Lnk negatively regulates self-renewal of
hematopoietic stem cells by modifying
thrombopoietin-mediated signal transduction
Jun Seita*†, Hideo Ema*, Jun Ooehara*, Satoshi Yamazaki*‡, Yuko Tadokoro*, Akiko Yamasaki*, Koji Eto*,
Satoshi Takaki§, Kiyoshi Takatsu§, and Hiromitsu Nakauchi*¶
*Laboratory of Stem Cell Therapy, Center for Experimental Medicine, and§Division of Immunology, Department of Microbiology and Immunology,
Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; and‡ReproCell, Inc., Imperial Hotel
Tower 12F, 1-1-1 Uchisaiwai-cho, Chiyoda-ku, Tokyo 100-0011, Japan
Edited by Irving L. Weissman, Stanford University School of Medicine, Stanford, CA, and approved December 7, 2006 (received for review July 22, 2006)
One of the central tasks of stem cell biology is to understand the
molecular mechanisms that control self-renewal in stem cells.
Several cytokines are implicated as crucial regulators of hemato-
poietic stem cells (HSCs), but little is known about intracellular
to clarify how self-renewal potential is enhanced in HSCs without
the adaptor molecule Lnk, as in Lnk-deficient mice HSCs are
expanded in number >10-fold because of their increased self-
renewal potential. We show that Lnk negatively regulates self-
renewal of HSCs by modifying thrombopoietin (TPO)-mediated
signal transduction. Single-cell cultures showed that Lnk-deficient
HSCs are hypersensitive to TPO. Competitive repopulation re-
vealed that long-term repopulating activity increases in Lnk-
deficient HSCs, but not in WT HSCs, when these cells are cultured
in the presence of TPO with or without stem cell factor. Single-cell
transplantation of each of the paired daughter cells indicated that
a combination of stem cell factor and TPO efficiently induces
symmetrical self-renewal division in Lnk-deficient HSCs but not in
WT HSCs. Newly developed single-cell immunostaining demon-
strated significant enhancement of both p38 MAPK inactivation
and STAT5 and Akt activation in Lnk-deficient HSCs after stimula-
tion with TPO. Our results suggest that a balance in positive and
negative signals downstream from the TPO signal plays a role in
the regulation of the probability of self-renewal in HSCs. In
general, likewise, the fate of stem cells may be determined by
combinational changes in multiple signal transduction pathways.
c-mpl ? p38 MAPK ? STAT5 ? Akt
plantation medicine. To this end, we need to understand mo-
lecular mechanisms underlying self-renewal in stem cells. In
hematopoietic stem cells (HSCs), the best-studied mammalian
stem cells, self-renewal has been demonstrated by in vivo assays
(1–4). However, molecular mechanisms regulating self-renewal
remain poorly understood. In particular, despite numerous
studies of cytokines and cytokine receptors, little is known about
intracellular signaling events in self-renewal of HSCs (5–7).
Major difficulties have been the paucity of HSCs and the in vitro
recapitulation of self-renewal (8, 9). We have approached this
issue by analyzing Lnk-deficient mice (Lnk?/?) in comparison
with WT mice.
Lnk is an adaptor protein containing a proline-rich domain, a
pleckstrin homology domain, and a Src homology 2 domain (10).
In Lnk?/? mice, long-term marrow repopulating activity is
markedly elevated because of increases in both absolute number
and self-renewal activity of HSCs (4, 11). These results suggest
that Lnk negatively regulates the key signaling pathways of HSC
self-renewal. Lnk is expressed in various hematopoietic lineages,
in which some of its functions have been reported (12–15). Lnk
is thought to regulate stem cell factor (SCF) signaling pathways
anipulation of stem cell self-renewal is a necessity for the
development of stem cell-based regenerative and trans-
negatively in immature B cells (12, 13). Recent reports indicated
that Lnk negatively regulates thrombopoietin (TPO) signaling in
megakaryocytes and erythropoietin signaling in erythroblasts
(14, 15). Although the functions of Lnk as a negative regulator
of cytokine signaling are shared by these lineages, the target
signaling pathways appear to differ among these lineages. We
therefore attempted to determine Lnk target signaling pathways
In both WT and Lnk?/? mice, CD34-negative or low, c-Kit-
positive, Sca-1-positive, lineage marker-negative (CD34?KSL)
cells within adult mouse bone marrow (BM) are highly enriched
in HSCs (4, 16). When single-cell transplantation with
CD34?KSL cells was performed, rates of long-term reconstitu-
tion were similar in WT and Lnk ?/? mice, indicating similar
degrees of HSC enrichment in this population. Using these
highly enriched HSC populations, we first studied cytokine-
induced division of CD34?KSL cells and found that Lnk is
involved in the TPO signaling pathway. We then investigated
how HSCs self-renew in culture with TPO by competitive
repopulation and paired daughter cell assays. Furthermore, we
developed single-cell immunostaining procedures for signal
transduction analysis to examine Lnk-interacting intracellular
signaling pathways in TPO-stimulated CD34?KSL cells.
