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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4118-4126
By
From Oakland University, Rochester, MI; and the Department of
Pediatrics and the Comprehensive Cancer Center, University of Michigan,
Ann Arbor.
Little is known concerning the interaction of thrombopoietin (TPO)
with other megakaryocyte-active cytokines in directing the early events
of megakaryocyte development. Culture of CD34+ cells in
interleukins (IL) -1, -6, -11, plus stem cell factor (SCF;
S) results in a 10- to 12-fold expansion in total cell
numbers, whereas total CD41+ megakaryocytes are expanded
~120-fold over input levels. Addition of TPO to IL-1, -6, -11, S
generates a biphasic proliferation of CD41+ cells,
accelerates their rate of production, and results in an ex vivo
expansion of more than 200-fold. The addition of Flt-3 ligand (FL)
increases CD41+ cell expansion to ~380-fold over input
levels. In the absence of TPO, ~95% of the expanded cells show the
phenotype of promegakaryoblasts; TPO and/or FL addition
increases CD41 antigen density and ploidy in a subpopulation of
promegakaryoblasts. A moderate (approximately sevenfold) expansion of
megakaryocyte progenitor cells (colony-forming unit-megakaryocyte) occurs in the presence of IL-1, -6, -11, S, and the addition of TPO to this cocktail yields an ~17-fold
expansion. We conclude that early proliferative events in megakaryocyte
development in vitro are regulated by multiple cytokines, and that TPO
markedly affects these early developmental steps. However, by itself,
TPO is neither necessary nor sufficient to generate a full
proliferative/maturational in vitro response within the megakaryocyte
compartment. TPO clearly affects terminal differentiation and the
development of (some) high-ploidy human megakaryocytes. However, its
limited in vitro actions on human cell polyploidization suggest that
additional megakaryocyte-active cytokines or other signals are
essential for the maximal development of human megakaryocytes.
MEGAKARYOCYTE development is a complex
process in which a wide variety of regulatory signals work in cohort to
direct a highly specific response to thrombopoietic
demand.1,2 Like other lineages, megakaryocytes show a
developmental hierarchy of cells ranging from the primitive, actively
proliferating progenitor cells to the postmitotic, polyploid
megakaryocytes undergoing maturational development.1 Within
this lineage, promegakaryoblasts represent a transitional stage between
the proliferating progenitor cells and the mature
megakaryocytes.3,4 Promegakaryoblasts are restricted (or
lacking) in proliferative potential, being the developmental stage at
which megakaryocytes cease to proliferate and begin to acquire an
increased DNA content. As such, they are endomitotic (a mechanism of
acquiring a polyploid DNA content) and contain a lower DNA content than
the earliest morphologically recognizable cell, the
megakaryoblast.5,6
Megakaryocyte development is controlled by growth factors that function
to regulate both the expansion of megakaryocyte numbers (proliferation)
and terminal differentiation (maturation).7-9 Numerous
pleiotropic cytokines are known to influence megakaryocytopoiesis, although their maturational and proliferative functions often overlap.
