Fig. 4: Establishment of the stem cell niche in the absence of embryonic myeloid cells.

a Dot plots display non-hematopoietic niche cells in the bone marrow of newborn (2 days) Rank^cre/+;Csf1r^fl/− and control mice. From dapi-negative singlets leukocytes (CD45^-) and red blood cells (Ter119^-) were excluded and endothelial cells (endo) identified by cell surface expression of CD31 and CD144. CD31 negative cells were further subdiveded by CD51 and PDGFRa to identify mesenchymal stromal cells, MSC and osteoprogenitors, OP. b Frequencies of niche cells within CD45- bone marrow cells identified as shown in a. A two-sided unpaired Student’s t-test was used. (n = 23, 5 biological replicates). c Representative photographs of histological sections of newborn (1-day-old) Rank^cre/+;Csf1r^fl/− and control mice stained with antibodies specific for MSCs (CD51, red), endothelial cells (CD31, green), and DNA (DAPI, blue)(left). Quantification of corrected total cell fluorescence (CTCF) of CD51 (right, top) and CD31 (right, bottom) positive cells. Each dot represents the value for one mouse. A Mann–Whitney U test was performed for statistical analysis. (n = 11 [7 wt, 4 mutant], 3 regions of interest were imaged in each bone, 3 independent experiments). d Photographs show CD31-stained cultivated metatarsals from newborn (3-day-old) Rank^cre/+;Csf1r^fl/− or control mice (left, 12 days). Plot shows the ratio vascularized area / bone area (right). A two-sided unpaired Student’s t-test was used. (n = 17, 3 biological replicates). e Photographs show alizarin red stained osteogenic cultures of unfractionated bone marrow cells from newborn (4-day-old) Rank^cre/+;Csf1r^fl/− and control mice (left, 11 days). Graph shows the quantification of osteogenic spots (alizarin red absorbance at 450 nm). A Mann–Whitney U test was performed for statistical analysis. (n = 12, 2 biological replicates). WBM whole bone marrow. f Photographs show Oil red O stained adipogenic cultures of unfractionated bone marrow cells from newborn (3-day-old) Rank^cre/+;Csf1r^fl/− and control mice (left, 7 days). Graph shows the quantification of Oil red O absorbance (510 nm). A Mann–Whitney U test was performed for statistical analysis. (n = 14, 2 biological replicates). g Frequencies of CXCL12 positive niche cells (intracellular staining) in bone marrow of newborn (2–4-day-old) Rank^cre/+;Csf1r^fl/− and controls. A two-sided unpaired Student’s t-test was used. (n = 13, 3 biological replicates).h Plots show numbers of KSL Slam HSCs in the newborn (0–4-day-old) bone marrow of mice of indicated genotypes. A two-sided unpaired Student’s t-test was used. (n = 16, 3 biological replicates for Prx^cre/+;Cxcl12^fl/fl, n = 16, 3 biological replicates for Cdh5^cre/+;Cxcl12^fl/fl, n = 13, 2 biological replicates for Vav^cre/+;Csf1r^fl/−). i Plots show numbers of KSL Slam HSCs in the newborn (0–4-day-old) liver of mice of indicated genotypes. A two-sided unpaired Student’s t-test was used. (n = 16, 3 biological replicates for Prx^cre/+;Cxcl12^fl/fl, n = 16, 3 biological replicates for Cdh5^cre/+;Cxcl12^fl/fl, n = 13, 2 biological replicates for Vav^cre/+;Csf1r^fl/−). Each dot represents the value for one mouse in figure parts c–i. Source data are provided as a Source Data file.