In Vitro Survival and Division of Single CD34?KSL Cells.Directeffects
of cytokines on both survival and proliferation of HSCs were
evaluated to see which cytokine signals are influenced by the
absence of Lnk. Serum-free culture of single WT or Lnk?/?
CD34?KSL cells was performed in the presence of various
cytokines at 100 ng/ml. Every 24 h after initiation of culture, cells
in each well were examined under the microscope. At 72 h of
culture, no CD34?KSL cells survived without a cytokine. In
contrast, ?70% of cells survived in the presence of SCF or TPO,
(Fig. 1A). When cells were cultured with SCF, frequencies of cell
division did not differ between Lnk?/? CD34?KSL cells
(47.9 ? 5.3%) and WT CD34?KSL cells (54.6 ? 9.1%) (P ?
Author contributions: J.S., H.E., and H.N. designed research; J.S., J.O., and A.Y. performed
K.E., S.T., and H.N. analyzed data; and J.S., H.E., and H.N. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: HSC, hematopoietic stem cell; SCF, stem cell factor; TPO, thrombopoietin;
†Present address: Department of Pathology, Stanford University School of Medicine,
Stanford, CA 94305.
¶To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
February 13, 2007 ?
vol. 104 ?
no. 7 ?
0.197). In contrast, when cells were cultured with TPO, the
frequency of cell division in Lnk?/? CD34?KSL cells (66.7 ?
8.0%) was significantly greater than that in WT CD34?KSL cells
(49.8 ? 7.6%) (P ? 0.009) (Fig. 1B). These data indicate that
SCF or TPO, but not the other cytokines studied, can support
survival and division of significant proportions of WT and
Lnk?/? CD34?KSL cells, and that TPO promotes division of
Lnk?/? CD34?KSL cells more efficiently than division of WT
Hypersensitivity of Lnk?/? CD34?KSL Cells to TPO Stimulation. Sen-
sitivity to SCF or TPO stimulation was compared between WT
and Lnk?/? CD34?KSL cells with respect to dose–response of
cell division. WT or Lnk?/? CD34?KSL cells were subjected to
single-cell serum-free culture with graded doses of SCF (1, 5, 10,
50, or 100 ng/ml) or TPO (0.1, 0.5, 1, 5, 10, 50, or 100 ng/ml).
After 72 h of culture, frequencies of cell division at each
concentration were determined. As shown in Fig. 2A, dose–
response curves were plotted by nonlinear regression. Midpoint
effective doses (ED50s) were obtained for SCF and TPO. When
WT or Lnk?/? CD34?KSL cells were cultured with SCF,
respective ED50s were 8.03 ? 0.92 and 7.17 ? 2.09 ng/ml (P ?
0.73). When WT or Lnk?/? cells were cultured with TPO,
respective ED50s were 1.89 ? 0.55 and 0.69 ? 0.06 ng/ml (P ?
0.04). SCF dose–response behaviors were quite similar in WT
and Lnk?/? CD34?KSL cells. In contrast, the concentrations of
TPO that induced cell division were lower for Lnk?/?
CD34?KSL cells than for WT CD34?KSL cells.