Both interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating
factor (GM-CSF) are mitogenic cytokines that primarily stimulate the
proliferative phases of megakaryocytopoiesis.10,11 In the
absence of other cytokines, IL-3, a potent megakaryocyte CSF, induces
early stages of megakaryocyte development in vitro. However, the
resulting cells are underdeveloped, and additional differentiation-inducing factors, such as IL-6 or IL-11, are required for full megakaryocyte maturation.12,13 Both of these ILs
primarily affect the later stages of megakaryocytopoiesis, and each
synergizes with IL-3 or GM-CSF to increase the number, size, and DNA
content of developing megakaryocytes.14 Interestingly, IL-l
enhances megakaryocyte proliferation in the presence of IL-3 and
IL-6,14 and also promotes long-term megakaryocytopoiesis,
possibly by driving commitment to this lineage.15 Stem cell
factor (SCF), the ligand for the c-kit proto-oncogene, is a
multipotent hematopoietic cytokine that also stimulates
megakaryocytopoiesis, and enhances IL-3- or GM-CSF-stimulated
progenitor cell (colony-forming unit-megakaryocyte [CFU-Mk] or
burst-forming unit-Mk [BFU-Mk]) proliferation, while having little effect on promoting megakaryocyte maturation (size or DNA
content).16 A similar cytokine, the ligand for the
Flt-3/Flk-2 tyrosine kinase receptor, is involved in
regulating growth of primitive hematopoietic cells.17
Initial reports indicated little or no effect of the Flt-3 ligand (FL)
on megakaryocytopoiesis.18 However, a recent study suggests
a role for FL in amplifying the megakaryocytic proliferative effects of
GM-CSF, IL-3, and SCF.19
A number of the cytokines that influence megakaryocyte development also
function in hematopoietic stem/progenitor cell proliferation. As
mentioned, both IL-6 and IL-11 synergize with IL-3; they also support
the proliferation of primitive hematopoietic cells, shortening the
G0/G1 period, and triggering dormant progenitor
cells into proliferation.20,21 Likewise, IL-1 supports the
proliferation of primitive hematopoietic cells,22 but may
do so indirectly.23 While having little colony stimulating
activity alone, SCF synergizes with other cytokines, stimulating
hematopoietic stem/progenitor cell growth and
differentiation.24 The Flt-3 receptor is preferentially expressed on primitive hematopoietic cells and, in humans, is largely
restricted to CD34+ cells; its ligand thus has an important
role in regulating growth of hematopoietic stem cells.25
Although the existence of a specific humoral regulator of platelet
production (thrombopoietin, TPO) was proposed as early as
1958,26 it was not until 1994 that several groups reported its isolation and/or cloning.27-32 TPO functions
not only to induce megakaryocyte progenitor cell proliferation, but
also to drive their terminal differentiation. Additionally, the
receptor for TPO (c-mpl) is expressed on primitive
hematopoietic progenitor cells, allowing TPO to regulate early
hematopoietic progenitor cells as well.33-35 Gene
inactivation studies have shown TPO to be the primary in vivo regulator
of thrombocytopoiesis.36,37 Mice deficient in either TPO or
its receptor (c-mpl) show a dramatic (~85%) decrease in
circulating platelets as well as bone marrow (BM) and splenic
megakaryocytes.36,37 Also, the megakaryocytes of the
TPO/c-mpl-deficient mice are smaller and exhibit a lower mean
ploidy than those of control mice, but are structurally
normal.38 The platelets in these animals also are reduced
in number, but are both structurally and functionally normal, and are
sufficient to prevent overt bleeding.36-38 The continued
platelet production in these animals suggests that megakaryocyte-active
cytokines other than TPO are capable of supporting, albeit at a reduced level, the in vivo processes of megakaryothrombocytopoiesis.
Although it is widely accepted that TPO is the primary regulator of
megakaryocytopoiesis, the other megakaryocyte-active cytokines undoubtedly contribute to the in vivo regulation of this process. Proliferation of primitive hematopoietic progenitor cells, as well as
early progenitor cells in the megakaryocyte lineage, are optimally
stimulated by the synergistic interaction of multiple cytokines.11,15,39,40 To examine the interactions of TPO with other megakaryocyte-active cytokines on developing megakaryocytes, we examined its actions in combination with those cytokines that exhibit the dual capacity to affect both primitive hematopoietic progenitor cells as well as cells of the megakaryocyte lineage. We
report that in serum-free, chemically defined media, the combination of
IL-1, -6, -11, and SCF, in conjunction with TPO, is capable of
significantly stimulating megakaryocytopoiesis using human BM
CD34+ progenitor cells as a starting cell population. As
well, Flt-3 ligand, when included in this cocktail of early acting
cytokines, exerts an additional megakaryocytopoietic effect, further
enhancing the total production of CD41+ cells.
Specifically, the combination of these cytokines regulates the
expansion of CD34+ cells into the megakaryocyte lineage,
generating a 20-fold increase in megakaryocyte progenitor cell numbers,
and a 200- to 300-fold increase in the number of CD41+
promegakaryoblasts generated ex vivo.
BM Samples
Cell Cultures
Ex vivo cell expansion in suspension-phase cultures.
