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270 Part IV: Molecular and Cellular Hematology Chapter 18: Hematopoietic Stem Cells, Progenitors, and Cytokines 271

only CFU-M−, CFU-G−, and CFU-GM–derived colonies in response to the erythroid lineage begin to express CD41 and the transferrin
to any of the growth factors, alone or in combination, and thus represent receptor (CD71), and as they mature lose CD41 expression but express
granulocyte/macrophage lineage-restricted progenitors (GMP). Finally, the thrombospondin receptor (CD36), glycophorin, and, ultimately,
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IL-7Rα−/Lin−/c-Kit+/Sca-1−/CD34−/FcRγ cells form only BFU-E−, globin. These and other cell-surface markers provide experimental
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CFU-MK−, and mixed megakaryocyte-erythroid colonies, leading to hematologists several strategies to purify committed MK 39,329 and ery-
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their designation as MEP. To demonstrate their capacity to differentiate throid progenitors. Another useful method to identify megakary-
in vivo, limiting numbers of each cell population were transplanted into oblasts is histochemical staining for von Willebrand factor, and in
congenic mice; in such studies, cell fate outcomes correspond strictly rodents, acetylcholinesterase. 331
with those of the in vitro colony assays. For example, 6 days after injec- The transcription factors expressed by erythroid and MK pro-
tion of 5000 CMPs, both donor-derived Gr-1+/Mac-1+ myelomono- genitors that allow for their commitment to the lineage are becoming
cytic cells and TER119+ erythroid cells were detectable in recipients. increasingly well understood. GATA1 is an X-linked gene encoding a
In contrast, when 5000 GMPs were transplanted, only Gr-1+/Mac-1+ 50-kDa polypeptide that contains two zinc fingers required for DNA
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cells were recovered, and only for a transient period of time. Likewise, binding. Genetic elimination of the transcription factor established
MEPs reconstituted only TER119+ cells in similar experiments, and the the critical role of this transcription factor in hematopoiesis; GATA1
genetically marked progeny from each of these progenitor populations −/− mice are embryonic lethal as a consequence of failure of ery-
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disappear within 4 weeks of transplantation, indicating their limited thropoiesis, and MK-specific elimination of GATA1 leads to severe
self-renewal capacity. thrombocytopenia as a consequence of dysmegakaryopoiesis. GATA1
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Erythroid/Megakaryopoietic, Erythroid, and Megakaryopoi- acts in concert with another protein that affects transcription without
etic Progenitors Culture conditions necessary for in vitro erythropoie- binding to DNA, friend of GATA (FOG). The importance of this
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sis have been known for nearly 35 years, 324,325 with colony morphologies interaction to megakaryopoiesis is clear: several different mutations
ranging from small compact clusters of 20 to 50 erythrocytes develop- of the site on GATA1 responsible for FOG binding lead to congenital
ing with 2 to 5 days in murine and human marrow plasma clot cultures thrombocytopenia. 335
(CFU-E), to large highly complex colonies containing up to thousands The Ets family of transcription factors includes about 30 members
of cells taking from 7 to 14 days to develop in methylcellulose or agar that bind to a purine box sequence, proteins that interact in both positive
(BFU-E). The cytokine requirement for the former is simple—EPO, and antagonistic ways. For example, PU.1, initially termed Spi-1 based
whereas a cytokine that stimulates earlier cells, such as IL-3 or SCF, is on its association with spleen focus-forming virus-induced erythroleu-
required for the latter progenitor cell type. kemias, blocks erythroid differentiation, although it appears important
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Culture conditions that support the proliferation of MK progeni- for MK development. Moreover, the Ets factor Fli-1 is essential for
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tors have been established for both mouse and humans. 29,31 Using either megakaryopoiesis and mutations in the transcription factor are also
methylcellulose, agar, or a plasma clot, two colony morphologies that associated with congenital thrombocytopenia in man. 271
contain exclusively MKs have been described. The CFU-MK is a cell Granulocyte/Monocytic, Granulocyte, and Monocytic
