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