Intravital microscopy of highly pure haematopoietic stem cells using a novel transgenic reporter strategy uncovers unexpected interaction dynamics within the stem cell niche

Clonal assays have recently highlighted a high degree of functional heterogeneity within the phenotypically defined HSC compartment. Platelet-biased HSCs express the megakaryocytic lineage gene Vwf and exhibit a platelet lineage biased reconstitution pattern (SanJuan-Pla et al., 2013). However, how the niche supports HSC function is still largely unknown. The Hoxb5 reporter identifies a population highly enriched for functional HSCs and multipotent progenitors (Chen et al., 2016). Here, we combined a Hoxb5-KuO reporter with the Vwf-GFP reporter (SanJuan-Pla et al., 2013) to identify lineage-biased HSCs in situ, characterize their native niches and investigate their kinetics in homeostasis and stress haematopoiesis. Within the LSK-SLAM compartment, expression of Hoxb5 and Vwf defined 3 populations. Importantly, gating on the 2 reporters alone provides an HSC purity of 88% and 40% within the Hoxb5+Vwf+ and the Hoxb5+Vwf- subsets, respectively. In vivo limiting dilution assays support this high HSC enrichment of the Hoxb5+Vwf+ and the Hoxb5+Vwf- populations and confirmed the platelet-biased pattern of reconstitution of the former. Intravital microscopy of the calvarium of this dual-reporter, followed by CellProfiler analysis to scale up the tracking of these rare cells, revealed very little motility of Hoxb5+Vwf+ cells while some Hoxb5+Vwf- cells were more motile. Remarkably, platelet depletion, which was previously shown to preferentially activate Vwf+ HSCs in a niche dependent manner (Luis et al., unpublished), did not increase the motility of either HSC subset. By using a new combination of transgenic reporters, the specific platelet depletion stress model and intravital microscopy, we shed a new light on the dynamics of HSC subsets in their native niche, in homeostasis and in response to specific demands in blood cell production. Clonal assays have recently highlighted a high degree of functional heterogeneity within the phenotypically defined HSC compartment. Platelet-biased HSCs express the megakaryocytic lineage gene Vwf and exhibit a platelet lineage biased reconstitution pattern (SanJuan-Pla et al., 2013). However, how the niche supports HSC function is still largely unknown. The Hoxb5 reporter identifies a population highly enriched for functional HSCs and multipotent progenitors (Chen et al., 2016). Here, we combined a Hoxb5-KuO reporter with the Vwf-GFP reporter (SanJuan-Pla et al., 2013) to identify lineage-biased HSCs in situ, characterize their native niches and investigate their kinetics in homeostasis and stress haematopoiesis. Within the LSK-SLAM compartment, expression of Hoxb5 and Vwf defined 3 populations. Importantly, gating on the 2 reporters alone provides an HSC purity of 88% and 40% within the Hoxb5+Vwf+ and the Hoxb5+Vwf- subsets, respectively. In vivo limiting dilution assays support this high HSC enrichment of the Hoxb5+Vwf+ and the Hoxb5+Vwf- populations and confirmed the platelet-biased pattern of reconstitution of the former. Intravital microscopy of the calvarium of this dual-reporter, followed by CellProfiler analysis to scale up the tracking of these rare cells, revealed very little motility of Hoxb5+Vwf+ cells while some Hoxb5+Vwf- cells were more motile. Remarkably, platelet depletion, which was previously shown to preferentially activate Vwf+ HSCs in a niche dependent manner (Luis et al., unpublished), did not increase the motility of either HSC subset. By using a new combination of transgenic reporters, the specific platelet depletion stress model and intravital microscopy, we shed a new light on the dynamics of HSC subsets in their native niche, in homeostasis and in response to specific demands in blood cell production.