Cell Stem Cell
Article
Contractile Forces Sustain and Polarize
Hematopoiesis from Stem and Progenitor Cells
Jae-Won Shin,1,2 Amnon Buxboim,1 Kyle R. Spinler,1 Joe Swift,1 David A. Christian,3 Christopher A. Hunter,3
Catherine Le´ on,4 Christian Gachet,4 P.C. Dave P. Dingal,1 Irena L. Ivanovska,1 Florian Rehfeldt,1 Joel Anne Chasis,5,6
and Dennis E. Discher1,2,*
1Biophysical Engineering Lab, University of Pennsylvania, Philadelphia, PA 19104, USA
2Cell and Molecular Biology and Pharmacology Graduate Groups, University of Pennsylvania, Philadelphia, PA 19104, USA
3Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
4UMR S949, Inserm, Universite´ de Strasbourg, E´ tablissement Franc¸ ais du Sang, Strasbourg, 67000, France
5Life Sciences Division, University of California, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
6Division of Hematology/Oncology, University of California, San Francisco, San Francisco, CA 94143, USA
*Correspondence: discher@seas.upenn.edu
http://dx.doi.org/10.1016/j.stem.2013.10.009
SUMMARY
Self-renewal and differentiation of stem cells depend
on asymmetric division and polarized motility processes
that in other cell types are modulated by
nonmuscle myosin-II (MII) forces and matrix mechanics.
Here, mass spectrometry-calibrated intracellular
flow cytometry of human hematopoiesis
reveals MIIB to be a major isoform that is strongly
polarized in hematopoietic stem cells and progenitors
(HSC/Ps) and thereby downregulated in
differentiated cells via asymmetric division. MIIA
is constitutive and activated by dephosphorylation
during cytokine-triggered differentiation of cells
grown on stiff, endosteum-like matrix, but not soft,
marrow-like matrix. In vivo, MIIB is required for generation
of blood, while MIIA is required for sustained
HSC/P engraftment. Reversible inhibition of both
isoforms in culture with blebbistatin enriches for
long-term hematopoietic multilineage reconstituting
cells by 5-fold or more as assessed in vivo. Megakaryocytes
also become more polyploid, producing
4-fold more platelets. MII is thus a multifunctional
node in polarized division and niche sensing.
INTRODUCTION
Stem cells must be able to self-renew and also give rise to
diverse cell types by asymmetric division in appropriate microenvironments
(Knoblich, 2010). This differential segregation
of cell fate determinants produces progenitors that expand
symmetrically to generate tissue. Hematopoietic stem cells
(HSCs, as a subset of CD34+
cells) exemplify these key properties
of stem cells in that they are often quiescent in niches
of the bone marrow (BM), but they and/or their daughter cells
polarize and divide asymmetrically in suitable niches to generate
progenitors that further divide and specialize to terminally
differentiated erythroid, megakaryocyte, and white cell lineages.
A number of models for marrow and soluble signal regulation
of HSC maintenance and differentiation have been
described (Trumpp et al., 2010), but many physical aspects of
hematopoiesis remain unclear. Many cell types apply forces
to the matrix that they adhere to, and the flexibility of extracellular
matrix is already known to modulate differentiation of
marrow-derived mesenchymal stem cells (MSCs) (Engler
et al., 2006) as well as the expansion of adult HSCs and progenitors
(HSC/Ps) (Holst et al., 2010). In both of these latter
studies, myosin-II (MII) inhibition revealed a key role for actomyosin
forces in adhesion and sensing of matrix. However, cell
contractile forces contribute to many processes in stem cell
and progenitor maintenance and division with likely relationships
to differentiation.
Cytokinesis is driven by nonmuscle MII in a cell’s cortex, and
the asymmetry of stem cell division in C. elegans is also established
by MII (Ou et al., 2010). Differentiation in embryogenesis
indeed requires active MII (Conti et al., 2004), and while inhibition
of MII in adherent embryonic stem cells (ESCs) increases
survival in culture by preserving intercellular contacts (Chen
et al., 2010), inhibition can also lead to multinucleated cells
(Canman et al., 2003). Actomyosin forces generally stabilize
the plasma membrane with an active cortical tension or rigidity
(Merkel et al., 2000), but these forces also drive cell rounding
in cytokinesis (Sedzinski et al., 2011) and can change dramatically
in differentiation (of MSCs) (Engler et al., 2006). Indeed,
while it has been known for many years that as granulocytes
differentiate they become soft to better traffic from marrow
through the endothelial barrier and into the circulation (Lichtman,
1970), any MII changes in such cells leaving the marrow or in
other hematopoietic cells is currently unknown.