The synergistic effects of SCF and TPO on cell division were
compared between WT and Lnk?/? CD34?KSL cells. Single-
cell serum-free cultures were performed in the presence of SCF
and TPO at various combinations of concentrations. At 72 h of
culture, frequencies of cell division were determined and dem-
onstrated by isobolography (17, 18) (Fig. 2B). When concentra-
tions of both SCF and TPO were ?5 ng/ml, ? 80% of both WT
concentration of SCF was greater than that of TPO, dose–
response behaviors of WT and Lnk?/? CD34?KSL cells were
similar. In contrast, when the TPO concentration was greater
than that of SCF and the SCF concentration was ?5 ng/ml,
Lnk?/? CD34?KSL cells divided more frequently than did WT
CD34?KSL cells. These results indicate that in CD34?KSL cells
the absence of Lnk causes hypersensitivity to TPO but not to
SCF even when both TPO and SCF are present.
In addition, the kinetics of cytokine-induced cell division was
compared between WT and Lnk?/? CD34?KSL cells [support-
ing information (SI) Fig. 5]. When cultured with SCF or SCF and
TPO, WT and Lnk?/? CD34?KSL cells showed quite similar
first-division kinetics. Of note is that the frequencies of cell
division were similar in WT and Lnk?/? CD34?KSL cells when
those cells were cultured with a combination of SCF and TPO
at saturating concentrations (Fig. 2B and SI Fig. 5). In contrast,
when cultured with TPO, it became apparent after 24 h of
culture that Lnk?/? CD34?KSL cells underwent a first division
more frequently than did WT CD34?KSL cells (SI Fig. 5).
Repopulating Activity Increases in Lnk?/? CD34?KSL Cells in Re-
sponse to TPO. We assumed that TPO might be able to induce
self-renewal in Lnk?/? HSCs more efficiently than in WT
HSCs. To address this issue, 40 WT or Lnk?/? CD34?KSL cells
for 72 h and underwent competitive repopulation assay (SI Fig.
6A). Twelve weeks after transplantation, repopulating units
none SCFTPOIL-3 IL-6IL-11
noneSCFTPO IL-3IL-6 IL-11
in the presence of a cytokine. WT or Lnk?/? CD34?KSL cells (n ? 96) under-
went single-cell serum-free culture in the presence of SCF, TPO, IL-3, IL-6, or
IL-11. At 72 h of culture, the number of cells in each well was counted under
an inverted microscope. Wells containing one or more cell(s) were judged to
exhibit ‘‘survival’’ and wells containing two or more cells were judged to
exhibit ‘‘division.’’ Frequencies of survival (A) and division (B) from five inde-
pendent experiments are shown as mean ? SD.*, P ? 0.009.
In vitro survival and division of single WT or Lnk?/? CD34?KSL cells
% division % division
Dose–response curves to SCF, TPO, or both are shown for WT and Lnk?/?
CD34?KSL cells. (A) (Left) When cultured with SCF, WT or Lnk?/? cells had
cultured with TPO, WT or Lnk?/? cells had ED50s of 1.89 ? 0.55 or 0.69 ? 0.06
of CD34?KSL cells. Percentages of cells having undergone division by 72 h of
culture among WT (Left) or Lnk?/? (Right) CD34?KSL cells in the presence of
SCF plus TPO are presented as isobolograms. Contours show percent division
at intervals of 10%.
Hypersensitivity of Lnk?/? CD34?KSL cells to TPO stimulation.
www.pnas.org?cgi?doi?10.1073?pnas.0606238104Seita et al.
(RUs) were determined by analyzing peripheral blood donor
chimerism. The RU is a quantitative index of repopulating
activity, and 1 RU is defined as the amount of repopulating
activity in 105unfractionated BM cells from WT mice, based on
competitive repopulation assay (19).
As shown in Fig. 3, RUs did not change in WT CD34?KSL
cells after a 72-h culture period in the presence of SCF, TPO, or
SCF plus TPO. RUs did not change significantly in Lnk?/?
CD34?KSL cells after culture with SCF alone. However, RUs
were significantly increased in Lnk?/? CD34?KSL cells after
culture with TPO alone or SCF plus TPO. These repopulating
activities were transplantable into secondary recipient mice (SI
Table 2), suggesting that self-renewal potential was maintained
in cultured cells. On average, RU increased 3-fold in both cases.
Based on the positive correlation between RUs and HSC
numbers (SI Fig. 6B), it was estimated that the number of
HSCs proportionately increased 3-fold. To increase in number,
HSCs should have undergone symmetrical self-renewal
Symmetrical Self-Renewal Division of Lnk?/? HSCs in Culture. To
know whether increased RUs in effect resulted from symmet-
rical self-renewal in Lnk?/? HSCs, paired daughter cell exper-
iments were performed (SI Fig. 6C). Single CD34?KSL cells
were directly transplanted into lethally irradiated mice or indi-
vidually cultured in the presence of SCF and TPO. When cells
in culture gave rise to two daughter cells, each daughter cell was
separated from the other by micromanipulation techniques.