that develops into a simple colony containing from 3 to 50 mature MKs, Progenitors As noted above, GMPs (CFU-GM) are IL-7Rα−/Lin−/c-
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larger, more complex colonies that include satellite collections of MKs Kit+/Sca-1−/CD34+/FcRγ , reflecting their beginning differentiation
and contain up to several hundred cells are derived from the burst- toward phagocytic cells (i.e., FcRγ-positive). In the human, GMP are
forming unit–megakaryocyte (BFU-MK). Because of the difference in CD34+/CD33+/CD13+ markers, which are of clinical significance. For
their proliferative potential and by analogy to erythroid progenitors, example, CD33 is also termed Siglec-2, a member of a family of sialic-
BFU-MK and CFU-MK are thought to represent primitive and mature acid-binding surface membrane proteins of the immunoglobulin super-
progenitors restricted to the MK lineage. And like their erythroid coun- family that are involved in cell–cell interactions and signaling. Although
terparts, the cytokine requirements for CFU-MK are simple: TPO stim- the role of CD33 is not yet known with certainty, it has become a ther-
ulates the growth of 75 percent of all CFU-MK, with IL-3 being required apeutic target because of its high-level expression on the blasts of sev-
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along with TPO for the remainder, whereas IL-3 or SCF is required eral forms of acute myelogenous leukemia ; the use of gemtuzumab
alone with TPO for more complex, larger MK colony formation from ozogamicin, in which a humanized anti-CD33 monoclonal anti-
their more primitive progenitors. body has been fused to N-acetyl-gamma calicheamicin 1,2-dimethyl
Progenitors for erythrocytes and MKs display many common hydrazine dichloride, a potent antitumor antibiotic, has resulted in a
features: they share a number of transcription factors (SCL, GATA1, complete remission rate of 15 to 20 percent as a single agent in patients
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GATA2, NF-E2), cell-surface molecules (TER119), and cytokine recep- with relapsed disease. These initial successes have prompted its testing
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tors (for IL-3, SCF, EPO, and TPO), and most erythroid and MK leu- in earlier stage disease along with other active agents. When marrow
kemia cell lines display, or can be induced to display, features of the grafts are purged of CD33-bearing cells durable engraftment occurs, but
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alternate lineage. Moreover, the cytokines most responsible for devel- is often quite delayed, indicating that CD33 is not present on the HSC,
opment of these two lineages—EPO and TPO—are the two most closely but that the presence of GMPs in a transplantation product is vital for
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related proteins in the hematopoietic cytokine family, and display rapid engraftment.
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synergy in stimulating the growth of progenitors of both lineages. For CD13 is also termed aminopeptidase N, an ectopeptidase present
these and other reasons it has been postulated that erythropoiesis and in many organs other than the marrow, a member of a family of pro-
megakaryopoiesis share a common progenitor cell, a hypothesis now teases that play an important role in cell growth by virtue of their cleav-
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36
established with the identification of IL-7Rα−/Lin−/c-Kit+/Sca-1−/ age of biologically important peptides, in some cases inactivating, and
CD34−/FcRγ cells. in some cases activating them, and by serving a cell adhesive function
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Like other primitive hematopoietic cells, bipotent MEP progen- as well. CD13 is present on early hematopoietic cells, including myeloid
itors resemble small lymphocytes but can be distinguished by a spe- and lymphoid lineage progenitors, but disappears from the latter class
cific pattern of cell-surface protein display. As noted above, MEPs are of cells and its expression rises as monocytes mature. Although it func-
IL-7Rα−/Lin−/c-Kit+/Sca-1−/CD34−/FcRγ . Cells committed to the tions to scavenge peptides in the intestinal brush border and degrade
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MK lineage then begin to express CD41 and CD61 (integrin αIIbβ ), endorphins and enkephalins in the synaptic cleft, its role in hemato-
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CD42 (glycoprotein Ib), and glycoprotein V. Those that are committed poiesis is less clear, although IL-8 is a substrate of its proteolytic activity.

Kaushansky_chapter 18_p0257-0278.indd 271 9/19/15 12:06 AM