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: Stem Cells and Development

Article Title: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors

doi: 10.1089/scd.2010.0017

Figure Lengend Snippet: List of Predesigned Taqman ® Probes for QPCR (Applied Biosystems)

Article Snippet: Hs00156373_m1 , CD34 , Homo sapiens CD34 molecule, mRNA.

Techniques: Variant Assay, Wilms Tumor Assay

Journal: Stem Cells and Development

Article Title: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors

doi: 10.1089/scd.2010.0017

Figure Lengend Snippet: Microarray analyses of undifferentiated, unfractionated, and sorted fractions. (A) Heat maps of undifferentiated, unfractionated, and sorted fractions. Combined heat map of 3 biological replicates shows down-regulation of stem cell genes (yellow) and up-regulation of developmental genes (blue) across the undifferentiated (UD), unfractionated (UF), +Low, and ++− fractions. All 3 replicates show similar expression patterns of pluripotent and developmental genes confirming the reproducibility of the array results. (B) Gene ontology analyses performed on the top 1,000 up-regulated genes (B-stat > 0) in the ++− compared with the undifferentiated fraction show a slight increase in percentage of kidney and vascular development-associated genes in the ++− fraction relative to either spontaneously differentiated embryonic stem (ES) cells or cell populations derived from varying stem cell marker expression-based fractionation (red arrowheads). Ontology analysis also indicated a reduced proportion of genes involved in neural and skeletal muscle development in the ++− cell fraction (black arrowheads). (C) Cluster analyses. (a) Cluster analysis of the top 200 up-regulated genes in the ++− fraction compared with undifferentiated ES cells cross-matched with a genitourinary development-specific gene expression database (GUDMAP) shows an overrepresentation of transcripts associated with E11.5 murine metanephric interstitium (green box) and E15.5 nephrogenic and cortical interstitium (yellow box). (b) This overrepresentation was not observed when a randomly generated list of genes was subjected to the same cross-match. (c) Cluster analysis performed on all genes up-regulated only in ++− compared with undifferentiated, spontaneously differentiated, and ++Low fractions shows these genes are associated with the MM at E11.5 (green box), podocytes at E13.5 and E15.5 (yellow box), and the collecting duct and proximal tubules (purple box) when cross-matched with the GUDMAP database.

Article Snippet: Hs00156373_m1 , CD34 , Homo sapiens CD34 molecule, mRNA.

Techniques: Microarray, Expressing, Derivative Assay, Marker, Fractionation, Gene Expression, Generated

Journal: Stem Cells and Development

Article Title: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors

doi: 10.1089/scd.2010.0017

Figure Lengend Snippet: Validation of microarray results by quantitative polymerase chain reaction (PCR). Results of the microarray were validated by quantitative PCR screen of genes involved in the development of each compartment of the mesoderm where expression levels were first normalized against 18S rRNA and calibrated using a CD30-expressing human embryonic stem (ES) cell line as an internal standard. The QPCR results demonstrated a slight up-regulation in transcript levels of all 3 intermediate mesoderm and 2 lateral mesoderm genes, CD34 and CDH5, in the ++− fraction compared with the other 3 fractions, indicating that cells expressing these transcripts are enriched from the unfractionated cell population by sorting based on CD24+/Podo+/GCTM2−. Conversely, an overall lower level of gene expression was observed for paraxial mesoderm genes, which also showed little variation in transcript abundance across the cellular fractions, indicating that the cells that express these genes are not selectively isolated by sorting with the above markers. Values are presented as means ± SEM, n = 3.

Article Snippet: Hs00156373_m1 , CD34 , Homo sapiens CD34 molecule, mRNA.

Techniques: Biomarker Discovery, Microarray, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Isolation

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: Journal of visualized experiments : JoVE

Article Title: Pan-Myeloid Differentiation of Human Cord Blood Derived CD34 + Hematopoietic Stem and Progenitor Cells

doi: 10.3791/59836

Figure Lengend Snippet: Table of Materials

Article Snippet: Human CD34 microbead kit , Miltenyi biotech , 130-046-702 , .