Individual daughter cells were transplanted into lethally irradi-
As shown in Table 1, both daughter cells were detected as
long-term repopulating cells in 3 of 20 pairs of daughters of
single Lnk?/? CD34?KSL cells and in none of 28 pairs of
daughters of single WT CD34?KSL cells. One of the two
daughter cells was detected as a long-term repopulating cell in
3 of 20 pairs of daughters of Lnk?/? CD34?KSL cells and in 3
of 28 pairs of daughters of WT CD34?KSL cells. When sym-
metrical self-renewal is defined as division resulting in genera-
tion of two daughter cells with long-term repopulation potential,
and asymmetrical self-renewal is defined as division resulting in
generation of one daughter cell with long-term repopulating
potential and another without long-term repopulation potential,
these data support the conclusion that in culture with SCF and
TPO, Lnk?/? HSCs undergo symmetrical self-renewal division
more frequently than do WT HSCs.
Development of Signal Transduction Analysis for HSCs. Because of
the paucity of HSCs (a single mouse yielded ?1,000 CD34?KSL
cells) to apply commonly used signal transduction assays to this
study was difficult. To circumvent this problem, we combined
multicolor fluorescence-activated cell sorting, fluorescent im-
munostaining, confocal laser scanning, and computational quan-
tification of fluorescent intensities. One of the two keys to
success in this assay was in-droplet immunostaining procedures.
Their principal steps are illustrated in SI Fig. 7. From the initial
cell purification step to the final analysis step, cells were main-
tained in a droplet of medium to avoid cell loss and cell damage.
The other key was measurement of signal intensity in individual
cells. In this study, we focused on phosphorylation kinetics of
signaling molecules because Lnk is an adaptor protein contain-
ing a Src homology 2 domain. As demonstrated in SI Fig. 8,
phosphorylation intensity of each CD34?KSL cell was evaluated
by using NIH ImageJ software. We named this method the
single-cell imaging of phosphorylation (SCIPhos) assay.
To validate the SCIPhos assay, signal transduction in a
cytokine-dependent cell line was examined simultaneously by
both SCIPhos assays and conventional Western blot analysis.
After cytokine deprivation, TPO-dependent 32D/Mpl cells were
stimulated with TPO, and phosphorylation of STAT5 and Jak2
was quantitatively measured. As representatively shown in SI
Fig. 9, SCIPhos and Western blot analyses gave similar kinetics
of STAT5 phosphorylation. Interestingly, STAT5 phosphoryla-
tion increased in a dose-dependent manner for TPO stimulation,
and SCIPhos data correlated well with Western blot data (SI Fig.
9C). Good correlation between results of these two assay tech-
niques was also observed for Jak2 phosphorylation (data not
Phosphorylation of Signaling Molecules in WT and Lnk?/? CD34?KSL
Cells. To identify the key signal pathways involved in HSC
Akt, p38 MAPK, and p44/42 MAPK after TPO or SCF stimu-
lation was compared between WT and Lnk?/? CD34?KSL
Fold increase in RU
day 0day 3
day 0 day 3
or Lnk?/? (Right) CD34?KSL cells were transplanted (n ? 8). Similar cells
alternatively were cultured with SCF (50 ng/ml) and/or TPO (50 ng/ml) for 3
days, and then transplanted (n ? 8). RUs before (n ? 8) and after culture were
compared 12 weeks after transplantation. Data are shown in terms of fold
increase (average RU before culture ? 1.0). RUs significantly increased in
0.046).*, P ? 0.05 vs. before culture.
RU increase in Lnk?/? CD34?KSL cells after culture. Forty WT (Left)
Table 1. Symmetrical self-renewal in culture of Lnk ??? CD34?KSL cells but not WT
pairs (%) % ChimerismOne The other
0.6, 0.6, 0.6
(2.0; 5.9), (50.9; 72.9), (42.9; 81.4)
0.7, 2.4, 23.0
Single WT or Lnk??? CD34?KSL cells were cultured with SCF and TPO. After a single cell gave rise to
paired-daughter cells, each daughter cell was individually transplanted (SI Fig. 6C). Twelve weeks after trans-
plantation reconstitution was observed in 4 of 25 mice or 3 of 16 mice that, respectively, had received single WT
or Lnk??? CD34?KSL cells at day 0. Successful reconstitution is shown as ?, and undetectable reconstitution is
shown as ?, for each transplanted daughter cell.