CD34+ cells were cultured in serum-free conditions at a
concentration of 5 × 104/mL in liquid medium plus
growth factors in 24-well plates (Costar, Cambridge, MA). The culture
medium consisted of serum-free McCoy's 5A (GIBCO, Life Technologies,
Grand Island, NY) nutrient-supplemented as previously
described,9 and containing 1% Nutridoma NS (Boehringer Mannheim, Indianapolis, IN). Cytokines, used in various combinations, included recombinant human (rhu) IL-1 (10 ng/mL; R & D, Minneapolis, MN), rhuIL-6 (25 ng/mL; R & D), IL-11 (25 ng/mL; R & D), SCF (25 ng/mL;
R & D), Flt-3 ligand (50 ng/mL; R & D), and TPO (50 ng/mL; a generous
gift from Dr Dan Eaton, Genentech, San Francisco, CA). Cultures were
incubated at 37°C in a humidified atmosphere containing 7%
CO2, and samples removed for analysis at the indicated time points. For all cell culture assays, viable cell number was determined by trypan blue exclusion, and cell counts determined using a
hemocytometer.
Hematopoietic progenitor cell assays.
Hematopoietic progenitor cells were cultured as described
previously.9,41 Briefly, 1 to 2 × 103
immunomagnetically separated CD34+ cells (as input cell
population), or suspension-culture expanded cells were placed in 1 mL
of semisolid media consisting of McCoy's 5A media (as described
above), and containing 30% FCS (HyClone Laboratories, Logan, UT),
2-mercaptoethanol (5 × 10-5 mol/L), rhuIL3 (25 ng/mL;
R & D), rhu erythropoietin (rhuEPO, 3 U/mL; Amgen Inc, Thousand Oaks,
CA), rhuSCF (25 ng/mL; R & D), rhuG-CSF (100 ng/mL; Immunex, Seattle,
WA), rhuGM-CSF (100 ng/mL; Immunex), penicillin/streptomycin (100 U/mL,
100 mg/mL, respectively; Life Technologies), and 0.9% methylcellulose
(Fluka Chemical, Ronkonkoma, NY). Three replicate cultures were set up
for each cytokine cocktail, for each marrow sample. Megakaryocyte
colonies (CFU-Mk) were scored after 14 to 16 days in culture and at 21 to 24 days for BFU-Mk. Cultures were incubated at 37°C in a
humidified atmosphere containing 7% CO2.
Flow Cytometry
Antibody labeling. A total of 105 cells were incubated with antibody (10 µg/mL) at room temperature for 30 minutes in Trisma-buffered phosphate-buffered saline (Tris-PBS), containing 1% bovine serum albumin, pH 7.6. Parallel samples were incubated with an isotype-specific, inappropriate antibody (MOPC; Becton Dickinson) to establish the background level of nonspecific staining. The incubations for CD41 antigen also included 5 mmol/L EDTA in the wash buffer to minimize selectin-mediated platelet binding that yields a falsely-elevated determination of CD41+ cells.42 CD41+ cells are defined as those cells whose antigenic content exceeds that expressed by 97% of the cells that labeled with inappropriate antibody. The ex vivo expansion of CD41+ cells was determined by evaluating the total number of CD41+ cells (thus defined) per culture compared with the input numbers of CD41+ cells for each cytokine cocktail. Unless otherwise stated, total cellularity and CD41+ cell numbers were determined on days 10 to 12 of culture. Megakaryocyte ploidy analysis. To determine megakaryocyte DNA content, anti-CD41- or mouse MOPC-IgG-labeled cells were fixed/permeabilized by gradual addition of iced methanol during mixing. The cells were incubated for 30 minutes on ice, centrifuged, and stained with propidium iodide (PI; 50 µg/mL in PBS, with 100 U/mL RNAse; Sigma Chemical). For evaluation, control populations of CD34+ cells were labeled with MOPC-FITC and PI, and 2C/4C DNA content cells gated to determine the upper 97% limit of nonspecific FITC-fluorescence. Positive fluorescence (ie, CD41 antigen-positive cells) is thus defined as any fluorescence above this limit. DNA analysis for ploidy determination was done on these CD41+ gated cells, and at least 10,000 to 20,000 cells were analyzed per condition. Cytocentrifugation, Cytological Staining, and Immunocytochemistry For morphological analysis, cells were centrifuged onto the slide surface using a Cytospin centrifuge (Shandon, Pittsburgh, PA), air-dried for 2 to 3 minutes and fixed in absolute methanol. Cells were stained with May-Grünwald-Giemsa (Sigma). For immunocytochemistry, fixed cytospin preparations were blocked with species-specific blocking serum, and incubated with unconjugated anti-CD41 (45 minutes, 22°C). Antibody-labeled megakaryocytic cells were visualized using an avidin-biotin-alkaline phosphatase system (ImmunoPure ABC Phosphatase Staining Kit; Pierce, Rockford, IL), according to manufacturer's instructions.Statistical Analysis Statistical evaluations were performed using the Student's t-test.