Mammals express three isoforms of MII: A (MYH9), B (MYH10),
and C (MYH14), and each is regulated transcriptionally as
well as posttranslationally. MIIA is found in most tissues
(Ma et al., 2010) including blood (Maupin et al., 1994) and is
essential to embryonic differentiation (Conti et al., 2004). MIIB
is particularly enriched in brain and cardiac tissues, and it is often
polarized to the rear of migrating cells (Vicente-Manzanares
et al., 2008; Raab et al., 2012). Recent studies have revealed
roles for MIIB in hematopoiesis, specifically in megakaryocyte
(MK) differentiation (Lordier et al., 2012) and in the asymmetric
process of erythroid enucleation (Ubukawa et al., 2012). MIIB
myofilaments are known to attach more strongly to and detach
Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc. 81
more slowly from F-actin than MIIA, resulting in higher force generation
per MIIB (Wang et al., 2003). However, MIIB in human
ESCs or stem cells in general has unknown functions. MIIC appears
restricted to epithelial cells (Ma et al., 2010) and serves
here as a useful negative control in expression analyses. Here,
we reveal critical roles of MIIA and MIIB in adult hematopoiesis
and use that understanding to enrich highly heterogeneous
HSC/Ps for long-term hematopoietic stem cells.
RESULTS
MII Isoforms Switch from B-and-A to Only A in Human
Adult Hematopoiesis
Immunofluorescence of human CD34+
cells reveals cortical
MIIB as well as MIIA (Figure 1Ai), but flow cytometry and immunoblots
show that myosin levels vary with surface markers and
also across differentiated lineages (Figure 1Aii, Figures S1A
and S1B, available online). Mass spectrometry-calibrated intracellular
flow (MS-IF) cytometry (Figure S1C, Supplemental
Experimental Procedures) was developed to quantify absolute
isoform stoichiometry, which is not possible by antibody
methods alone due to differential sensitivities of antibodies to
isoforms. MS-IF cytometry of diverse hematopoietic cell types
reveals that MIIB is no more than $30% of total MII across cell
types and has a large dynamic range of $5,000-fold compared
to $80-fold for MIIA (Table S1). However, MS also revealed
MIIA phosphorylation at S1943 (pS1943), which deactivates
MIIA through myofilament disassembly (Dulyaninova et al.,
2007), and so a phosphospecific antibody was used to estimate
the pS1943 stoichiometry of MIIA through a calibration scheme
using mutant GFP-MIIA (see Supplemental Experimental Procedures).
Based on this, $50%–60% of MIIA is phosphorylated as
pS1943 in the three key HSC/P subpopulations of CD34+
cells
(Figure S1D) per standard surface markers (Majeti et al., 2007;
Novershtern et al., 2011):
{‘‘HSC enriched’’ cells: CD34+
, CD38À
, CD90+
, CD45-RAÀ
,
CD133+
}
{Multipotent progenitor (‘‘MPP’’): CD34+
, CD38À
, CD90À
,
CD45-RAÀ
, CD133+
},
A
B
Figure 1. Two-Component Lineage Trajectories
of MII Isoform States in Hematopoie-
sis
(A) MIIB relative to active fraction of MIIA (nonphosphorylated
MIIA), transformed to a measurable
B:A ratio versus sum total intensity (a.u.). (Ai)
Images of coimmunostained MIIA and MIIB (bars =
5 mm). (Aii) Representative intracellular FACS dot
plots show expression of MIIA, pS1943, and MIIB
(y axis) across subpopulations (markers indicated
in x axis). (Aiii) Mean fluorescent intensity of MIIs
for each subpopulation from flow cytometry was
normalized to an internal fluorescence control
(A549), and B:A was calibrated to an absolute ratio
from mass spectrometry analyses of MSCs (B:A =
6:94). The perforated endothelium schematically
illustrates the permeable barrier between bone
marrow and circulating cells. MKP, MK Progenitor
1 (CD34+
CD41+
), 2 (CD34À
CD41+
); ProE, Proerythroblast
(CD44+
GPAÀ
); EryP, Erythroid Progenitor
1 (CD44+
GPA+
), 2 (CD44À
GPA+
); Plt,
Platelet; T, B, Lymphoid; Myemid
, Myehi
, Bone
marrow CD33+
myeloid. WBC: Mean result for PB.