Seita et al.
February 13, 2007 ?
vol. 104 ?
no. 7 ?
cells. As shown in Fig. 4, after stimulation with TPO JAK2 was
similarly phosphorylated in WT and Lnk?/? CD34?KSL cells.
However, STAT5 and Akt were more intensely phosphorylated
in Lnk?/? CD34?KSL cells than in WT CD34?KSL cells. In
contrast, phosphorylation levels of p38 MAPK in Lnk?/?
CD34?KSL cells were down-regulated after TPO stimulation,
whereas those in WT CD34?KSL cells remained unchanged.
Significant phosphorylation of p44/42 MAPK was not detected
we detected no significant difference between WT and Lnk?/?
CD34?KSL cells in phosphorylation patterns of these signaling
molecules (data not shown). Because these comparisons were
made with actively self-renewing HSCs, enhanced up-regulation
of STAT5 and Akt pathways and enhanced down-regulation of
p38 MAPK pathways can be inferred to be associated with
initiation of HSC self-renewal.
To understand why self-renewal potential is greater in HSCs
without Lnk, we focused our attention on a first division of
CD34?KSL cells because self-renewal is likely to be progres-
sively reduced in the following divisions (7, 8). Analysis of their
first division revealed that Lnk?/? CD34?KSL cells are more
sensitive to TPO than are WT CD34?KSL cells. SCF and TPO
synergistically acted on both WT and Lnk?/? CD34?KSL cells
and efficiently induced their division. Over 80% of both WT and
Lnk?/? CD34?KSL cells divided once by day 3 of culture in the
presence of saturating amounts of SCF and TPO (Fig. 2 and SI
Fig. 5). Finding that the frequency of dividing cells among
Lnk?/? CD34?KSL cells did not differ from that among WT
CD34?KSL cells led us to the hypothesis that outcomes of a first
division might differ between WT and Lnk?/? CD34?KSL
cells. This hypothesis was supported by competitive repopulation
data and was verified by paired-daughter cell experiment data.
During 3-day culture with TPO or with SCF and TPO, RUs
increased 3-fold in Lnk?/? cells, whereas RUs did not signifi-
cantly increase in WT cells (Fig. 3A). To increase RUs, Lnk?/?
CD34?KSL cells must have undergone symmetrical self-renewal
division during the culture period. As far as first divisions were
examined, both symmetrical and asymmetrical self-renewal di-
visions were detected in Lnk?/? CD34?KSL cells (Table 1). In
contrast, symmetrical self-renewal division was not detected in
WT CD34?KSL cells. The results clearly indicate that Lnk?/?
HSCs self-renew better than do WT HSCs in response to TPO.
Nolan and colleagues (20–22) have reported an intracellular
phospho-protein analysis technique using flow cytometry (Phos-
pho Flow). This technique enables signal transduction analysis of
target cells in heterogeneous cell populations. The cells that
surround CD34-KSL cells, however, have produced many cyto-
kines before a particular experimental stimulus is applied to
CD34-KSL cells. The unavoidable prestimulatory effects of
these cytokines impair the utility of the Phospho Flow method
in the analysis of CD34?KSL cells. Furthermore, internalization
of c-Kit in response to SCF makes phenotypic identification of
CD34-KSL cells difficult. To overcome these problems, in the
SCIPhos assay cells are first sorted and then stimulated with
cytokines under defined conditions, followed by single-cell im-
munostaining. The SCIPhos assay enables quantitative measure-
ment of phosphorylation levels of signal transduction molecules
in individual cells, as verified by comparison with Western
blotting data using only 50 cells (SI Fig. 5). Moreover, intracel-
lular localization of signal molecules can be examined simul-
Using SCIPhos assays, we attempted to identify signal trans-
duction pathways that in self-renewing Lnk?/? HSCs are
activated or inactivated differently from those in self-renewing
p-p44/42 MAPK (Erk1/2)
Duration of TPO stimulation (min)
Relative fluorescence intensity
0 10 3060
0 10 3060
010 30 60
0 10 30 60
0 1030 60
0 10 3060
0 10 30 60
0 10 3060
0 10 3060
0 1030 60
0 10 30 60
in TPO-stimulated WT and Lnk?/? CD34?KSL cells. All fluorescence intensities of individual cells were computationally quantified, normalized, and compared
against the mean intensity of unstimulated cells. Each dot shows normalized fluorescence intensity of individual cells (n ? 50). Error bars indicate mean ? SD.
