CD5+ cells. D, The CD7+ (proT1) cell numbers on day 14 were different between groups without AA under ambient air and groups with AA under physioxia. CD7+ cell number showed differences between physioxia and ambient air groups (n = 9, two-way ANOVA, *P < .05, **P < .01). E, CD7+CD5+ (proT2) cell numbers showed differences between ambient air and physioxia groups (n = 9, two-way ANOVA, *P < .05). F, Folds of numbers of CD33+ (myeloid) cells on day 14 (n = 4, *P < .05). Cell numbers were normalized as cell numbers plated into one well (4000 CD34 + HSC/HPCs). Plots are presented as mean ± SEM. H, physioxia; HA, physioxia with AA; N, ambient air (non-physioxia); NA, ambient air with AA; NS, not significant

Article Snippet: CD34+ cell fractions were further isolated through a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, California).

Techniques: Expressing, Flow Cytometry

Journal: Stem cells (Dayton, Ohio)

Article Title: Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

doi: 10.1002/stem.3259

Figure Lengend Snippet: HSC/HPC populations in physioxia vs ambient air groups. Cell numbers of (A) HSC/HPC (CD34+CD38−), (B) HSC (CD34+CD38−CD45RA−CD90+), and (C) multipotent progenitor (MPP, CD34+CD38−CD45RA−CD90−) were not different between physioxia and ambient air groups. D, Higher numbers of Lymphoid-primed multipotent progenitors (LMPP, CD34+CD38−CD45RA+CD90lo/−CD10−) and multi-lymphoid progenitors (MLP, CD34+CD38−CD45RA+CD90lo/−D10+) were observed in physioxia groups than in ambient air groups (P = .056) on day 7 (n = 3). E, Gene expression of the Notch1 signaling pathway (Notch1, HES1, DELTEX, TCF7, and GATA3) and PU.1 were not differently expressed between physioxia and ambient air groups at 48 hours (n = 4). Data are expressed as mean ± SEM. H, physioxia; HA, physioxia with AA; N, ambient air; NA, ambient air with AA; NS, not significant

Article Snippet: CD34+ cell fractions were further isolated through a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, California).

Techniques: Gene Expression

Journal: Stem cells (Dayton, Ohio)

Article Title: Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

doi: 10.1002/stem.3259

Figure Lengend Snippet: Maturation of progenitor T cells on artificial thymic organoid cultures (ATO). A, Experimental schema. B, ATO at week 6. Images of ATOs divided by four or nine pieces at ×10 magnification were taken using inverted microscope (Eclipse Ts2R, Nikon, Tokyo, Japan), then merged those into one images using a large imaging stitching tool with NIS-elements Basic Research software (Nikon). C, Representative flow cytometry plot showing progenitor T cells and T-cell development on ATOs. Week 2 is the timing of placing progenitor cells onto ATOs, and week 6 is the timing for harvesting ATO cells for further analysis. More abundant expression of progenitor T and mature T-cell markers on cells were noted at week 6 than at week 2, with a comparison of cells maintained under ambient air (21% O2) vs under physioxia (5% O2) (n = 3)

Article Snippet: CD34+ cell fractions were further isolated through a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, California).

Techniques: Inverted Microscopy, Imaging, Software, Flow Cytometry, Expressing, Comparison

Journal: Stem cells (Dayton, Ohio)

Article Title: Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

doi: 10.1002/stem.3259

Figure Lengend Snippet: Effect of physioxia vs ambient air during the maturation phase in ATOs. A, Experimental schema. B, ATO cell numbers under physioxia (5% O2) were increased more than those under ambient air (21% O2). Cell numbers were normalized by cell numbers placed onto one ATO (7500 progenitors/ATO) and cell numbers plated into one well (4000 CD34+ cells/well) (n = 3, two-way ANOVA, *P < .05). C, Cell numbers of CD7+CD1a+ precursor T cells per 4000 CD34+ cells between physioxia vs ambient air groups in ATOs (n = 3, two-way ANOVA, **P < .01). D-F, Numbers of T-cell subsets per 4000 CD34+ cells between physioxia vs ambient air groups in ATOs on week 6 (n = 3, two-way ANOVA, *P < .05). Data are presented as mean ± SEM. Diff., Differentiation; Mat., Maturation; NS, not significant

Article Snippet: CD34+ cell fractions were further isolated through a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, California).

Techniques:

