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

Journal: The Journal of Immunology Author Choice

Article Title: Adaptive from Innate: Human IFN-γ + CD4 + T Cells Can Arise Directly from CXCL8-Producing Recent Thymic Emigrants in Babies and Adults

doi: 10.4049/jimmunol.1700551

Figure Lengend Snippet: CXCL8-producing T cells decline with age in humans and in vivo in humanized mice and are enriched in RTEs. ( A ) CXCL8 production was determined in naive CD4 + T cells (4 h PI + BFA) obtained from 45 individuals (aged between 3 mo and 57 y). ( B ) Irradiated NSG mice were reconstituted with human CD34 + cells (CB derived). The graph shows reconstitution of T cells over time in individual mice (gray lines), with a reciprocal decline in T cell production of CXCL8 (black lines). ( C ) FACS plots show an example of this reciprocal change in one reconstituted NSG mouse; percentage of cells expressing CD3 within human CD45 + cells (upper panels) and percentage of cells expressing CXCL8 among CD3 T cells (lower panels; PI + BFA or BFA alone for 4 h). CD4 + T cells from adults ( D and F ) or children aged 1–12 y ( E ) were activated with PI (4 h), and cytokine production was determined by intracellular staining. Individual (cytokine + ) subsets were then sorted, and TREC content was determined by qPCR. Results are shown as TREC levels per million naive CD4 + cells or TREC levels per million total CD4 + cells in (F), because very few naive CD4 + cells express IFN-γ. Trend lines depict the differences in TREC levels among the four sorted populations in three patients. ( G ) CXCL8 production was determined following activation in vitro with PI (4 h, in the presence of BFA) in primary T-ALL samples ( n = 12); later stages (T-III/T-IV) are represented by gray diamonds.

Article Snippet: For CB, mononuclear cells were enriched in CD34 + cells (STEMCELL Technologies), and the CD34 − fraction (on occasion from pooled donors) was used for experiments.

Techniques: In Vivo, Irradiation, Derivative Assay, Expressing, Staining, Activation Assay, In Vitro

Journal: The Journal of Immunology Author Choice

Article Title: Adaptive from Innate: Human IFN-γ + CD4 + T Cells Can Arise Directly from CXCL8-Producing Recent Thymic Emigrants in Babies and Adults

doi: 10.4049/jimmunol.1700551

Figure Lengend Snippet: CXCL8 production is imprinted in the thymus. CXCL8 production was determined following activation in vitro with PI (4 h, in the presence of BFA) in whole thymocytes versus peripheral blood from humanized mice (37 wk post–hematopoietic stem cell inoculation, n = 7) ( A ), sorted human thymocyte subsets ( n = 15, CD4 − CD8 − DN, CD4 + CD8 + double positive, CD4 − CD8 + SP CD8, CD4 + CD8 − SP CD4 + ) ( B ), and enriched T-lineage–committed DN thymocytes (defined as CD4 − CD8 − CD34 + CD7 + CD5 + CD1a + , n = 6) ( C ). Example and cumulative data are shown. Stimulation: BFA only in dark gray and PI+BFA in light gray. ( D ) Comparison of CXCL8 production between paired human SP CD4 + thymocytes and naive (CD31 + CD45RA + ) peripheral CD4 + T cells ( n = 10). * p < 0.05, ** p < 0.001.

Article Snippet: For CB, mononuclear cells were enriched in CD34 + cells (STEMCELL Technologies), and the CD34 − fraction (on occasion from pooled donors) was used for experiments.

Techniques: Activation Assay, In Vitro, Comparison