Journal:

Article Title: PlexinD1 controls migration of positively-selected thymocytes into the medulla

doi: 10.1016/j.immuni.2008.10.008

Figure Lengend Snippet: A. Thymocytes of N15TCRtg+/+ RAG2−/− mice were stained with anti-CD8α-FITC and anti-CD4-PE. Three different populations were sorted based on the CD4 cell surface expression level: DP is CD8highCD4high, Int is CD8high/CD4int (intermediate population) and CD8 SP is mature CD8 single positive. Total RNA was isolated from sorted thymocytes and used for microarray analysis as described in the supplementary methods. B. Hierarchical clustering was performed with genes that show significant changes (LCB >2.0). C. Gene expression Patterns I and II as noted in B. The 391 genes showing an increase during maturation were clustered in pattern I. The 328 genes that decreased (LCB <−2.0) with maturation cluster to make up pattern II. R: Receptor. Pattern III and IV are altered in the Int population only and documented in Fig. S1 and http://cvc.dfci.harvard.edu/share_folder/N15RAG-2ko_microarray_data/.

Article Snippet: Antibodies and reagents Anti-FcγR (2.4G2), anti-TCRβ-APC, anti-CD69-FITC, anti-CD25-PE, anti-CD44-biotin, streptavidin-APC-Cy7, anti-CD4-Alexaflour647 and anti-Ly-CD45.2-FITC were purchased from BD-Pharmingen (San Jose, CA).

Techniques: Staining, Expressing, Isolation, Microarray

Journal:

Article Title: PlexinD1 controls migration of positively-selected thymocytes into the medulla

doi: 10.1016/j.immuni.2008.10.008

Figure Lengend Snippet: A. Migration assays were performed in 24-well transwell plates with a 5μm pore size as depicted. Cells were added to the insert and the indicated chemokines were added to the bottom well with purified sema3E-Fc or Fc-only protein. B. After 2 hrs, cells that had migrated to the bottom well were harvested and stained with anti-CD4, anti-CD8α, anti-CD69, and anti-TCRβ mAbs. Mock in the top panel is a sample without chemokine treatment. CCL25 was added to the bottom well in the left set of panels and CCL21 to the right set. The concentration of S3E (sema3E-Fc) is indicated on the right of each panel. In the middle panels, the CD4/CD8 patterns of the CD69+ cells that migrated to the bottom of the well are shown for the CCL25- and CCL21-treated cultures. More than two-thirds of CD69+ thymocytes were DP in CCL25-treated and less than one-quarter were DP in CCL21-treated cultures. C. An aliquot of cells harvested from B was counted and the number of CD69+ thymocytes migrating in response to CCL25 or CCL21 in the presence of various concentrations of sema3E was determined. Error bars are the standard error of one triplicate experiment. D. Single cell suspensions were prepared from B6 thymus, stained with the indicated mAbs and analyzed on a flowcytometer. Data were analyzed using FlowJo (TreeStar, Or). The upper left panel is a CD4/CD8 profile. CD69 expression in each gated population is depicted with dark gray above control staining in the upper right panels. In the lower four sets of panels, CCR7 and CCR9 expression in each population (gated on CD69, CD4 and CD8 expression) are shown. The upper row in each set of four shows samples stained with isotype control mAbs while the lower row shows those stained with anti-CCR7 and anti-CCR9 mAbs. The numbers in the panels are % of gated populations.

Article Snippet: Antibodies and reagents Anti-FcγR (2.4G2), anti-TCRβ-APC, anti-CD69-FITC, anti-CD25-PE, anti-CD44-biotin, streptavidin-APC-Cy7, anti-CD4-Alexaflour647 and anti-Ly-CD45.2-FITC were purchased from BD-Pharmingen (San Jose, CA).

Techniques: Migration, Purification, Staining, Concentration Assay, Expressing

Journal:

Article Title: PlexinD1 controls migration of positively-selected thymocytes into the medulla

doi: 10.1016/j.immuni.2008.10.008

Figure Lengend Snippet: A. Cryosections of reconstituted mice were stained with anti-CD4/CD8α/CD69 mAbs. Stained sections were analyzed using a Carl Zeiss LSM510 confocal microscope. The blue dashed line follows the corticomedullary junction and solid white line shows the thymic capsule. This figure is representative of four independent transplantation experiments. CD69 is depicted as white spots in the upper left section of each quartet and as blue spots in the merged image in the lower left section of the same quartet. CD4 is green and CD8 is red. (Note that CD69+ cells co-expressing CD4 and/or CD8 appear in different colors.) The sections are from B6 mice, mice reconstituted with Plxnd1+ or Plxnd1−/− fetal liver cells (left, middle and right panels, respectively). Cryosections were stained with anti CD4 (Green)/CD8α (Red)/ER-TR5 (Blue; a marker of mTEC) mAbs. B. Stained sections were analyzed as described in A and represent magnified images of inserts (a) and (b) shown in Fig. S6A. The solid line is the corticomedullary junction and the dashed line is the boundary of SP thymocytes. The graph depicts the distribution of SP thymocytes in the cortex (ER-TR5− area), analyzed by plotting SP thymocyte numbers based on the size of SP thymocyte clusters in the cortex. SP thymocyte clusters and number of cells per cluster were counted from 5 different slides. The error bar is the standard error. C. Medullary structures were visualized by staining paraffin sections of transplanted mice with hematoxylin and eosin. The indicated inserts in the chimeric thymi from Plxnd1−/− fetal liver cell donors are magnified and the arrows highlight contact between the medulla and the thymic capsule.