*, P ? 0.001 vs. unstimulated cells (t ? 0).
www.pnas.org?cgi?doi?10.1073?pnas.0606238104Seita et al.
WT HSCs. Because RUs in cultured Lnk?/? cells increased in
the presence of TPO alone, but not SCF alone, to the same
extent as that seen in the presence of SCF plus TPO (Fig. 3), we
simply compared SCF- or TPO-mediated signal transduction
between Lnk?/? and WT HSCs. Lnk?/? HSCs in the process
of self-renewal revealed enhancement of combinatorial change
in signal transduction, i.e., activation of both STAT5 and Akt
signal transduction and inactivation of p38 MAPK (Fig. 4).
Phoshorylation of p38 MAPK in freshly isolated HSCs was
possibly caused by stress in cell preparation procedures. How-
ever, its dephosphorylation was significantly enhanced in
Lnk?/? HSCs. Kato et al. (24) have recently reported that
continuous activation of STAT5, but not STAT3, in CD34?KSL
cells results in myeloproliferative disease accompanied with
increase of a broad range of myeloid progenitors in addition to
suppression of apoptosis (25). The p38 MAPK pathway has been
implicated as regulating cell cycle progression negatively by
activating transcription of the Ink4a-Arf locus and inhibiting the
expression of D-type cyclins (26). A p38 MAPK inhibitor
reportedly prevents decline of self-renewal potential in HSCs
through serial transplantation (27). These signal modifications
together may give HSCs advantages in maintaining self-renewal
potential. How Lnk controls the probability of symmetrical
self-renewal in HSCs is extremely intriguing.
No hematopoietic malignancy has been observed to date in
Lnk-deficient mice (11, 28). Lnk may provide a suitable molec-
ular target for enhancement of self-renewal capacity of HSCs.
Lnk inhibitors should give selective advantage to HSCs and
should be useful for stem cell transplantation or gene therapy
targeting HSCs. Alternatively, Lnk inhibitors can be used for ex
vivo expansion of HSCs. Takizawa et al. (29) have reported that
transient inhibition of endogenous Lnk activity by introduction
of a dominant-negative form of Lnk can increase engraftment
rates of HSCs. Interestingly, inhibition of Lnk function increases
cell adhesion ability in HSCs so that HSCs can efficiently home
to a BM niche. These results suggest that Lnk interacts with
multiple signaling cascades related to cytokine signal transduc-
tion and cell mobility.
In this study, we showed that Lnk negatively regulates TPO-
mediated signaling pathways in HSCs. Comparison of HSC
has recently been reported (28). Increases in numbers of HSCs
in Lnk?/? mice have been shown to be overridden by decreases
in numbers of HSCs in TPO?/? mice (28). Interestingly, HSC
numbers are greater in Lnk?/?TPO?/? mice than in TPO?/?
mice, suggesting that not only TPO signaling is involved. We
conclude that Lnk negatively interacts with signaling pathways
downstream of TPO/c-Mpl that play an important role in HSC
fate decision, namely, whether or not HSCs self-renew. We
in positive and negative signals from multiple pathways rather
than by self-renewal-specific signals. In this regard, self-renewal
Materials and Methods
Mice. C57BL/6 mice congenic for the Ly5 locus (B6-Ly5.1) and
Lnk?/? B6-Ly5.1 mice were bred and maintained at the Animal
Research Center of the Institute of Medical Science, University
of Tokyo. The Animal Experiment Committee of the Institute of
Medical Science, University of Tokyo, approved animal care and
use. B6-Ly5.1/5.2 (B6-F1) mice were obtained from mating pairs
of B6-Ly5.1 and B6-Ly5.2 mice. B6-Ly5.2 mice were purchased
from Nihon SLC (Shizuoka, Japan).