Techniques: Sterility, Staining, Modification, Concentration Assay, Fractionation, Recombinant, Flow Cytometry, Light Microscopy

Journal: Journal of visualized experiments : JoVE

Article Title: Pan-Myeloid Differentiation of Human Cord Blood Derived CD34 + Hematopoietic Stem and Progenitor Cells

doi: 10.3791/59836

Figure Lengend Snippet: (A) Cells were gated on forward and side scatter to select a single cell population. Dead and lineage positive cells were eliminated by staining with 7-AAD and antibodies to CD3, CD7, CD10, CD11b, CD19, and CD235a (all FITC stained). The Lin−/live cells were analyzed with antibodies to CD34 (APC-Cy7), CD38 (PE), CD123 (APC), and CD45RA (PE-Cy7). The progenitors were distinguished from the CD34+/CD38+ cells. CMPs are CD123lo/CD45RA−, GMPs are CD123lo/CD45RA+ and MEPs are CD123−/CD45RA−. Representative scatter plots from an experiment are shown. (B) Percentages of total HSPCs (total CD34+ cells), CMPs, GMPs, and MEPs in cord blood are represented. Data presented are averages with standard error from at least 3 experiments.

Article Snippet: Human CD34 microbead kit , Miltenyi biotech , 130-046-702 , .

Techniques: Staining

Journal: Journal of visualized experiments : JoVE

Article Title: Pan-Myeloid Differentiation of Human Cord Blood Derived CD34 + Hematopoietic Stem and Progenitor Cells

doi: 10.3791/59836

Figure Lengend Snippet: Single cells were gated based on forward and side scatter. CD34− cells were selected by staining with antibody to CD34 (APC-Cy7). Myeloid lineages in the CD34− population were analyzed with antibodies to CD14 (PE), CD66b (PE-Cy7), CD41 (PerCP-Cy5.5), and CD235a (APC) on day 1 (A) and day 21 (B). Monocytes are CD14+/CD66b−, granulocytes are CD14−/CD66b+, megakaryocytes are CD41+/CD235a− and erythroid cells are CD41−/CD235a+. Representative scatter plots from an experiment are shown. Fractions of monocytes, granulocytes, megakaryocytes, and erythroid cells in the CD34− population on day 1 and day 21 are represented (C). Data presented are averages with standard error from at least 3 experiments. This figure has been modified from Bapat et al.11.

Article Snippet: Human CD34 microbead kit , Miltenyi biotech , 130-046-702 , .

Techniques: Staining, Modification

Journal: Journal of visualized experiments : JoVE

Article Title: Pan-Myeloid Differentiation of Human Cord Blood Derived CD34 + Hematopoietic Stem and Progenitor Cells

doi: 10.3791/59836

Figure Lengend Snippet: CD34+ HSPCs (A) and cells from myeloid cultures at day 21 (B) were stained with the Wright-Giemsa stain. Cells corresponding to granulocytes (black arrows), monocytes (blue arrows) and erythroid cells (red arrows) are shown. Scale bars represent 10 μM. This figure has been modified from Bapat et al.11.

Article Snippet: Human CD34 microbead kit , Miltenyi biotech , 130-046-702 , .

Techniques: Staining, Giemsa Stain, Modification

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis

doi: 10.1186/ar2916

Figure Lengend Snippet: Analysis of total synovium-derived cell population with four-color flow cytometry: representative sample . Cells are stained with 7-aminoactinomycin (7-AAD), CD45- fluoro-isothiocyanate (FITC), one phycoerythrin (PE)-conjugated antibody and one allophycocyanin (APC)-conjugated antibody or their isotypes. (a) Dye exclusion of debris and dead cells in forward scatter (FSC)/FL3 in contour plot (top) and dot plot (bottom). G1 gates for live cells. (b) Subgating of CD45-negative stromal (G2) and CD45-positive (G3) hematopoietic cell populations based on isotype staining with IgG1-FITC and cell granularity (side scatter). (c) Quantification of CD34-expression and CD90-expression within the stromal fraction. The gating for CD90-positive cells was based on IgG1-PE specifically measured on CD45-negative cells. (d) Quantification of CD14, CD3, and CD20-expression within G3. The gating for CD20-positive cells was performed on IgG1-PE measured on total cells. (e) Plots showing CD105-APC, CD73-PE, CD146-PE, and human leucocyte antigen (HLA)-DR-PE staining in the stromal fraction with their appropriate isotype controls.

