Journal: Blood

Article Title: Adult human circulating CD34 − Lin − CD45 − CD133 − cells can differentiate into hematopoietic and endothelial cells

doi: 10.1182/blood-2010-10-316596

Figure Lengend Snippet: PB CD34−Lin−CD45− cells give rise to hematopoietic and endothelial-lineage cells on LTC-HE. (A) Sorting criteria used to separate fresh adult human CD34−Lin−CD45− and CD34−Lin−CD45+ cells from PB. (B) RT-PCR showing CD34 and CD45 mRNA expression in freshly sorted CD34−Lin−CD45− and CD34−Lin−CD45+ cells, and hematopoietic stem cells (HSCs) as a control. (C) Time-course of immunophenotypic changes in CD34−Lin−CD45− cells during LTC-HE: progressive appearance of CD34+CD45+ and CD34−CD45+ cells. Data shown as mean ± SE of 6 independent experiments. (D) Representative plots showing the expression of hematopoietic markers by CD34−Lin−CD45− compared with CD34−Lin−CD45+ cells after 6 weeks in LTC-HEs. The mean ± SE of 20 independent experiments is shown in each quadrant. (E) After 6 weeks in LTC-HEs, both cell populations showed similar CFU capacity. Represenative bright-field microscopy images (original magnification 40×). (F) Time-course of immunophenotypic changes in CD34−Lin−CD45− cells during LTC-HE: progressive appearance of CD45−KDR+, CD45+KDR+, and CD45+KDR− cells. Data shown as mean ± SE of 6 independent experiments. (G) Representative plots showing that after 6 weeks of culture (under LTC-HE conditions) CD34−Lin−CD45− but not CD34−Lin−CD45+ cells gave rise to CD45+ cells and to CD45− cells coexpressing molecules linked to endothelial development. The mean ± SE of 9 independent experiments is shown in each quadrant. (H) Percentage of CD45−KDR+, CD45+KDR+, and CD45+KDR− cells in LTC-HEs of CD34−Lin−CD45− cells after 6 weeks. The graph shows the results obtained in each experiment. FSC indicates forward scatter.

Article Snippet: Further characterization of the engrafted cells was obtained by standard flow cytometry of BM suspensions with the use of following anti–human mAbs: PCy5-conjugated anti-CD45; PE-conjugated anti-CD34, CD19; FITC-conjugated anti-CD33 (all from BD Bioscience).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Microscopy

Journal: Blood

Article Title: Adult human circulating CD34 − Lin − CD45 − CD133 − cells can differentiate into hematopoietic and endothelial cells

doi: 10.1182/blood-2010-10-316596

Figure Lengend Snippet: Single CD34−Lin−CD45−CD133− cells give rise to hematopoietic and endothelial lineage cells on LTC-IC-HE culture. (A) Strategy used to sort human CD34−Lin−CD45−CD133− (1), CD34−Lin−CD45−CD133+ (2), CD34−Lin−CD45+CD133− (3), and CD34−Lin−CD45+CD133+ (4) cells. (B) Flow cytometric analysis showing that after 6 weeks in LTC-IC-HEs only CD34−Lin−CD45−CD133− cells gave rise, starting from a single cell, to both CD45−KDR+ (endothelial) and CD45+KDR− (hematopoietic) cells. (C) Poisson analysis of 5 independent experiments, showing the frequency of wells containing CD45+KDR− only, CD45−KDR+ only, or CD45+KDR− and CD45−KDR+ cells, obtained after LTC-IC-HE of the 4 distinct sorted cell populations. (D) Confocal images of cells derived from single CD34−Lin−CD45−CD133− cells after 6-week culture (under LTC-IC-HE conditions). Round CD45+ (hematopoietic-like) and spindle-shaped VE-cadherin+ (endothelial-like) cells in intimate contact with MS-5 murine stromal cells are shown. (E) May-Grunwald-Giemsa staining showing typical myeloid cells generated in CFU assay of LTC-IC-HEs started from single CD34−Lin−CD45−CD133− cells. Original magnification 40×. (F) RT-PCR showing the expression of the hematopoietic CD45 marker and the endothelial VE-cadherin marker in the 4 sorted cell populations at day 0 and after 6 weeks in LTC-IC-HE. Three representative LTC-IC-HE wells (a-c) per cell population after 6 weeks are shown. Only the endothelial-positive control HUVEC line and LTC-IC-HE wells derived from CD34−Lin−CD45−CD133− contained cells expressing VE-cadherin. The concomitant expression of VE-cadherin and CD45 in a single well indicated that, starting from a single cell, CD34−Lin−CD45−CD133− cells gave rise to cells of both endothelial and hematopoietic lineages. (G) Representative flow cytometric histogram showing the expression of VE-cadherin (green line) by cells derived from CD34−Lin−CD45−CD133− single cells (gray line indicates isotype control). At the end of the culture, 11 of 60 seeded wells contained a sufficient number of cells for analysis. Of these 11 wells, 3 tested positive for VE-cadherin expression. APC indicates allophycocyanin.

