Proc Natl Acad Sci U S A 100(1): 205-210

Characterization of mouse clonogenic megakaryocyte progenitors

Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305

^{To whom correspondence should be addressed at: Beckman Center B261, Stanford, CA 94305. E-mail:}ude.drofnats.dnalel@nrokanan.

^{Present address: First Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.}

Contributed by Irving L. Weissman

Accepted 2002 Oct 23.

Abstract

Although it has been shown that unfractionated bone marrow, hematopoietic stem cells, common myeloid progenitors, and bipotent megakaryocyte/erythrocyte 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^IL7Rα^Thy1.1^Lin^{fraction of adult C57BL/Ka-Thy1.1 bone marrow. The CD9}^{fraction contained a subset of CD41}^FcγR^CD34^CD38^{cells that represent ≈0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit–megakaryocytes and occasionally burst-forming unit–megakaryocytes, 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 ≈3 weeks. Common myeloid progenitors and megakaryocyte/erythrocyte 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.

Abstract

Hematopoiesis is a complex developmental process in which a rare population of bone marrow cells called hematopoietic stem cells (HSC) continuously generate all blood cells for the life of an animal. According to the current model of hematopoiesis, HSC give rise to progenies in each lineage through multiple steps of committed progenitors (1, 2). This paradigm is based historically on the results of in vitro clonogenic assays of bone marrow cells in semisolid media (3, 4). Unfractionated mouse bone marrow has been shown to contain multiple types of colony-forming units (CFU) such as (i) multipotent CFU for all myeloid cells, (ii) bipotent CFU for granulocytes and macrophages (CFU-GM), for megakaryocytes and erythrocytes (CFU-MegE), as well as (iii) monopotent CFU for granulocytes (CFU-G), macrophages (CFU-M), erythrocytes (CFU-E), or megakaryocytes (CFU-MK). Accordingly, they are believed to form different compartments of the myeloid progenitors in bone marrow (5–10). By using the same methodology developed for the isolation of HSC (11, 12), we have prospectively identified three different populations of murine myeloid progenitors: common myeloid progenitors (CMP), granulocyte/monocyte progenitors (GMP), and megakaryocyte/erythrocyte progenitors (MEP). CMPs give rise to all myeloid lineages, whereas GMPs and MEPs give rise to cells in the granulocyte/monocyte and megakaryocyte/erythrocyte lineages, respectively. The restricted differentiation capacity and lineage commitment of these cells were clearly demonstrated by both in vitro culture and in vivo transplantation assay (13, 14). Whether monopotent progenitors for each myeloid lineage exist in the same manner is yet to be proven, because they have not been prospectively isolated and tested for the in vitro and in vivo differentiation potentials at the clonal level.

In the megakaryocytic lineage, it is widely believed that CFU-MK are derived from monopotent megakaryocyte-committed progenitors (MKP) in the bone marrow (15–18). However, at the single-cell level, CMPs and MEPs can also give rise to pure megakaryocyte colonies (13). This can be explained by stochastic or deterministic cell fate decisions taken by CMPs or MEPs that could differentiate first into MKPs, which then give rise to CFU-MK. Alternatively, it is also possible that these progenitors may remain in the multipotent or oligopotent stage, but the culture conditions do not allow them to differentiate into cells of the other lineages. Therefore, the presence of CFU-MK in vitro does not necessarily indicate the existence of the MKP population in the bone marrow.

To address this question, we prospectively searched for a population of myeloid progenitors in the bone marrow that can give rise only to megakaryocytes. Because the megakaryocytic and erythroid lineages are closely related and have been shown to share some common precursors, even at the late stages (19), we speculated that, to isolate MKPs successfully, one needs to use cell-surface markers that are differentially expressed in the megakaryocytic lineage but not in the erythroid lineage. CD9 is such a marker and has been shown to be involved in the differentiation of human megakaryocytes (20, 21). In fact, we found CFU-MK activity to be highly enriched in the CD9^CD41^FcγR^c-kit^Sca-1^IL7Rα^Thy1.1^Lin^{fraction of bone marrow. These cells can give rise mainly to megakaryocytes and platelets both}in vitro and in vivo; thus, they represent the population of MKPs in mouse bone marrow.

One hundred cells were sorted directly onto 35-mm dishes containing methylcellulose medium supplemented with SCF, Flt3-ligand, IL-3, IL-11, GM-CSF, Tpo, and Epo. Colonies were counted at day 10. Each experiment was done in triplicate. The data are presented here as the average number of colonies per dish ± SEM. BFU-MK/E, burst-forming unit–megakaryocytes/erythrocytes. BFU-E, burst-forming unit-erythrocytes.

BFU-E, burst-forming unit–erythrocytes.

Acknowledgments

We thank S.-I. Nishikawa for anti-IL-7Rα antibody, Julie Christensen and Amy Wagers for technical advice, Susan Prohaska and Motonari Kondo for critical evaluation of the manuscript, Libuse Jerabek for excellent laboratory management and assistance with animal procedures, Stephanie Smith for antibody preparation, the Stanford Shared FACS facility for flow cytometer maintenance, and Lucino Hidalgo, Diosdado Escoto, and Bert Lavarro for animal care. This research was supported by U.S. Public Health Service Grant CA42551 and the Leukemia Society de Villier's International Achievement Award (to I.L.W.). T.N.N. was supported by the Anandamahidol Foundation under the royal patronage of His Majesty the King of Thailand.

Acknowledgments

Abbreviations

HSC	hematopoietic stem cells
CFU	colony-forming unit
CFU-G	CFU for granulocytes
CFU-M	CFU for macrophages
CFU-GM	CFU for granulocytes and macrophages
CFU-MK	CFU–megakaryocytes
CMP	common myeloid progenitors
GMP	granulocyte/monocyte progenitors
MEP	megakaryocyte/erythrocyte progenitors
MKP	megakaryocyte progenitors
SCF	stem cell factor
Epo	erythropoietin
Tpo	thrombopoietin
GM	granulomonocytic
BFU-MK/E	burst-forming unit–megakaryocytes/erythrocytes

Abbreviations

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