Article Snippet: Antibodies and reagents Anti-FcγR (2.4G2), anti-TCRβ-APC, anti-CD69-FITC, anti-CD25-PE, anti-CD44-biotin, streptavidin-APC-Cy7, anti-CD4-Alexaflour647 and anti-Ly-CD45.2-FITC were purchased from BD-Pharmingen (San Jose, CA).

Techniques: Staining, Microscopy, Transplantation Assay, Expressing, Marker

Journal: PLoS ONE

Article Title: Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

doi: 10.1371/journal.pone.0042838

Figure Lengend Snippet: ( a ) Schematic summarizes the experimental strategy for determining 4F reprogramming efficiencies of FACS-purified hematopoietic progenitors of ( b ) BMSC-primed lineage-committed or ( c ) +/− BMSC-primed transgene-enriched myeloid populations. Experimental details are provided in . The %CD34 + gradient symbol above the schematic reflects the concept that multipotent primitive CD34+CD38lo stem-progenitors are enriched during Days -3 to -2, but differentiate rapidly in culture thereafter (see also Fig. 1c and Fig. S2a) . CD34 + CD38 lo stem-progenitors give rise to lineage-committed CD34 + CD38 + progenitors which subsequently differentiate further to CD34-negative CD33 + CD45 + myeloid cells ( e.g ., promyelocytes). Post-sort analysis of FACS-purified Day 0 CD34 + CD38 + CB fractions verified that >95% of this populations consisted of CD33 + CD13 + CD45 + myeloid cells (see also Fig. 1c ). Both reprogramming efficiency (AP and live TRA-1-81 staining of hESC-like colonies) and reprogramming completion (bulk SSEA4 + TRA-1-81 + and NANOG + FACS staining) assays were conducted 4 weeks following 4F nucleofections on P 0 MEF cultures. ( b ) Shown is a representative AP staining (plates done in triplicate, with indicated average number of hESC-like colonies emerging per the number of single sorted day 3 BMSC-primed CB cells plated on MEF (i.e., unsorted CB vs. CD34 + CD38 lo vs. CD34 + CD38 + fractions. The averaged results of two independent experiments are indicated. In lower panels are shown representative FACS staining of surface TRA-1-81 and intracellular NANOG pluripotency markers of the same cultures demonstrating that 50–80% of AP + hESC-like colonies possessed a Type III TRA-1-81 + NANOG + phenotype . ( c ) To determine the more accurate reprogramming efficiency of purified +/− BMSC-primed episome-expressing myeloid populations, CB progenitors were co-nucleofected on day 0 with both the 4F pEP4 EO2S EM2K episome, as well as a pCEP4-GFP episomal GFP reporter construct. Episomal transgene expressing-only populations were subsequently FACS-purified by GFP expression prior to plating on day 3 MEF following with (+BMSC) or without (-BMSC) stromal co-culture. This was done by staining +/− BMSC-primed CB cells on day 3 with CD34-PE and FACS-purifying them into episome-expressing (GFP + CD34 + , GFP + CD34 − ), and non episome-expressing (GFP − ) populations prior to MEF plating. The results of AP stained plates shown are representative of independent sorting experiments using pooled donor CB samples for each 4F nucleofection, with the averaged results of two independent experiments indicated below. FACS analysis of these same experiments (data not shown) revealed 60% TRA-1-81 and NANOG expression for +BMSC AP + colonies (GFP + CD34 − ), and 55% TRA-1-81 expression for –BMSC AP + colonies (GFP + CD34 − ).

Article Snippet: Antibodies included APC conjugated SSEA4 (R&D Systems), PE Mouse anti-Human TRA-1-60 antigen (BD Biosciences) and PE Mouse anti-Human TRA-1-81 antigen (BD Biosciences).