Article Snippet: Further characterization of the engrafted cells was obtained by standard flow cytometry of BM suspensions with the use of following anti–human mAbs: PCy5-conjugated anti-CD45; PE-conjugated anti-CD34, CD19; FITC-conjugated anti-CD33 (all from BD Bioscience).

Techniques: Derivative Assay, Staining, Generated, Colony-forming Unit Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Positive Control

Journal: Blood

Article Title: Adult human circulating CD34 − Lin − CD45 − CD133 − cells can differentiate into hematopoietic and endothelial cells

doi: 10.1182/blood-2010-10-316596

Figure Lengend Snippet: Immunophenotype of the cells obtained after 6 week LTC-IC-HE of adult human purified CD34 − Lin − CD45 − CD133 − compared with CD34 − Lin − CD45 − CD133 + cells

Article Snippet: Further characterization of the engrafted cells was obtained by standard flow cytometry of BM suspensions with the use of following anti–human mAbs: PCy5-conjugated anti-CD45; PE-conjugated anti-CD34, CD19; FITC-conjugated anti-CD33 (all from BD Bioscience).

Techniques: Purification

Journal: Blood

Article Title: Adult human circulating CD34 − Lin − CD45 − CD133 − cells can differentiate into hematopoietic and endothelial cells

doi: 10.1182/blood-2010-10-316596

Figure Lengend Snippet: Freshly isolated CD34−Lin−CD45−CD133− cells have a stem cell profile compatible with hematopoietic and endothelial potential. (A) May-Grunwald-Giemsa staining showing that freshly isolated CD34−Lin−CD45−CD133− cells have blastoid structure typical of stem cells. (B) Flow cytometric analysis showing that freshly isolated CD34−Lin−CD45−CD133− cells express c-Kit and CXCR4, according to their progenitor nature. (C) RT-PCR showing that freshly isolated CD34−Lin−CD45−CD133− cells express selected stem cell markers as well as markers shared by progenitors of hematopoietic and endothelial lineages. HSCs and primary human endothelial cells (HUVECs) were used as positive controls. One representative of 8 independent experiments is shown. (D) Failure to detect expression of Oct4 (red) and Nanog (green) in freshly isolated CD34−Lin−CD45−CD133− cells and CD34+ HSCs, as assessed by confocal microscopy after immunostaining with specific Abs. Nuclei (blue) are stained with DAPI. iPSC served as a positive control (results not shown). FSC indicates forward scatter. Original magnification in panels A and D is 20×.

Article Snippet: Further characterization of the engrafted cells was obtained by standard flow cytometry of BM suspensions with the use of following anti–human mAbs: PCy5-conjugated anti-CD45; PE-conjugated anti-CD34, CD19; FITC-conjugated anti-CD33 (all from BD Bioscience).

Techniques: Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Confocal Microscopy, Immunostaining, Positive Control

Journal: Blood

Article Title: Adult human circulating CD34 − Lin − CD45 − CD133 − cells can differentiate into hematopoietic and endothelial cells

