Complete Spatial Mapping of Steady-State Erythropoiesis

Lack of markers to image the different progenitors has limited analyses of interactions between HSPC and their offspring. To overcome this we analyzed the expression of 250+ cell surface molecules for which antibodies are commonly available in all hematopoietic progenitors. We found 76 differentially expressed markers in at least one HSPC showing medium to bright fluorescence suggesting that many commonly used cell surface markers can be used to prospectively image HSPC in the bone marrow. We focused in erythropoiesis as it has not been possible to image step-wise red blood cell maturation in vivo. We found that all erythroid progenitors can be defined as Ly6C-CD27-ESAM-CD117+ cells and then Pre-MegE (earliest erythroid progenitor Cell Stem Cell. 2007 1(4):428-42) are CD150+CD71-. These give rise to CD71+CD150+ Pre-CFU-E that differentiate into CD71+CD150- CFU-E that then generate early erythroblasts. All BM BFU-E activity was restricted to Pre-MegE and CFU-E (70 and 30% of all BFU-E) whereas all CFU-E colonies were spread between Pre-MegE (44%), pre-CFU-E (10%) and CFU-E (46%). We also confirmed previously published data showing that CD71 and Ter119 can be used to image step-wise terminal erythropoiesis; CD71+Ter119dim early erythroblasts, CD71+Ter119bright late erythroblasts, CD71dimTer119bright reticulocytes and CD71-Ter119bright erythrocytes. Importantly, all populations were detected at identical frequencies using FACS or confocal imaging indicating that our imaging strategy detects all erythroid cells in the BM (Pre-CFU-E: 0.022 vs 0.027 %; CFUE: 0.32 vs 0.30%; Early-Ery: 0.62 vs 0.66%; Late-Ery: 32.05 vs 32.12%; Reticulocyte: 5.98 vs. 3.36%; Erythrocytes: 12.49 vs. 13.47%). We mapped the 3D location of every erythroid lineage cell in the murine sternal BM and interrogate the spatial relationships between the different maturation steps and with candidate niches. We compared the interactions found in vivo with those found in random simulations. Specifically, we used CD45 and Ter119 to obtain the spatial coordinates of every hematopoietic cell in a mouse sternum. Then we randomly placed each type of erythroid lineage cell at identical frequencies as those found in vivo to generate random simulation. We found that Pre-MegE and Pre-CFU-E are closer to each other than predicted from random (average Pre-CFU-E to Pre-MegE distance= 92.3 µm vs. 156.7 µm random, p=0.028) but never adjacent indicating that Pre-CFU-E migrate away from Pre-MegE (0% of Pre-CFU-E adjacent to PreMegE). We also found that CFU-E were not adjacent to pre CFU-E, instead 7-8 CFU-E align to form “CFU-E strings” along the central BM (74% of CFU-E found in strings vs. 17.5% in random p In summary we have found 76 differentially expressed markers that can be combined to detect most HSPC; validated a 5-color stain to image all steps of red blood cell maturation; demonstrated that erythropoiesis takes place in highly organized 4-cluster structures emerging from strings of sinusoidal CFU-E, and demonstrated that sinusoids are the exclusive site of erythropoiesis. Disclosures No relevant conflicts of interest to declare.