cd56 ncam1 af647  (R&D Systems)

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A ) Stacked violin plots of stromal, osteochondrogenic, and fibroblastic gene expression in different stromal clusters. Corresponding stromal cell groups are indicated on the x-axis legend. The color bar indicates gene expression in each cluster. ( B ) mRNA fold change of CD81 in FACS soreted A1-A4 subsets (n=3). Results are shown as mRNA fold change after standardization with GAPDH levels. For comparison, expression level of CD81 in A4 is set as one fold. *: p<0.05 (Kruskal-Wallis test). ( C ) The relative contribution of each sorted stromal cell populations in ( B ) to total CFU-F. Data are given as a normalized mean percentage as indicated in the figure (n=3). The sum of CFU-F of all populations is defined as 100%. ( D ) Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for CD271 (pink), CD81 (green), NCAM1 (cyan) and CD45 (yellow) and scanned with the OlympusVS120 slide scanner. Single staining for each marker is shown as indicated under each picture. Scale bars represent 50 µm. Red arrows: CD271 + CD81 ++ cells; white arrows: arteriolar walls; white lines: bone lining regions. Bone ( b ), adipocytes ( a ), and capillaries (*) are indicated. ( E ) FFPE human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), CD81 (green), CD271 (pink), and NCAM1 (cyan) and scanned with the OlympusVS120 slide scanner. Scale bars represent 20 µm.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Gene Expression, Comparison, Expressing, Formalin-fixed Paraffin-Embedded, Staining, Marker

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A ) Dot plot of surface marker gene expression in different stromal cell clusters. Cluster numbers and corresponding stromal cell groups are indicated on the y-axis legend. Dot sizes represent the percentage of cells expressing a certain gene in each cluster and dot colors represent the scaled average expression of that gene. ( B ) FACS plots illustrating the gating strategy for the isolation of different stromal subsets. The displayed cell populations are indicated on top of the plot. Following exclusion of doublets, dead cells, CD45- and CD235a-expressing cells, CD45 low/- CD235a - CD71 - CD271 + cells (left panel) were gated based on CD52 and NCAM1 expression (middle panel). The resulting three populations were labelled as A-C (middle panel), corresponding to the stromal cell groups in ( A ) (A, CD52 - NCAM1 - ; B, CD52 - NCAM1 + ; C, CD52 + NCAM1 - ). CD45 - CD235a - CD71 - CD271 + CD52 - NCAM1 - (group A) cells were further divided based on CD81 expression and four populations were identified ( A1–A4 ) (right panel). A1, CD81 ++ ; A2, CD81 + ; A3; CD81 +/- ; A4, CD81 - . ( C ) CFU-F frequencies of sorted stromal cell populations as shown in ( B ). Data are presented as individual data (dots) and median (horizontal lines) from independent experiments (n=3–6). Symbol colors and x-axis labels correspond to the cell population colors in ( B ). *: p<0.05; ****: p<0.0001 (Kruskal-Wallis test). ( D ) In vitro differentiation capacity of sorted stromal cell populations (as indicated in B) towards the adipogenic, osteoblastic, and chondrogenic lineage. Non-induction controls are shown in the left panel. Scale bars represent 200 µm. A representative set of pictures from a total of three independent experiments is shown. ( F ) Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), CD81 (green), CD271 (pink), and NCAM1 (cyan) and scanned with the OlympusVS120 slide scanner. Different staining combinations are shown as indicated under each picture to provide better visualization of individual staining obtained from the same FFPE slide. Scale bars represent 50 µm. Red arrows: CD271 + CD81 ++ cells; white arrows: arteriolar walls; white lines: bone lining regions. Bone ( b ), adipocytes ( a ), and capillaries (*) are indicated.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Marker, Gene Expression, Expressing, Isolation, In Vitro, Formalin-fixed Paraffin-Embedded, Staining

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A ) CFU-F frequency of single-cell and bulk sorted MSSCs, i.e. sorted A1 cells (CD45 low/- CD235a - CD71 - CD271 + NCAM1 - CD52 - CD81 ++ ). Data are shown as individual data points and medians (horizontal lines), n=3. **: p<0.01 (Mann-Whitney test). ( B ) A dot plot overview of top-ranked ligand-receptor pairs between different bone marrow stromal clusters. P values are indicated by circle color. The means of the average expression levels are indicated by circle size. Scale bars are provided on the right side of the plot. Stromal cell cluster pairs are indicated by ‘cluster number_cluster number’ (y-axis labels). Dashed lines separate the different stromal clusters. HAGEP, highly adipocytic gene-expressing progenitors; Bal., balanced progenitors; Pre-ob, pre-osteoblasts; Pre-fb, pre-fibroblasts. Interacting molecule pairs are indicated in the x-axis labels. ( C ) UMAP (as in ) illustration of the normalized expression of genes involved in alpha 1 beta 1 integrin complex (a1b1 complex). ( D ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor genes. ( E ) Fold change of total number of CD34 + cells (left) and CD34 + CD90 + cells (right) produced after seven days in culture. Results are shown as fold change relative to the cell number of standard CD34 + culture without stroma support (No stroma). Data are presented as individual data (dots) and median (horizontal lines) from independent experiments (n=2).