doi: 10.1182/blood-2010-10-316596

Figure Lengend Snippet: Adult human CD34−Lin−CD45−CD133− cells give rise to both hematopoietic and endothelial lineage cells in NOD/SCID mice. (A) Example of multilineage human hematopoietic (CD45+) reconstitution of BM cells from the IBMI of NOD/SCID recipients receiving CD34−Lin−CD45−CD133− cells that include CD34 (stem/progenitor hematopoietic cells), CD33 (myeloid), and CD19 (B lymphoid) lineage markers. Representative results from 4 injected mice. (B) Frequency of cells positive for human CD45 staining in the BM of mice that received a transplant. Graph showing the results obtained in each experiment; m indicates mouse. (C) Coexpression of human CD34, CD33, and CD19 by CD45+ in BM cells from the same mice. Graph showing the results obtained in each experiment; m indicates mouse; LB, left bone; and OB, other bones. (D) Mouse CD31, human CD31, and human CD45 immunostaining of MOPC315 tumor tissues from mice injected 4 weeks earlier with 2 × 102 CD34−Lin−CD45−CD133− cells. (Top) Confocal microscopy images (3-dimensional rendering) showing the colocalization of mouse tumor vessels (mouse CD31+) and human CD31+ cells, which fail to costain for human CD45. (Bottom) Confocal microscopy images showing human CD31+/CD45− and human CD45+/CD31− cells proximal or distant from mouse CD31+ vessels. The asterisk points to a human CD45+ cell (pink) adjacent to a human CD31+ cell (red); the cells are shown at a higher magnification in the inset. The arrowheads point to the colocalization (yellow) of mouse CD31 (green) and human CD31 (red) immunostaining. The arrow in the far right panel points to a human CD45+/CD31− cell. Scale bars = 50 μm. (E) Images from clipping and rotation of a 3-dimensional confocal image of a tumor vessel from a mouse injected 4 weeks earlier with CD34−Lin−CD45−CD133− cells showing the spatial relationship between the murine endothelial cells (green, mCD31+) and the human endothelial cells (red, hCD31+) contributing to the vessel wall. Scale bar = 50 μm. (F) MOPC315 tumor tissue from a mouse injected with 1 × 103 CD34−Lin−CD45+ cells shows vascular structures expressing mouse but not human CD31. Clusters of human CD45+ cells are observed. Results from immunostaining with specific anti–mouse CD31 (green), anti–human CD31 (red), and anti–human CD45 (pink) are shown. Nuclei are detected by DAPI staining (blue). FSC indicates forward scatter.

Article Snippet: Further characterization of the engrafted cells was obtained by standard flow cytometry of BM suspensions with the use of following anti–human mAbs: PCy5-conjugated anti-CD45; PE-conjugated anti-CD34, CD19; FITC-conjugated anti-CD33 (all from BD Bioscience).

Techniques: Injection, Staining, Immunostaining, Confocal Microscopy, Expressing

Journal: medRxiv

Article Title: Identification and validation of heterotypic cell-in-cell structure as an adverse prognostic predictor for young patients of resectable pancreatic ductal adenocarcinoma

doi: 10.1101/2020.07.08.20148825

Figure Lengend Snippet: ( A ) Unprocessed composite core multiplex stained with E-cadherin, CD68, CD45, and DAPI. ( B ) Unmixed composite core pseudo-colored with yellow for E-cadherin, green for CD68, red for CD45, and blue for DAPI. ( C ) Spectral parameters for image unmixing. Fluorescent signals for different targets were captured based on the spectra indicated. ( D ) Images of single or merged channels displaying the boxed region in ( C ). Scale bar: 20 μm. ( E ) Representative images for the 4 CIC subtypes as indicated; scale bars: 5 μm;

Article Snippet: Samples were first stained with anti-CD45 antibody (mouse mAb from Boster, BM0091) at a dilution of 1:400 using Opal Multiplex tissue staining kit (Perkin Elmer, NEL791001KT) according to the manufacturer’s standard protocol.

Techniques: Multiplex Assay, Staining

Journal: medRxiv

Article Title: Identification and validation of heterotypic cell-in-cell structure as an adverse prognostic predictor for young patients of resectable pancreatic ductal adenocarcinoma

doi: 10.1101/2020.07.08.20148825

Figure Lengend Snippet: (A) CIC structures indicated by arrows in PDAC tissues stained by H&E. Boxed regions are zoomed in at the right. (B) CIC structures in PDAC tissues stained with antibodies for CD68 or CD45, respectively, by IHC. Boxed regions are zoomed in at the bottom. (C) Number of tissues positive in each CIC subtype. (D) The compositions of CIC subtypes in different TNM stages.

Article Snippet: Samples were first stained with anti-CD45 antibody (mouse mAb from Boster, BM0091) at a dilution of 1:400 using Opal Multiplex tissue staining kit (Perkin Elmer, NEL791001KT) according to the manufacturer’s standard protocol.

Techniques: Staining