Article Snippet: Partial enrichment for CD34-positive fraction was performed with an EasySep CD34 selection kit using the clone QBend10 (Stem Cell Technologies, Grenoble, France).

Techniques: Derivative Assay, Flow Cytometry, Staining, Expressing

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis

doi: 10.1186/ar2916

Figure Lengend Snippet: Influence of the isolation procedure on the detection of stromal markers . (a) Dot plot showing CD34- allophycocyanin (APC) and isotype staining of viable CD45-positive CD34-enriched cord blood mononuclear cells (CB-MNCs) before and after exposure to the digestion method. (b) Plots showing (top panel) human leucocyte antigen (HLA)-DR- phycoerythrin (PE) staining and (lower panel) forward scatter (FSC)/side scatter (SSC) of viable, CD45-positive peripheral blood mononuclear cells (PBMCs) before and after exposure to the digestion method. (c) Dot plots showing CD73-PE, CD105- fluoro-isothiocyanate (FITC) and isotype stainings of cultured passage five synovium-derived mesenchymal stem cells. (d) Plots showing CD146-PE and isotype staining in viable (negative for 7-aminoactinomycin (7-AAD) in peridinin chlorophyll protein channel) human osteosarcoma cells before and after exposure to the digestion method.

Article Snippet: Partial enrichment for CD34-positive fraction was performed with an EasySep CD34 selection kit using the clone QBend10 (Stem Cell Technologies, Grenoble, France).

Techniques: Isolation, Staining, Cell Culture, Derivative Assay

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis

doi: 10.1186/ar2916

Figure Lengend Snippet: Quantification of surface marker expression in synovial digests

Article Snippet: Partial enrichment for CD34-positive fraction was performed with an EasySep CD34 selection kit using the clone QBend10 (Stem Cell Technologies, Grenoble, France).

Techniques: Marker, Expressing

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis

doi: 10.1186/ar2916

Figure Lengend Snippet: Semi-quantitative assessment of vascularization in synovial tissue by immunohistochemistry . Magnification 100×. (a to c) von Willebrand Factor staining. (a) Positive endothelial cells are detected. (b) Isotype control. (c) Positive (sub)endothelial cells and fibroblasts are detected. (d to f) CD34 staining. (e) Isotype control. (c and f) Scores in different patient groups.

Article Snippet: Partial enrichment for CD34-positive fraction was performed with an EasySep CD34 selection kit using the clone QBend10 (Stem Cell Technologies, Grenoble, France).

Techniques: Immunohistochemistry, Staining

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis

doi: 10.1186/ar2916

Figure Lengend Snippet: Surface marker phenotype of cultured synovium-derived cells

Article Snippet: Partial enrichment for CD34-positive fraction was performed with an EasySep CD34 selection kit using the clone QBend10 (Stem Cell Technologies, Grenoble, France).

Techniques: Marker, Cell Culture

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis

doi: 10.1186/ar2916

Figure Lengend Snippet: Detection and quantification of CD271 and CD34 in digests and cultured cells . (a) Plots of two representative samples. The CD45-negative fraction is gated in G1. Within this fraction, a portion of cells is positive for CD271 or CD34. Double positive cells are sometimes well clustered (lower plot). To be uniform, the quantification is performed using quadrant analysis. (b) Data points representing the expression levels in percentage positivity on the cell surface detected in the fresh digests. (c) Plots of cultured cells after staining. Cells are gated according to scatter properties (G1), viability (G2) and the signal for the markers is compared with the isotype controls. No double positive cells are recognized here. 7-AAD = 7-aminoactinomycin; APC = allophycocyanin; FITC = fluoro-isothiocyanate; FSC = forward scatter; PE = phycoerythrin; SSC = side scatter.

Article Snippet: Partial enrichment for CD34-positive fraction was performed with an EasySep CD34 selection kit using the clone QBend10 (Stem Cell Technologies, Grenoble, France).

Techniques: Cell Culture, Expressing, Staining

Journal: Arthritis Research & Therapy

Article Title: Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis

doi: 10.1186/ar2916

Figure Lengend Snippet: Comparing marker expression in the digests and cultures from the same samples

Article Snippet: Partial enrichment for CD34-positive fraction was performed with an EasySep CD34 selection kit using the clone QBend10 (Stem Cell Technologies, Grenoble, France).

Techniques: Marker, Expressing, Cell Culture

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