Techniques: Purification, Staining, Expressing, Construct, Co-Culture Assay

Journal: PLoS ONE

Article Title: Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

doi: 10.1371/journal.pone.0042838

Figure Lengend Snippet: ( a ) Illumina microarray expressions of ESC-like gene modules (MYC, PRC1, PRC2, and Core; see for gene lists). Shown are donor cell populations with (+) and without (−) 4F episomal nucleofections, and with (+) and without (−) BMSC-priming: adult fibroblasts (F), Day -3 unstimulated CB cells (D-3 CB), Day 0 FTK GF-stimulated CB cells (D0), Day 3+/− BMSC-primed (D3), and bulk Day 23 early CB-iPSC culture (D23 iPSC). These early D23 iPSC populations were already composed of >50–60% populations with fully-reprogrammed TRA-1-81 + NANOG + phenotypes. Undifferentiated H9 hESC samples served as control (hESC). Module expressions represent log2 mean-subtracted normalized values of signal intensities from averaged, independent biological replicate microarray samples (n = 3 per condition). Although they possessed a transcriptionally inactive Core module, day 0 GF-activated CB progenitors expressed active ESC and MYC modules, and inactive PRC1, and PRC2 modules at mean expression levels that were already comparable to levels in hESC. The annotation and references of all genes in each module is provided in . ( b ) Partially reprogrammed ESC module expression in CB progenitors. Legend for each sample is the same as above. Unsupervised hierarchical clustering heatmaps of ( b ) expression and ( c ) Box plots of log2 mean-normalized values of the ESC module gene signal intensities in somatic target populations, hESC, and reprogrammed cell lines. The heatmap’s color scale was chosen to emphasize subtle mid-range change. The resulting values emphasize relative expression across cell types rather than relative absolute expression across genes. This box and whisker plots (right panels) depict the log2 mean-subtracted normalized values of signal intensities of genes comprising the module set for each cell type indicated from Illumina array data. The top and bottom of a box mark the 75 th and 25 th percentile log2 signal values, respectively, while the bar at the middle denotes the median. The whiskers above and below each box mark the upper 90 th and lower 10 th percentiles. Paired tests with significance p <0.05 (*) or without significance ( NS ; p>0.05) with values of control hESC are indicated.

Article Snippet: Antibodies included APC conjugated SSEA4 (R&D Systems), PE Mouse anti-Human TRA-1-60 antigen (BD Biosciences) and PE Mouse anti-Human TRA-1-81 antigen (BD Biosciences).

Techniques: Microarray, Expressing, Whisker Assay

Journal: PLoS ONE

Article Title: Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

doi: 10.1371/journal.pone.0042838

Figure Lengend Snippet: ( a ) Emergence of surface pluripotency markers (SSEA4, TRA-1-81) were assayed by FACS at 3 weeks in bulk cultures of 4F episomally-reprogrammed somatic cells briefly co-cultured with (+) or without ( − ) irradiated BMSC ( Fig. S1 ). Fetal fibroblasts (FFB), adult fibroblasts (AdFib), adult keratinocytes (Ker), and GF-activated Day 0 CB (CB) were nucleofected with 4F or 7F on Day 0 ( Fig. S1 ), and reprogrammed bulk cultures were analyzed by FACS 3 weeks later. AP stains of hESC-like colonies were done in parallel of these same experiments, and are presented in Fig. 1d . Shown are the averaged results of 2–5 experiments with averages, and significances ( * ) designated at peak of bar graphs. ( b,c ) The kinetics of pluripotency marker emergence of 4F reprogrammed CB progenitors with (+) and without (−) BMSC priming. ( b ) SSEA4 + , and ( c ) SSEA4 + TRA-1-60 + expressions. ( d ) Enhancement of 4F CB reprogramming with (+BMSC) and without (- BMSC) stromal priming was due to signals that were partially cell contact-dependent, and partially soluble factor-mediated. GF-activated CB cells were cultured as described in Fig. S1 from Day 0 until Day 3 without BMSC co-culture (-BMSC), with BMSC co-culture (+BMSC), or with BMSC co-culture but physically separated from CB cells with a Transwell insert that prevented cell-cell contact between BMSC and CB cells, but allowed diffusion of soluble stromal factors (+BMSC (T)). Shown is the relative fold-increase of reprogramming efficiency (enumerated AP+ hESC-like colonies) from two averaged 4F-reprogramming experiments from baseline efficiencies (-BMSC conditions). Reprogramming efficiency was determined at 3 weeks post-nucleofection with 4F, determined by AP staining of hESC-like colonies (as described in ). ( e ) Gene specific enrichment analysis (GSEA) computation of pathways activated in 4F-nucleofected CB cells by stromal signals. The GSEA algorithm was used to identify curated pathways over-represented among genes with significant (p<0.05, FDR<0.05) differential expression between Day 3 (D3) +BMSC-primed CB samples, vs . D3 unprimed (-BMSC) CB samples that were nucleofected at Day 0 with 4F episome. summarizes the MSigDB v. 3.0 gene set categories that were enriched with FDR <0.05 and nom p <0.05 for these two paired gene set computations. Shown in ( e ) are the major categories of enriched pathways that were significantly and differentially activated in Day 3 BMSC-primed and 4F-nucleofected CB samples.

Article Snippet: Antibodies included APC conjugated SSEA4 (R&D Systems), PE Mouse anti-Human TRA-1-60 antigen (BD Biosciences) and PE Mouse anti-Human TRA-1-81 antigen (BD Biosciences).

Techniques: Cell Culture, Irradiation, Marker, Co-Culture Assay, Diffusion-based Assay, Staining, Expressing