Journal: Leukemia

Article Title: Extracellular vesicles from human iPSCs enhance reconstitution capacity of cord blood-derived hematopoietic stem and progenitor cells

doi: 10.1038/s41375-021-01325-y

Figure Lengend Snippet: CD34 + cell population highly enriched in HSPCs was purified from CB units and cultured in a dedicated expansion medium containing hiPSC-EVs. A Visualization of hiPSC-EVs internalization into CB-derived CD34 + cells. Cells were incubated with copGFP+ hiPSC-EVs for 2 h. After the removal of unbound hiPSC-EVs, cell nuclei were stained with Hoechst 33342 dye (Hoe). Cell images were captured by Leica DMI6000B microscope with 100× NA-1.47 oil immersion objective. A representative merged image for differential interference contrast module and fluorescence channels for copGFP and Hoe is presented. White arrows indicate copGFP+ hiPSC-EVs accumulated inside the CB-HSPCs. White asterisks indicate aggregated copGFP+ hiPSC-EVs attached to the cells. Scale bar indicates 5 µm. B Kinetics of CB-HSPCs ex vivo expansion. Data are expressed as fold expansion compared to the number of isolated CB-HSPCs. Each dot represents data obtained from individual experimental repetition ( N = 4) for cells expanded in the control medium (Ctrl) or hiPSC-EVs (+hiPSC-EVs) medium. C The effect of hiPSC-EVs on the metabolic activity of CB-derived HSPCs. Expanding CB-HSPCs were treated with hiPSC-EVs for 2, 24, or 48 h. Subsequently, a luminescence assay was performed to measure the concentration of ATP produced by the cells. Data on the graph present the ATP level in hiPSC-EVs-treated cells expressed as the percentage of the control (cells untreated with hiPSC-EVs) in individual experimental repetitions ( N = 5). Black lines represent the mean value, whereas the red line indicates the level of the control (100%). * p < 0.05 for the control vs. hiPSC-EVs-treated cells, two-tailed Student’s t -test. D Kinetics of phenotypic changes in CB-HSPCs during ex vivo expansion. Cells were expanded for 14 days in control medium (Ctrl) or medium containing hiPSC-EVs (+hiPSC-EVs). On the indicated day of the expansion, cells were harvested and stained with fluorescent-conjugated antibodies against CD34 and hematopoietic lineage markers. The analysis of antigen expression was performed with the BD LSRFortessa flow cytometer. Data are presented as mean ± SD ( N = 3). * p < 0.05 for control vs. hiPSC-EVs-treated cells, two-tailed Student’s t -test.

Article Snippet: Fraction highly enriched in HSPCs was sorted by triple-step protocol including: (i) red blood cells lysis with BD Pharm Lyse buffer (BD Bioscience, San Jose, CA, USA), (ii) initial purification of CD34 + fraction using magnetic-activated cell sorter AutoMACS Pro system (Miltenyi Biotec, Bergisch Gladbach, Germany), and (iii) final re-purification by fluorescence-activated cell sorter (FACS) BD FACSAria III (BD Bioscience), following staining with antibodies against selected lineage (Lin) markers, CD45 and CD34 (BD Bioscience).

Techniques: Purification, Cell Culture, Derivative Assay, Incubation, Staining, Microscopy, Fluorescence, Ex Vivo, Isolation, Control, Activity Assay, Luminescence Assay, Concentration Assay, Produced, Two Tailed Test, Expressing, Flow Cytometry

Journal: Leukemia

Article Title: Extracellular vesicles from human iPSCs enhance reconstitution capacity of cord blood-derived hematopoietic stem and progenitor cells

doi: 10.1038/s41375-021-01325-y

Figure Lengend Snippet: Prior experiment, CB-HSPCs were expanded for 7 days in the control medium without hiPSC-EVs and were subsequently treated with hiPSC-EVs for 2 h. Next, cells were stained with calcein AM and seeded onto 96-well plates coated with fibronectin ( A ) or covered with the monolayer of hMSCs ( B ), and HUVECs ( C ). After the incubation, unbound cells were washed out, and fluorescence from the wells was measured by a plate reader. Data on the graphs present the averaged fluorescent signal from attached calcein AM-stained CD34 + cells calculated as a percentage of the control (cells untreated with hiPSC-EVs) in individual experimental repetitions ( N = 7). Black lines represent the mean value, whereas the red line indicates the level of the control (100%). * p < 0.05 for control vs. hiPSC-EVs-treated cells, two-tailed Student’s t -test. D Representative images of calcein AM-labeled CB-HSPCs attached to fibronectin captured in brightfield (BF) and green fluorescence channel by Leica DMI6000B fluorescent microscope with 10× objective magnification. Scale bars indicate 250 µm. E Expression of adhesive molecules on CB-HSPCs treated with hiPSC-EVs for 2, 6, or 24 h. After the incubation, cells were stained with fluorescent-conjugated antibodies against CD49d, CD49e, and LFA-1 and acquired by flow cytometer. Data were calculated as a percentage of the median fluorescent intensity for the control (cells untreated with hiPSC-EVs) and is presented as the mean ± SD ( N = 3). The red line indicates the level of the control (100%). * p < 0.05 for control vs. hiPSC-EVs-treated cells, two-tailed Student’s t -test. F Analysis of LFA-1 expression on hiPSC-EVs. Representative dot plots of hiPSC-EVs stained with RNASelect dye and anti-LFA-1 fluorescent antibody. The percentage of objects positive for the analyzed marker is shown in the red gate. MALS-medium angle light scatter parameter corresponds to the relative size of analyzed particles. In gating strategy, LFA-1 expression was analyzed only on hiPSC-EVs positive for RNASelect.

