Journal: bioRxiv

Article Title: Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

doi: 10.1101/2024.03.14.585083

Figure Lengend Snippet: A ) Schematic describing the sample collection and cell isolation strategy. Magnetic-activated cell sorting (MACS) separation of hematopoietic, stem/progenitor, and mesenchymal fractions were performed and then pooled into one scRNA-Seq reaction per patient. B ) UMAP representation of 82,742 single-cell transcriptomes from bone marrow of 12 individuals. AEC, arterial endothelial cell. SEC, sinusoidal endothelial cell. VSMC, vascular smooth muscle cell. Ba, Basophil. Eo, Eosinophil. Ma, Mast Cell. RBC, red blood cell. pDC, plasmacytoid dendritic cell. CLP, common lymphoid progenitor. MEP, megakaryocyte erythroid progenitor. GMP, granulocyte monocyte progenitor. MPP, multipotent progenitor. HSPC, hematopoietic stem and progenitor cell. HSC, hematopoietic stem cell. Meg/E, megakaryocyte/erythroid. MSC, mesenchymal stromal cell. C ) Bar plots showing the cell counts for each lineage captured (left) and the per sample cell lineage frequencies normalized by the total cells in each sample (right). D ) Heatmap with normalized gene expression scaled by column (cell type) showing the gene expression of key cell type marker genes. Rows were clustered such that genes expressed in similar cell types cluster together. The top two most significant genes by adjusted p-value comparing each cell type to all other cell types were plotted. Selected marker genes for each lineage were labeled. EC, endothelial cell. M, vascular smooth muscle.

Article Snippet: 20uL of RBC depleted cells were put on ice, while 67% of the remaining cells were used for immunomagnetic CD45 depletion using EasySep Human CD45 Depletion Kit II (STEMCELL # 17898) to isolate non-hematopoietic cells in an unbiased fashion, while 33% of cells were subjected to immunomagnetic CD34 enrichment using EasySep Human CD34 Positive Selection Kit II (STEMCELL #17856).

Techniques: Cell Isolation, FACS, Gene Expression, Marker, Labeling

Journal: bioRxiv

Article Title: Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

doi: 10.1101/2024.03.14.585083

Figure Lengend Snippet: A ) UMAP computed from 19,257 mesenchymal cells from 12 individuals showing different mesenchymal subsets, with RNAlo MSCs being excluded. B ) Dot plot showing normalized expression of key mesenchymal marker genes in MSC subsets. Rows and columns were manually ordered. C ) Dot plot showing the normalized expression of literature-derived marker genes for human MSCs including NT5E (CD73), THY1 (CD90), and ENG (CD105). NGFR (CD271) and MCAM (CD146) have also been described as canonical MSC markers. D ) CytoTRACE analysis projected onto the MSC UMAP showing the predicted differentiation score, where higher values imply the cell is more primitive. E) Boxplots showing the relative colony forming potential (number of colonies produced by each cell type divided by total colonies from each sample, each data point is one sample) of sorted Fibro-MSC (CD45-CD38-CD235ab-VECAD-PDPN+), THY1+ MSC (CD45-CD38-CD235ab-VECAD-PDPN-LEPR+ CD90+), Adipo-MSC (CD45-CD38-CD235ab-VECAD-PDPN-LEPR+ CD90-), and osteolineage cells (CD45-CD38-CD235ab-VECAD-PDPN-CD56+). P-values were computed as Fibro-MSC vs. all by Dunnett’s Multiple Comparisons test. F) Line plot showing the population doublings over the course of cell culturing for eight passages, where all cells were passaged every 7 days. P-values are computed as Fibro-MSC vs other MSCs by two-way ANOVA. G ) UMAP showing 3,874 endothelial cells from the 12 individuals. SEC – Sinusoidal Endothelial Cells, AEC – Arterial Endothelial Cells. H ) Dot plot showing normalized expression of top differentially expressed genes by adjusted p-value between endothelial subsets, as well as CDH5 , CD34 , and KDR which were manually selected as pan-endothelial markers. I ) MSCs from human bone marrow aspirate reported from De Jong et al., Nature Immunology , 2021 were reference mapped to our scRNA-Seq atlas and the cell type labels were predicted. J ) Human fetal bone marrow mesenchymal cells from Jardine et al., Nature , 2021 were reference mapped to our scRNA-Seq atlas and the cell labels were predicted.

Article Snippet: 20uL of RBC depleted cells were put on ice, while 67% of the remaining cells were used for immunomagnetic CD45 depletion using EasySep Human CD45 Depletion Kit II (STEMCELL # 17898) to isolate non-hematopoietic cells in an unbiased fashion, while 33% of cells were subjected to immunomagnetic CD34 enrichment using EasySep Human CD34 Positive Selection Kit II (STEMCELL #17856).

Techniques: Expressing, Marker, Derivative Assay, Produced, Cell Culture

Journal: bioRxiv

Article Title: Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

doi: 10.1101/2024.03.14.585083

Figure Lengend Snippet: A) Schematic depicting the CODEX experimental and computational workflow leading to cell type identification. B) Diagram showing the 54-plex CODEX panel (53 antibodies + DAPI) split by target cell population. C) Heatmap showing average centered-log-odds ratio normalized expression per cell type scaled and clustered by protein showing the marker protein expression is consistent with literature knowledge (Left). Cell types were annotated by unsupervised clustering and manual gating as described in Materials and Methods. Boxes were manually drawn to highlight coordinate marker expression. UMAP showing the 803,131 single cells in the CODEX atlas from 12 individuals colored by cell type (Right). Adipocytes were labeled with /Artifact because this population contained a mixture of true adipocytes and artifactual staining. HSPC – Hematopoietic Stem and Progenitor Cell. Meg/E-Megakaryocyte/Erythroid, Endo-Endothelial, M-Vascular Smooth Muscle, Sh-Schwann Cells. D) Stacked bar plot showing cell type frequencies normalized by total cells per sample. E) CODEX image (left) is paired with the cell phenotype map (CPM,right). An arteriolar structure and hematopoietic cells are shown using selected relevant fluorescent markers, which is juxtaposed with the same image with the cellular segmentation masks colored by cell type showing how CODEX allows single-cell mapping of the bone marrow microenvironment.

Article Snippet: 20uL of RBC depleted cells were put on ice, while 67% of the remaining cells were used for immunomagnetic CD45 depletion using EasySep Human CD45 Depletion Kit II (STEMCELL # 17898) to isolate non-hematopoietic cells in an unbiased fashion, while 33% of cells were subjected to immunomagnetic CD34 enrichment using EasySep Human CD34 Positive Selection Kit II (STEMCELL #17856).

Techniques: Expressing, Marker, Labeling, Staining