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: MANN-WHITNEY, Expressing, Produced

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A–D, F–G ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. Red dashed lines in figures A, B and D mark the erythroid clusters. The blow-up shown in ( G ) is to better visualize PDGFA- and PDGFB-expressing cells. ( E ) Left panel: Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), SPP1 (red), CD271 (pink), and NCAM1 (cyan) and scanned with an OlympusVS120 slide scanner. Left upper image: all markers are shown; left lower image: just NCAM1 channel is shown. Middle panel: confocal laser scanning analysis of BM biopsies co-stained with CD271 (red), NCAM1 (green), SPP1 (white), and DAPI (blue) presented as 3D orthographic cross-section view. Right panel: Single staining channel data for CD271 (red), NCAM1 (green), SPP1 (white), and corresponding blow-ups for indicated areas. NCAM1, SPP1 and CD271(NGFR). White scale bars represent 50 μm and yellow scale bars represent 25 μm. White dashed lines indicate the trabecular bone surface lining regions.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Staining

Journal: eLife

Article Title: Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing

doi: 10.7554/eLife.81656

Figure Lengend Snippet: ( A–E, G–H, J ) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. ( F ) Flow cytometric analysis of SPP1 expression of primary CD45 low/- CD235a - CD71 - CD271 + CD56 + cells (red histogram), CD45 low/- CD235a - CD71 - CD271 + CD56 - CD81 ++ cells (blue histogram) and corresponding isotype control (orange histogram). ( I ) CFU-F assay of sorted CD45 low/- CD235a - CD71 - CD271 + cells (100 cells/well) in the presence (upper panel) or absence (lower panel) of sorted bone marrow CD45 + cells (3x10 5 cells/well), respectively.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56 (NCAM1)-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Single-cell RNA sequencing identifies phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow

doi: 10.1101/2022.01.26.477664

Figure Lengend Snippet: (A) Dot plot of surface marker gene expression in different stromal cell clusters. Cluster numbers and corresponding stromal cell groups are indicated on the y-axis legend. Dot sizes represent the percentage of cells expressing a certain gene in each cluster and dot colors represent the scaled average expression of that gene. (B) FACS plots illustrating the gating strategy for the isolation of different stromal subsets. The displayed cell populations are indicated on top of the plot. Following exclusion of doublets, dead cells, CD45- and CD235a-expressing cells, CD45 low/- CD235a - CD71 - CD271 + cells (left panel) were gated based on CD52 and NCAM1 expression (middle panel). The resulting three populations were labelled as A-C (middle panel), corresponding to the stromal cell groups in (A) (A, CD52 - NCAM1 - ; B, CD52 - NCAM1 + ; C, CD52 + NCAM1 - ). CD45 - CD235a - CD71 - CD271 + CD52 - NCAM1 - (group A) cells were further divided based on CD81 expression and four populations were identified (A1-A4) (right panel). A1, CD81 ++ ; A2, CD81 + ; A3; CD81 +/- ; A4, CD81 - . (C) CFU-F frequencies of sorted stromal cell populations as shown in (B). Data are presented as individual data (dots) and median (horizontal lines) from independent experiments (n=3-6). Symbol colors and x-axis labels correspond to the cell population colors in (B). *: p<0.05; ****: p <0.0001 (Kruskal-Wallis test). (D) In vitro differentiation capacity of sorted stromal cell populations (as indicated in B) towards the adipogenic, osteoblastic, and chondrogenic lineage. Non-induction controls are shown in the left panel. Scale bars represent 200 µm. A representative set of pictures from a total of three independent experiments is shown. (F) Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), CD81 (green), CD271 (pink), and NCAM1 (cyan) and scanned with the OlympusVS120 slide scanner. Different staining combinations are shown as indicated under each picture to provide better visualization of individual staining obtained from the same FFPE slide. Scale bars represent 50 µm. Red arrows: CD271 + CD81 ++ cells; white arrows: arteriolar walls; white lines: bone lining regions. Bone (b), adipocytes (a), and capillaries (*) are indicated.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Marker, Gene Expression, Expressing, Isolation, In Vitro, Formalin-fixed Paraffin-Embedded, Staining

Journal: bioRxiv

Article Title: Single-cell RNA sequencing identifies phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow

doi: 10.1101/2022.01.26.477664

Figure Lengend Snippet: (A-D, F-G) UMAP (as in ) illustration of the normalized expression of selected ligand and receptor gene pairs. Red dashed lines in figures A, B and D mark the erythroid clusters. The blow-up shown in (G) is to better visualize PDGFA- and PDGFB-expressing cells. (E) Formalin-fixed, paraffin-embedded (FFPE) human BM slides were sequentially stained for DAPI (blue), CD45 (yellow), SPP1 (red), CD271 (pink), and NCAM1 (cyan) and scanned with an OlympusVS120 slide scanner. Lower panel: Single staining for each marker is shown as indicated under each image. Scale bars represent 50 µm. White lines indicate bone lining regions. b, Bone.

Article Snippet: Antibodies used were CD45-AlexaFlour 647 (Bio-Rad, MCA87A647T), CD56-AF647 (Biolegend, 318313), mouse anti-human CD271 (R&D Systems, MAB367), and rabbit anti-human CD81 (Novus Biologicals, NBP1-77039) with secondary goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-166) and goat anti-rabbit AF488 (Jackson ImmunoResearch, 111-545-003) with DAPI (Sigma, D956410MG) as a nuclear counterstain, respectively.

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Staining, Marker