Fig. 7
Monocle trajectory analysis and sequential cultures identify a novel megakaryocyte-committed progenitor population. a Pseudo-temporal ordering of cells using Monocle [37]. Trajectories are shown for Population 1 to 2 (Pre-MEP to E-MEP; left plot) and Population 1 to 3 (Pre-MEP to MK-MEP; right plot). b CD71 + 41- 42- (Population A), CD71 + 41 + 42- (Population B), and CD71midCD41hiCD42+ (Population C) were FACS-isolated from day 4 in vitro megakaryocyte cultures for secondary culture and re-plated in either TPO-based (no EPO, left) or EPO-based (no TPO, right) cultures. c Left: 3 and 7 days after re-plating in TPO medium, Populations A, B, and C demonstrated progressive megakaryocytic maturity. Population A gave rise to CD41 + CD42-/+ megakaryocytes; b and c showed progressive CD42 acquisition supporting a unidirectional differentiation hierarchy. Photographs show representative cell cytospins 3 days after secondary culture. Population A shows early megakaryoblasts; Population B shows CD41+ CD42+/- megakaryocytes with single/bi-lobulated nuclei; Population C shows mature, proplatelet-forming megakaryocytes, multilobulated nuclei. Right: In parallel, Populations A, B, and C were re-plated into EPO-based secondary cultures (without TPO) and methylcellulose to test erythropoietic potential. A and B gave rise to CD71 + GlyA+ progeny with typical erythroblast morphology and BFU-Es; C were unable to differentiate into erythroid cells and were immunophenotypically/morphologically identifiable as CD41 + 42+ polyploid megakaryocytes (with abnormal nuclear lobe separation). n = 4. d Expression of selected genes in the MK-MEP subpopulation stratified according to CD42 cell surface expression. CD71 + 41 + CD42+ cells showed significantly lower expression of erythroid genes (e.g. ANK1, CD71, MYB), genes associated with more primitive HSC/progenitors (e.g. CD34, CD44) and higher expression of megakaryocyte genes (e.g. VWF, CD61, CLU, NF1B) than CD71 + 41 + CD42- cells. e A revised model of the megakaryocyte-erythroid differentiation hierarchy showing replacement of classically defined MEP with three novel subpopulations (yellow box). Arrows represent weighted differentiation capability; dashed arrows represent the potential for alternate-lineage differentiation. CLP, common lymphoid progenitors; CMP, common myeloid progenitors; E, erythroid; GMP, granulocyte/macrophage progenitors; HSC, hematopoietic stem cells; LMPP, lymphoid-primed multipotent progenitors; MEP, megakaryocyte-erythroid progenitors; MK, megakaryocyte; MPP, multipotent progenitors