Mean ± SEM of n R 3, with errors bars omitted if
<5% of mean.
(B) Key genes correlated with MYH10 and ranked
by jPearson correlationj > 0.75 or fit with a powerlaw.
(Bi) Data sets were derived from RMA summarized
microarray analyses of fresh populations
of HSC-enriched, MPP, CPP, and cultured CD34+
derived
cells control or treated with Blebb (see
Supplemental Experimental Procedures). Colors in
bar graphs and gene symbols respectively represent
power law exponents or gene intensities, and
they are normalized by minimum levels (green: 0 or
log23) and maximum levels (red: 3 or log211) of
correlated genes using MYH10 as a reference
(black: 1 or log26). Representative correlation plots
between MYH10 and HSC/P (Bii) or differentiation
(Biii) gene markers are shown (mean ± SEM of
n R 2).
See also Figure S1, Table S1, and Table S2.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
82 Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc.
{‘‘Common potent progenitor (‘‘CPP’’): CD34+
, CD38+
,
CD90À
, CD45-RA+
, CD133À
}.
In differentiation, pS1943 generally decreases as MIIA is activated
(Figure S1D), except for MKs in which pS1943 is similarly
high as in HSC/Ps.
Phosphorylation at S1943 is equivalent to inhibiting $50% of
MIIA activity (Raab et al., 2012), so that for any cell type,
active-MIIA = (50% of total MIIA) 3 (1 + MIIA fraction of nonpS1943).
The result for active-MIIA proves to be nearly constant
throughout hematopoietic differentiation and makes a useful
common denominator. As such, a myosin map of hematopoiesis
shows the highest stoichiometric ratio of (MIIB: active-MIIA) z
1:2 (50%) in ‘‘HSC-enriched’’ (Figure 1Aiii) and a ratio of (MIIB:
active-MIIA) R 1:10 for the other marrow-restricted cells that
include nucleated erythroid progenitors, MKs, and nonhematopoietic
MSCs. For cell types that predominantly exit the marrow,
the ratio (MIIB: active-MIIA) generally decreases as MIIB is specifically
repressed—except for red blood cells (RBCs), which
repress both myosins in similar proportion. Hematopoietic differentiation
thus involves a major switch from MIIB + MIIA to active
MIIA alone.
The programmatic nature of MII changes in hematopoiesis
is evident in the fact that all nine of the marrow-resident cell
types measured here cluster together in the MII map (Figure
1Aiii). Based on the ‘‘marrow’’ space in such a map being
roughly half as large as that spanned by all measured cell types,
we estimate a clustering probability p z (1
/2)11
(1
/2)5
= 0.000015.
This high significance p provides a metric of the systematic
consistency of our MII measurements in addition to revealing a
broad MII program.
Since MIIB was highest at the protein level in CD34+
subpopulations,
microarray profiling of the different stem/progenitor/
differentiated cells allowed us to identify genes that correlate
with expression of MYH10 (Figure 1Bi). CD34 correlated strongly
with MYH10 in showing a power law exponent of 1.8 (Figure 1Bii),
whereas the differentiation gene ELANE is strongly anticorrelated
with a power law of À1.8 exponent (Figure 1Biii). MYH9
shows no correlation with MYH10. Transcripts that fit well were
further assessed with a second, novel algorithm for a robust
list (Table S2). A key subset is ranked on the correlation with
MYH10 and color-coded for the power law. Consistent with
protein-level analyses, both MYH9 and MYH10 transcripts are
of similar (midrange) intensity. About 1% of MYH10-correlated
genes are known HSC/P markers, while >10% of the most anticorrelated
genes are specific lineage markers. The profiling
indeed shows maintenance of HSC/P markers (e.g., CD34 and
ALDH1A1) and suppression of differentiation genes (e.g.,
ELANE) (Figures 1Bii and 1Biii). Transcriptome dynamics thus
align well with protein dynamics along with the differentiation
trajectory.