Purification of CD34?KSL Cells. CD34?KSL cells were purified
from BM cells of 2-month-old WT or Lnk?/? B6-Ly5.1 mice as
described (16, 30). In brief, low-density cells were isolated on
Ficoll-Paque PLUS (Amersham Bioscience, Uppsala, Sweden).
The cells were stained with an antibody mixture consisting of
biotinylated anti-Gr-1, anti-Mac-1, anit-B220, anti-CD4, anti-
CD8, and anti-Ter-119 antibodies (Pharmingen, San Diego,
CA). The cells were subsequently labeled with MACS goat
anti-rat IgG microbeads. Lineage-positive cells were then de-
pleted by using the Midi-MACS system (Miltenyi Biotec, Ber-
gisch Gladbach, Germany). The cells were further stained with
FITC-conjugated anti-CD34, phycoerythrin-conjugated anti-
Sca-1, and allophycocyanin (APC)-conjugated anti-c-Kit anti-
bodies (Pharmingen). Biotinylated antibodies were detected
with streptavidin-APC-Cy7 (Molecular Probes, Eugene, OR).
Four-color analysis and sorting were performed on a MoFlo Cell
Sorter (DakoCytomation, Glostrup, Denmark).
Single-Cell Serum-Free Culture. Single-cell cultures of CD34?KSL
cells were performed under serum-free conditions as described
(9, 31). Cells were individually deposited into single wells of a
96-well round-bottom microtiter plate and cultured in S-clone
SF-O3 medium (Sanko-Junyaku, Tokyo, Japan) supplemented
with 0.5% BSA and the following cytokines: 0.1–100 ng/ml
mouse SCF, 0.1–100 ng/ml human TPO, 100 ng/ml mouse IL-3,
100 ng/ml human IL-6, and 100 ng/ml human IL-11 (PeproTech,
Rocky Hill, NJ). After cell sorting, the presence of one cell per
well was verified under an inverted microscope. The cells were
incubated at 37°C in a humidified atmosphere with 5% CO2in
air. At several time points, numbers of cells per well were
counted under an inverted microscope. Each frequency of cell
division was obtained from 96 wells.
Transplantation Assays. Competitive repopulation assays were
performed with the Ly5 congenic mouse system. Forty
CD34?KSL cells from WT B6-Ly5.1 mice were transplanted into
a WT B6-Ly5.2 mouse irradiated at a dose of 9.5 Gy with 4 ?
105competitor cells from WT B6-F1 mice. Forty CD34?KSL
cells from Lnk?/? B6-Ly5.1 mice were transplanted into a WT
B6-Ly5.2 mouse irradiated at a dose of 9.5 Gy with 8 ? 105
competitor cells from WT B6-F1 mice. Concurrently, 40 WT or
Lnk?/? CD34?KSL cells were cultured in the presence of
cytokines for 3 days, and then transplanted, with 4 ? 105or 8 ?
105WT B6-F1 competitor cells, respectively, into a lethally
irradiated (9.5 Gy) WT B6 mouse. When one CD34?KSL cell or
each one of its paired-daughter cells generated in culture was
transplanted, 2 ? 105competitor cells were used. Twelve weeks
after transplantation, peripheral blood cells of the recipients
were stained with FITC-conjugated anti-Ly5.2 (104) and biotin-
ylated anti-Ly5.1 (A4) (Pharmingen). The cells were simulta-
neously stained with phycoerythrin (PE)-Cy7-conjugated anti-
B220 antibody and a mixture of allophycocyanin-conjugated
anti-Mac-1 and -Gr-1 antibodies and a mixture of PE-conjugated
anti-CD4 and -CD8 antibodies (Pharmingen). The biotinylated
antibody was detected with streptavidin-Texas red. Cells were
analyzed on a FACS Vantage (Becton Dickinson, San Jose, CA).
Percentage chimerism was calculated as (percent Ly5.1-donor
cells) ? 100/(percentage Ly5.1-donor cells ? percentage F1-
competitor cells). RUs were calculated with Harrison’s method
(19) as follows: RU ? (percentage chimerism) ? (number of
competitor cells) ? 10?5/(100 ? percentage chimerism). In the
case of single-cell transplantation, when percentage chimerism
was ?0.5, test donor cells were considered to be long-term
repopulating cells. Secondary transplantation was performed 4
months after primary transplantation. After peripheral blood
was analyzed for chimerism once again, 2 ? 106BM cells from
selected recipient mice were transplanted into mice irradiated at
a dose of 9.5 Gy.