Cytokine Stimulation of CD34+ Cells Megakaryocytic precursor cells are known to respond to a variety of growth factor signals, showing a moderate expansion of megakaryocytes when stimulated with two to three growth factors.7,11 We reasoned that, as in other systems,39,43 multiple growth factor stimulation might better augment megakaryocyte development in vitro, and that specific combinations of megakaryocyte-active cytokines might yield a preferential ex vivo expansion of early cells of the megakaryocyte lineage. Our initial studies of two factor combinations yielded a modest proliferation of megakaryocytic precursor cells. For example, SCF plus TPO gave a 10- to 20-fold expansion of CD41+ cells (data not shown). Therefore, we decided to evaluate combinations of four to six growth factors, choosing those known to affect both hematopoietic stem/progenitor cells as well as megakaryocytes.
CD34+ Cells Stimulated by Multiple Cytokines
Generate Large Numbers of CD41+
Promegakaryoblasts
Human Megakaryocyte Progenitor Cells Respond to Combinations of
Megakaryocyte-Active Cytokines
Evaluation of the actions of multiple megakaryocyte-active cytokines on
the ex vivo expansion of promegakaryoblasts from human CD34+ BM cells indicates that combinations of IL-1 ,-6, -11, SCF, FL, and/or TPO markedly affect this process. Each of
these cytokines contributes to the ex vivo expansion of megakaryocytes,
with TPO and SCF playing a predominant role. Although the addition of
multiple cytokines increases the quantity of megakaryocytes, the
overall quality of these cells remains the same. The use of
multiple-cytokine combinations preferentially stimulates megakaryocyte
expansion from CD34+ cells, as combinations of one to three
cytokines gave only a modest (10- to 20-fold) increase in megakaryocyte
numbers, whereas combinations of four to six megakaryocyte-active
growth factors induced an 80- to 300-fold increase in CD41+
cells. In sharp contrast, the respective increase in total nucleated cells generated using the same growth factor combinations ranged from
10- to 20-fold. The addition of TPO to any cytokine combination modestly increased the number of total nucleated cells while
significantly increasing the total number of CD41+ cells,
CD41 antigen-density, and the DNA content of a small subpopulation of
CD41+ cells, as well as altering megakaryocyte
developmental kinetics. These data are consistent with the concept that
these cytokine cocktails act by influencing early (proliferative)
events in megakaryocytic development, working predominately at the
progenitor cell level. As a result, the predominant cell type produced
in these cultures is the CFU-Mk progeny, ie, the promegakaryoblast, a
transitional cell of relatively smaller size, lower antigen expression,
and lower DNA content.
Submitted October 28, 1997;
accepted January 29, 1998.
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Abstract
Introduction
Abstract
.05 compared with IL-1,6,11,S-driven CD41+ cell expansion in the absence of
TPO.
), CD41+ cell expansion in
1,6,11,S; (
), CD41+ cell expansion in IL-1,6,11,S,T.
8C. May-Grünwald-Giemsa staining showed
that these cells have the features of promegakaryoblasts, being
relatively small, with a high nucleus:cytoplasm ratio, a (variably)
lobulated nucleus, and basophilic cytoplasm. Immunocytochemical
analysis confirmed the expression of low amounts of CD41 (data not
shown). In contrast, the addition of TPO generated a population of
cells expressing a markedly higher CD41 antigen level
(CD41High cells) and increased the frequency of cells with
higher DNA content (
(TGF-
(MIP-I
15%) are
morphologically mature and have a DNA content