MIIB Polarization in Marrow-Derived CD34+
Cells
Some of the MYH10-correlated genes (CEP70, DOCK1, MACF1,
MAP7, and TUBB4) not only polarize but also have roles in
motility via the microtubule system. MIIB might be more cortical
than MIIA in round, uncultured CD34+
cells (Figure 2A), and in
the $50% of CD34+
cells that are polarized in culture, MIIB is
clearly enriched in the uropod by 75%, while MIIA is diffuse
and uniform. The microtubule organizing center (MTOC) was
also in the uropod as expected (Giebel et al., 2004). However,
because cytoskeletal polarization in MSCs is suppressed by
soft microenvironments (Raab et al., 2012), it became important
to measure the elasticity of intact BM. Therefore we sectioned
fresh bone lengthwise to expose the marrow for probing by
atomic force microscopy. We determined a microscale elasticity
of $0.3 kilopascal (kPa) (Figure 2B), which is >106
-fold softer
than rigid bone and plastic. HSC/Ps have also been seen to
localize at or near the osteoblastic endosteum of bone, which
we had previously measured to have an elasticity >30 kPa
(Engler et al., 2006). The softness of marrow is likely a product
of both high cellularity and low extracellular matrix density:
fibronectin (FN) is ubiquitous in marrow but is denser near the
endosteum where collagen-I is also high (Nilsson et al., 1998).
CD34+
cells clearly anchor specifically to FN versus collagen-I
(Figures S2A–S2C), and so we FN-functionalized gels of polyacrylamide
to test matrix elasticity effects on MIIB polarization.
Similar to our findings for marrow-derived MSCs (Raab et al.,
2012), MIIB polarization in CD34+
cells is suppressed in cultures
on marrow-mimetic soft matrix but promoted in cultures
on endosteum-mimetic stiff matrix (Figure 2C). Blebb rapidly
abolishes MIIB polarization on stiff matrix without affecting
membrane intensity of MIIB. MII activity and stiff matrix
(including rigid plastic if cells adhere) thus drive polarization in
CD34+
cells.
Local stresses independent of adhesion can polarize MII
in Dictyostelium cells that are pulled into micropipettes by
aspiration (Ren et al., 2009). Hematopoietic cells were likewise
aspirated at low stress (<1 kPa) after transfection of GFPMIIA
or MIIB, and within just 20 min, MIIB polarized by more
than 10-fold into the stressed projection (Figure 2D), while
MIIA polarized much less. Importantly, receptors such as
integrins do not engage the micropipette wall, and so polarization
can indeed be independent of adhesion. Partial knockdown
of MIIB in CD34+
cells followed by aspiration also showed
greater distension of the membrane as well as membrane
fragmentation (Figure 2E), and knockdown cells also showed
an $20% decrease in migration through a 3 mm pore filter (Figure
S2D). Functionally, MIIB polarization in CD34+
cells is thus
protective of membrane shape changes produced by cell
forces.
Asymmetric Division Is Biophysically Regulated by MIIB
Large cortical tensions are generated in cells as they round up
and divide during asymmetric division (Sedzinski et al., 2011).
Because MYH10 correlates with a half-dozen genes involved
in asymmetric division of hematopoietic cells (Ting et al., 2012)
(Table S3), confocal imaging and partial knockdown (Figure 3Ai)
were used to assess MIIB in asymmetric division of CD34+
cells
(Figure 3Aii), which occurs in $30% of cells (consistent with
Lordier et al., 2012). MIIB enriches toward the CD34hi
daughter
cell, concentrating near the cleavage furrow by $3-fold (Figure
3B), whereas CD34 appears segregated between cells but
otherwise locally homogenous, consistent with lateral mobility
of this membrane protein. The results suggest that high cortical
tensions in the cleavage furrow have a similar effect on receptorindependent
localization of MIIB as that of local stressing by a
micropipette.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc. 83
Partial knockdown of MIIB abolishes the asymmetry and also
the segregation of CD34 (Figure 3B, bottom). Whereas asymmetric
division of CD34+
cells results in 6-fold higher MIIB in
the CD34+
daughter than in the CD34À
daughter, knockdown
decreases the MIIB level in CD34+
to that in CD34À
and suppresses
asymmetric division (Figure 3C). Prolonged cultures of
MIIB knockdown cells increase the relative number of CD34+
progenitors with more colony forming unit-granulocyte and
macrophage (CFU-GM) (Figure 3D), consistent with MIIB
regulating asymmetric division when late CD34+
progenitor
cells transition to CD34À
cells and when CD34 molecularly
segregates between daughter cells. Tracking of division
using carboxyfluorescein diacetate succinimidyl ester (CFSE)
(Hawkins et al., 2007) shows that partial knockdown of MIIB
increases the number of CD34+
CD38À
cells by 2-fold (Figure 3Ei)
or for CD34+
CD38+
cells by 1.5-fold (Figure 3Eii), whereas
CD34À
numbers remain unaltered (Figure 3Eiii). On the other
hand, partial MIIA knockdown ($30%) did not alter numbers of
any subpopulations (Figure S2E). The data reveal MIIB as a major
factor in asymmetric division and differentiation of CD34+
cells to
CD34À
cells.
