3043 – A PIPELINE TO IMAGE HEMATOPOIETIC DIFFERENTIATION REVEALS THE ARCHITECTURE OF ERYTHROPOIESIS IN THE BONE MARROW

The anatomy of differentiation in the BM is poorly understood due to a lack of markers to image stepwise HSPC differentiation. We immunophenotyped all types of multipotent and lineage committed HSPC and identified 56 cell surface markers that can be “mixed and matched” to prospectively image HSPC. We used this data to develop a pipeline to map erythropoiesis in whole-mounted sternum. Pre-Meg-E are ESAM-Ly6C-CD117+CD150+CD71-, Pre-CFU-E are Ly6C-CD117+CD150+CD71+ and CFU-E are Ly6C-CD117+CD150-CD71+ cells. We found that all three types of erythroid progenitors show no specific association with LT-HSC/MPP2/MkP, indicating that Pre-Meg-E or more primitive progenitors leave the HSC niche after differentiation. Both Pre-Meg-E and Pre-CFU-E are found as single cells through the central BM space and do not specifically associate with other progenitors, or components of the microenvironment. In contrast almost all CFU-E locate to strings (28 strings per sternum) containing 6±0.28 CD117brightCD71dim CFU-E that are selectively recruited to sinusoids (mean CFU-E to sinusoid distance=2.2µm). As soon as CFU-E detach from sinusoids they show sharp reductions in CD117 and upregulation of CD71 giving rise to a cluster of early erythroblasts that buds from the vessel. These progressively upregulate Ter119 to generate large clusters of late erythroblasts that in turn differentiate into clusters of reticulocytes and erythrocytes. To understand how the architecture of erythropoiesis changes in response to stress we performed phlebotomy. Two days later we found Pre-Meg-E and Pre-CFU-E numbers and locations was unaltered. The number of CFU-E strings remained constant (30 CFUE strings/sternum) but all strings contained more CFU-E (2-fold) that then differentiated as above. Our imaging pipeline can be quickly adapted to image all other HSPC during normal, stress, and diseased hematopoiesis.