Article Snippet: Fraction highly enriched in HSPCs was sorted by triple-step protocol including: (i) red blood cells lysis with BD Pharm Lyse buffer (BD Bioscience, San Jose, CA, USA), (ii) initial purification of CD34 + fraction using magnetic-activated cell sorter AutoMACS Pro system (Miltenyi Biotec, Bergisch Gladbach, Germany), and (iii) final re-purification by fluorescence-activated cell sorter (FACS) BD FACSAria III (BD Bioscience), following staining with antibodies against selected lineage (Lin) markers, CD45 and CD34 (BD Bioscience).

Techniques: Control, Staining, Incubation, Fluorescence, Two Tailed Test, Labeling, Microscopy, Expressing, Adhesive, Flow Cytometry, Marker

Journal: Nucleic Acids Research

Article Title: A promoter DNA demethylation landscape of human hematopoietic differentiation

doi: 10.1093/nar/gkr685

Figure Lengend Snippet: DNA methylation changes during hematopoietic differentiation. ( A ) Matrix showing the number of demethylated CpG sites in each hematopoietic cell subset and demethylated CpG sites shared between distinct hematopoietic cell types. ( B ) Illumina array methylation clustering heatmap of SHEF-1 hESC line hypermethylated genes, demethylated in at least one of the six hematopoietic cell types analyzed: CD34 + HSPCs, neutrophils, B cells, NK cells, CD8 + T cytotoxic cells (CD8 + ) and CD4 + T helper cells (CD4 + ). Methylation levels are indicated as in B. ( C ) Box plots of microarray-based gene expression data (log scale). In each blood cell type, specific demethylated genes exhibited higher expression levels compared to other cell types. P -values are shown. n = number of genes analyzed.

Article Snippet: The CB-derived CD34 + -enriched fraction (2 × 10 3 cells/cm 2 ) was plated in methycellulose-based medium supplemented with SCF (50 ng/ml), GM-CSF (10 ng/ml), IL-3 (10 ng/ml) and erythropoietin (3 U/ml; Methocult GF H4434; StemCell Technologies).

Techniques: DNA Methylation Assay, Methylation, Microarray, Gene Expression, Expressing

Journal: Nucleic Acids Research

Article Title: A promoter DNA demethylation landscape of human hematopoietic differentiation

doi: 10.1093/nar/gkr685

Figure Lengend Snippet: Promoter methylation and expression levels of hematopoietic genes in CD34 + HSPCs, cells differentiated from CD34 + and iPSCs generated from CD34 + HSPCs. ( A ) High purity sorted CB-derived CD34 + HSPCs (top middle panel) were differentiated in vitro (Diff-CD34) for 14 days in the presence of SCF, GM-CSF, IL3 and EPO. Granulocyte (CFU-G), monocyte (CFU-M), granulo-monocyte (CFU-GM) and erythroid (BFU-E) colony forming units were scored by light microscopy (right panels). Additionally, CB-derived CD34 + HSPCs were induced to travel back in development by generating iPSCs through ectopic expression of Oct4, Klf4, Sox2 and c-Myc (left panel shows a phase contrast image of a CD34-iPSC). The bottom panel shows a scatter plot of remethylated genes in CD34-iPSC (red) ( Supplementary Table S13 ) and demethylated genes in the differentiated CD34 progeny (Diff–CD34) (green) ( Supplementary Table S12 ). ( B ) Promoter methylation and expression of HLA-DR, CD31 and PIK3CD. Methylation and expression for each gene is indicated as in . Expression by flow cytometry of each gene in CD34-iPSC (top panel) and CD34 + HSPCs (bottom panel). For PIK3CD, WB analysis was performed in CD34 + HSPCs, CD34-iPSC and Diff-CD34 (β-actin was used as loading control).

Article Snippet: The CB-derived CD34 + -enriched fraction (2 × 10 3 cells/cm 2 ) was plated in methycellulose-based medium supplemented with SCF (50 ng/ml), GM-CSF (10 ng/ml), IL-3 (10 ng/ml) and erythropoietin (3 U/ml; Methocult GF H4434; StemCell Technologies).

Techniques: Methylation, Expressing, Generated, Derivative Assay, In Vitro, Light Microscopy, Flow Cytometry, Control

Journal: Nucleic Acids Research

Article Title: A promoter DNA demethylation landscape of human hematopoietic differentiation

doi: 10.1093/nar/gkr685

Figure Lengend Snippet: Cartoon depicting the overall methylation levels of hematopoietic genes at different developmental/differentiation stages: hESC, CD34 + HSPCs and mature hematopoietic cell types (undifferentiated stages, purple nuclei; neutrophils, pink nucleus; lymphoid cells, blue nuclei). For each differentiation stage, the heatmap shows the methylation levels of a selected group of hypermethylated genes in hESCs that are demethylated in that cell type ( Supplementary Table S11 ). Names of some key blood genes are mapped at the right of each methylation heatmap.

Article Snippet: The CB-derived CD34 + -enriched fraction (2 × 10 3 cells/cm 2 ) was plated in methycellulose-based medium supplemented with SCF (50 ng/ml), GM-CSF (10 ng/ml), IL-3 (10 ng/ml) and erythropoietin (3 U/ml; Methocult GF H4434; StemCell Technologies).

Techniques: Methylation