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Erythropoiesis at a clonal level
Discussion
The cellular differentiation pathways supporting ongo- ing adult erythropoiesis from primitive marrow HSPC are still not completely clear, with some contradictory find- ings in murine and human studies, particularly regarding whether erythroid production is supported by multipo- tent then bipotent intermediate steps downstream from the most primitive HSC, as in the classical model.1-3 Recent findings in murine model human cells studied in robust single cell in vitro assays, and pseudotemporal ordering of single cell RNA-Seq gene expression patterns have raised the possibility that the erythroid and megakaryocytic lin- eages, along with eosinophils and basophils in some stud- ies, may represent the earliest branch point during hematopoiesis from self-renewing LT-HSC.5,8-10,12,13,17,38,39 The RNA-Seq study most relevant to erythropoiesis came from Tusi and co-workers, enriching for cell populations previously linked to erythropoiesis to study differentia- tion trajectories, reporting that HSC/MPP first bifurcate towards erythroid-basophil-megakaryocytes versus myeloid-lymphoid pathways before constricting to ery- throid or towards myeloid and lymphoid fates. Murine lineage tracing studies using platelet lineage-specific pro- moters have uncovered evidence for long-lasting and self- renewing megakaryocyte-restricted HSC, but to date, ery- throid-restricted engrafting HSPC have not been uncov-
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ered, despite the suggestion, based on gene expression studies, that they may exist.15-17,40 These observations led us to ask whether we could detect long-lived erythroid- restricted or highly erythroid-biased HSPC in our RM bar- coded clonal tracking model.
Techniques allowing long-term tracking of hematopoiesis in vivo at a single cell level via fate map- ping or clonal tags are powerful tools to provide answers to these questions and have been utilized primarily in murine models over the past decade. Yamamoto et al.15 used single cell murine HSPC transplants along with lin- eage-specific markers and demonstrated lineage-restrict- ed engrafting progenitors producing platelets, platelets and red cells, or all myeloid/erythroid lineages, and sin- gle cells able to produce both megakaryocyte and multi- potent engrafting daughter cells, but no other uni-lineage daughter HSC. Using a non-transplant “naïve hematopoiesis” murine transposon-tagging model, Rodriguez et al. came to similar conclusions regarding LT- HSPC heterogeneity, showing megakaryocytic-restricted long-term contributing HSPC clones, but did not exam- ine erythroid output at a clonal level.17 The Jacobsen lab- oratory also reported lineage-restricted long-term self- renewing potential initially only for the platelet lineage in mice,40 more recently adding an erythroid-specific tracer that did not reveal long-term erythroid-restricted HSPC, but did uncover very rare platelet-erythroid
Figure 6. Impact of erythropoietin (EPO) administration on erythroid clonal patterns. (A) Timeline of EPO stimulation and sampling in ZL40, EPO was administrated at 11.5m post transplantation. (B) The reticulocyte (RETIC) percentage during EPO stimulation. (C) Heatmap of the top ten contributing clones from bone marrow (BM) monocytes (Mono), granulocytes (Gr) and nucleated red blood cell (NRBC) DNA barcodes and peripheral blood (PB ) red blood cell (RBC) RNA barcodes samples before and post EPO stimulation. Heatmap was constructed as described in Figure 2B. day -35: baseline, day 6: reticulocyte peak, and day 87: recovery after EPO stimulation are shown.
haematologica | 2020; 105(7)
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