Hematopoietic progenitor cells are the progeny of hematopoietic stem cells (HSC) that coordinate the production of precise number of mature blood cells of diverse functional lineages. Megakaryocytes (Meg) are mapped at the downstream of bilineage progenitors for erythroid and megakaryocyte (MEP) in the most widely accepted scenarios, although different notions have also been suggested. Thrombopoietin (TPO) is thought to be the master cytokine for megakaryopoiesis. In mice lacking cMpl, the receptor for TPO, production of platelets and Meg is severely impaired. However, Meg are known to be still present in the bone marrow of these mice. These findings suggested that TPO independent signaling for Meg differentiation would exist.
To clarify the differentiation pathway of the Meg lineage, we focused on GPIb (CD42)-V-IX complex, expression of which has not been characterized in any progenitor cells whereas it is well known to be expressed on mature Meg and platelets. We also investigated how TPO-cMpl signaling would affect at MEP or pure megakaryocyte progenitor (MKP) stage using the cMpl deficient mice.
GPIb alpha (CD42b) was expressed on 3–6 % of a mouse bone marrow population characterized as common myeloid progenitors (CMP), i.e., Lin-c-Kit+Sca1-CD34+CD16/32low cells. The GPIb alpha+ CMP (thereafter designated 34-alpha) population also expresses CD9, SLAM1, and CD41. These 34-alpha cells showed a restricted differentiation capacity to the mature Meg in in vitro culture. By intravenously infusing 34-alpha cells derived from CAG promoter-driven GFP-expressing mice into sublethally irradiated syngenic mice, GFP-expressing platelets were generated in vivo. Thus, we designate the 34-alpha cells as 34-alpha MKP. Gene expression analysis also supported that 34-alpha MKP has a restricted capacity of megakaryopoiesis.
In vitro colony-forming assay and short-term liquid culture assay suggested that they are not derived from MEP but from the SLAM1+Flt3-c-Kit+Sca1+Lin- population, which highly contain HSC. When experimental thrombocytopenia was induced by injecting 5-fluorouracil into mice, the frequency of 34-alpha MKP was rapidly increased compared to that of MEP. These data imply a distinct pathway of Meg differentiation, which originates at the proximity of HSC.
We next investigated whether generation of 34-alpha MKP and MEP is differently impaired in cMpl-deficient mice. The frequency of MEP was only mildly reduced. In contrast, 34-alpha MKP were much severely reduced. Notably, in vitro Meg differentiation was markedly impaired from both MEP and 34-alpha MKP derived from cMpl-deficient mice. These data suggested that discordance between Meg and platelet production is caused by the different dependence on TPO-cMpl signaling between the pathways generating MEP and 34-alpha MKP from HSC.
We also found that Hes1, a transcription factor that is the best characterized effector functioning downstream of the Notch signaling pathway, is highly expressed in 34-alpha MKP. Conversely, Meg differentiation was abrogated by retroviral transduction of a dominant-negative mutant of Hes1.
Taken together, our data imply the presence of two distinct Meg differentiation pathways from HSC and further suggest that the dependency of TPO-cMpl signaling is different in these pathways and Notch-Hes signaling plays an additional role in them.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.