Cell Lines. 32D cells (ATCC, Manassas, VA) were maintained in
RPMI medium 1640 with 10% FCS and 10% conditioned
Seita et al.
February 13, 2007 ?
vol. 104 ?
no. 7 ?
medium from the culture of WEHI-3B cells. Human c-MPL Download full-text
cDNA was ligated to the pMY-IRES-EGFP retroviral vector (a
gift of T. Kitamura, University of Tokyo, Tokyo, Japan). The
recombinant viruses were produced by transfecting pMY-Mpl-
IRES-EGFP into 293gp cells with pcDNA3-VSV-G. 32D cells
were then infected with these viruses. TPO-dependent 32D
(32D/Mpl) cells were selected, cloned, and maintained with 10
SCIPhos Assay. Phosphorylation of cytokine signaling molecules
was analyzed by fluorescent immunocytostaining (SI Fig. 7). WT
or Lnk?/? CD34?KSL cells were directly sorted by flow
cytometry into droplets of medium on poly-L-lysine-coated glass
slides (Matsunami Glass, Osaka, Japan). Cells were stimulated
with cytokine(s) at indicated concentrations for indicated times,
and then fixed with 4% paraformaldehyde and permeabilized
with 0.1% Triton X-100. Subsequently, the cells were stained
with phosphorylation-specific anti-JAK2 (Tyr-1007/1008), anti-
STAT3 (Tyr-705), anti-STAT5 (Tyr-694), anti-Akt (Ser-473),
anti-p38 MAPK (Thr-180/Tyr-182), and anti-p44/42 MAPK
(Thr-202/Tyr-204) antibodies (Cell Signaling Technology, Bev-
erly, MA). After washing with PBS containing 2% goat serum,
cells were stained with Alexa Fluor 488- or 647-conjugated goat
anti-rabbit IgG antibody (Molecular Probes) and DAPI.
To avoid quenching of fluorescence, cells were scanned only
once, at the cells’ centers, by a TCS SP2 AOBS confocal
laser-scanning microscope (Leica, Wetzlar, Germany). Cell im-
ages were obtained at 100 ? 100 pixels resolution by using a ?63
objective lens. Fluorescence intensities of individual cells (n ?
50) were computationally quantified by using ImageJ 1.33 soft-
ware (http://rsb.info.nih.gov/ij) and were normalized against the
mean intensity of unstimulated cells.
Western Blot Analysis. 32D/Mpl cells were starved in RPMI
were stimulated with 50 ng/ml TPO for 0, 1, 5, 10, or 60 min or
0, 0.8, 1.6, 3.2, 6.4, or 12.8 ng/ml TPO for 10 min. Cells were lysed
in buffer consisting of 50 mM Tris?HCl, 5 mM EDTA, 150 mM
inhibitor in water (Roche Molecular Biochemicals, Basel, Swiz-
erland). Cell lysates in 2? SDS sample buffer (BioRad Labo-
ratories, Hercules, CA) were subjected to SDS/PAGE, blotted,
and probed with antiphosphorylated STAT5 (Tyr-694) or anti-
phosphorylated JAK2 (Tyr-1007/1008) antibodies. Primary an-
tibodies were detected with the SuperSignal West Pico system
(Pierce Biotechnology, Rockford, IL).
Statistical Analysis and Nonlinear Regression. Mean values of two
groups were compared by two-tail unpaired t testing. Nonlinear
regression by the four-parameter logistic method was performed
for the dose–response and division kinetics curves. The ED50s of
the two groups were compared by F testing. All statistical
analyses were performed on Prism 4 software (GraphPad, San
We thank T. Kitamura for providing human c-MPL cDNA and pMY-
IRES-EGFP retroviral vector, A. S. Knisely for critical reading of the
manuscript, A. Iwama for helpful discussions, and Y. Yamazaki for
excellent assistance in analysis and sorting on a flow cytometer. This
work was supported in part by grants from the Naito Foundation, the
Terumo Lifescience Foundation, and the Ministry of Education, Cul-
ture, Sport, Science, and Technology of Japan.
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