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. Author manuscript; available in PMC: 2022 May 4.
Published in final edited form as: Nature. 2021 Sep 29;598(7880):327–331. doi: 10.1038/s41586-021-03929-x

Extended Data Figure 9. Stromal cell heterogeneity in FBM.

Extended Data Figure 9

9a) Expression of IF microscopy markers in the FBM (total) CITE-seq dataset; genes (left) and their protein equivalent (right). Methods/interpretation as in Extended Data Fig. 1d (upper limits of 3/15 applied for gene/protein expression).

9b) Left: Scatterplot of CD34 and CD117 co-expression in FBM leukocytes by flow cytometry and expression of CLEC9A in gated fractions (representative from n=2). Right: Scatterplots of CD34, CD38 and CD117 protein expression in the FBM (total) CITE-seq dataset (Supplementary Table 48). Values are ln(DSB-normalised) and scaled to a lower limit of 0. Quadrants denote manual ‘gating’ thresholds (see Methods). Kernel density gradient is overlaid.

9c) Longitudinal section of fetal bone stained with hematoxylin and eosin. Left: location of haematopoietic tissue (10x magnification), scale bar; 1mm. Right top: haematopoietic architecture (20x magnification), scale bar; 200µm. Right bottom: identification of morphologically distinct cell types (50x magnification), scale bar; 20µm. Abbreviations: Eryth. = erythroid Neut. = neutrophil lineage; Eo. = eosinophil. Representative from n=4 samples (14-15 PCW).

9d) Longitudinal section of fetal femur with multiplex IF staining. Yellow boxes = regions of interest. Representative image from n=4 samples (14-15 PCW) at 4x magnification. Scale bar; 3mm.

9e) Sections of fetal femur, each stained with a single primary antibody from the multiplex and co-stained with DAPI. Representative fields of view from n=2 samples (14-15 PCW) at 20x magnification. Scale bars; 100µm.

9f) Identification of HSC/MPP and progenitors as cells co-expressing CD34 and CD117 (white arrow): left = all multiplex channels, middle = CD117 only, right = CD34 only. Representative image at 100x magnification. Replicates as per panel g. Scale bars; 50µm.

9g) Scatterplot showing proportions of CD34+ CD117+ HSC/MPP and progenitors per nucleated cells in metaphyseal (M) versus diaphyseal (D) regions of interest. Bars display mean and error bars SD of 522 HSC/MPP locations across 127 regions of interest in n=4 biologically independent FBM samples (14-15 PCW). Difference in frequency of CD34+CD117+ HSC/MPP and progenitors relative to cellular density was assessed by Wald test (p= 0.431) (Supplementary Table 28).