Characterization of mouse clonogenic megakaryocyte progenitors

Abstract

Although it has been shown that unfractionated bone marrow, hematopoietic stem cells, common myeloid progenitors, and bipotent megakaryocyte͞erythrocyte progenitors can give rise to megakaryocyte colonies in culture, monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here, we use a monoclonal antibody to the megakaryocyte-associated surface protein, CD9, to purify MKPs from the c-kit ؉ Sca-1 ؊ IL7R␣ ؊ Thy1.1 ؊ Lin ؊ fraction of adult C57BL͞Ka-Thy1.1 bone marrow. The CD9 ؉ fraction contained a subset of CD41 ؉ Fc␥R lo CD34 ؉ CD38 ؉ cells that represent Ϸ0.01% of the total nucleated bone marrow cells. They give rise mainly to colonyforming unit-megakaryocytes and occasionally burst-forming unitmegakaryocytes, with a plating efficiency >60% at the single-cell level. In vivo, MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for Ϸ3 weeks. Common myeloid progenitors and megakaryocyte͞erythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures, indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns.

Loss-of-function Additional sex combs like 1 mutations disrupt hematopoiesis but do not cause severe myelodysplasia or leukemia

“…21 In 2 independent experiments, we found no differences in CFU-S 12 colony numbers between Asxl1 mutant and control samples within either experiment (data not shown).…”

Hematopoietic stem/progenitor cell commitment to the megakaryocyte lineage

“…In the myeloid branch, CMPs gave rise to 2 more restricted progenitors, megakaryocyte-erythrocyte progenitors (MEPs) and granulocyte-macrophage progenitors (GMPs), 5 with MEPs giving rise to erythroid progenitors and unipotent megakaryocytic precursors ( Figure 1A). 8,9,28 Although the classical model of hematopoiesis is simple in its binary conception of lineage potential, this model of hematopoiesis has been challenged by the identification of new progenitor populations. On the basis of their prior observation that loss of long-term HSC (LT-HSC) self-renewal coincided with the upregulation of FMS-like tyrosine kinase 3 (Flt3) 29 32 Because megakaryocytes and erythrocytes were thought to share a common progenitor, 5 the authors investigated the erythroid potential of LSKFlt3 1 cells and showed that these cells also exhibited limited ability to form erythroid progeny at a clonal level.…”

“…[5][6][7] Moreover, unipotent megakaryocytic progenitor cells were placed downstream of bipotent MegE progenitors, suggesting that all megakaryocytes arise from committed precursors that are formed after requisite intermediate states. 8,9 Although the hierarchical model has been very useful for understanding hematopoiesis, it has become increasingly clear that this model is inadequate for capturing all the complexities of early commitment steps in hematopoiesis and especially in megakaryopoiesis. With advances in the ability to prospectively separate HSCs and committed progenitors and the development of functional and molecular assays to assess the development potential of single cells in vitro and in vivo, a more complex picture of HSC commitment to the megakaryocytic lineage has emerged in which megakaryocytes may arise directly from HSCs as well as from multi-, bi-, and unipotent progenitors.…”

Extrinsic and intrinsic control by EKLF (KLF1) within a specialized erythroid niche

“…To address whether we could also observe the same results from a definitive erythroid source, we developed a protocol to prospectively sort dispersed cells from differentiating EBs at day 6 (and thus enriched for definitive erythroid cell production) into lin-Kit+sca1- Akashi et al, 2000;Nakorn et al, 2003;Terszowski et al, 2005), and plated these individually sorted cells at low density onto methylcellulose under conditions optimal for definitive colony analysis. It has been difficult to obtain colonies from such prospectively sorted populations when derived from differentiating EBs (Drissen et al, 2010); indeed, we found that use of an ESC-based medium rather than the standard media conditions was absolutely crucial for success (see Materials and Methods).…”