MIIA Dephosphorylates in Differentiation
to a Mechanically Active State
MIIA is often the dominant MII isoform and can influence MIIB
(Raab et al., 2012), which implies that phosphoregulation of
MIIA can in principle influence hematopoiesis (Figure 4A). We
therefore examined regulation by niche factors of pS1943 in
CD34+
cells and found the highest levels of pS1943 in uncultured
CD34+
cells (Figure 4Bi; Figure S1D), with levels systematically
decreased upon differentiation with Thrombopoietin (Tpo) and
Granulocyte-Colony Stimulating Factor (G-CSF), but not Stem
Cell Factor (SCF) alone (Figure 4Bii). Transforming Growth Factor-b
(TGF-b) promotes HSC hibernation (Yamazaki et al.,
2011) and blocks the decrease in pS1943 with cytokines (Figure
4B). Remarkably, Blebb mimics TGF-b (Figure 4B). In
contrast, very low pS1943 in myeloid CD33+
cells (Figure S1D)
and in the human monocytic cell line (THP-1) are consistent
with rapid proliferation of THP-1 in suspension (which can be
blocked by Blebb). Both CD34+
and CD34À
cell numbers anticorrelate
with the level of pS1943, and the half-maximal effect
was observed at $35% pS1943-MIIA (Figure 4B). MIIA is thus
modulated by cytokines critical to precirculation differentiation.
A
B
C
D
E
Figure 2. MIIB Is Polarized in Motile CD34+
Cells and Localizes to Where Cells Are
Stressed
(A) MIIB is more membrane-localized than MIIA in
CD34+
cellsbeforeandafterserum-freeculture,but
not in CD34À
cells. Bar = 5 mm. For the left and right
panels, protein intensity was measured across
the cell diameter (representative images and diameters
are shown). For the middle panel, cortical
intensity was measured clockwise (arrow) from
the front of the cell origin (‘‘0’’). ‘‘Front’’ is the summed
intensity over contour in the first and fourth
quadrants($50%ofthetotalcontour),while‘‘Rear’’
is thesummed intensityoverthe remainingcontour.
(B) Measurement of in situ marrow elasticity by
atomic force microscopy (AFM). Young’s modulus
Emarrow is obtained from indentation measurements
performed at different locations across
the exposed marrow samples. The force versus
indentation curves are fit by F = E d2
(2/p) (tan a)/
(1À n2
), where d is the indentation, n is the Poisson
ration (assumed to be 0.5), and a is the half
opening angle of the AFM tip (Sneddon, 1965).
From 88 measurements done on four mouse
tibia or femur samples, Emarrow = 0.32 ± 0.07 kPa.
(C) Arrows indicate direction of intensity measurement
as described for Figure 2A middle panel.
(D) Stress-induced localization of mCherry-MIIB
and less so GFP-MIIA are demonstrated in micropipette
aspiration of MEG01 cells. Yellow region
in cartoon represents MII accumulation and white
regions represent MIIlo
areas. Representative
images and intensity measurements across cell
midline are shown with the inset kinetics of MIIA
and MIIB intensities normalized by the intensity at
t = 0 min. (Pressure Dp = 1 kPa; bar = 10 mm).
(E) MIIB maintains cortical stability of CD34+
cells,
based on aspiration of cells with or without
knockdown with si-MIIB. (Dp = 1 kPa; bar = 5 mm).
For all image analyses, n R 10 each sample, two
donors, mean ± SEM, and *p < 0.05. See also
Figure S2.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
84 Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc.
Fresh CD34+
CD38À
are softer than CD34+
CD38+
(Figure 4Ci),
and consistent with the high pS1943-MIIA in CD34+
cells, cells
transfected with a site-specific MIIA phosphomimetic mutant
(S1943D) fragment more often (from a weak cortex) and also
divide more slowly compared to wild-type controls (Figures
4Cii and 4Ciii). These functional results all indicate that high
pS1943-MIIA impacts cell mechanics and limits cell division,
and hence, differentiation. Transcriptional profiles reveal perturbation
of pathways that regulate MIIA phosphorylation in CD34+
cells by Blebb (Table S4). Matrix mechanics therefore have an
understandable effect on pS1943-MIIA as well (Figure 4Di):
soft FN-coated gels (20 mg/ml) maximize pS1943 in CD34+
cells
treated with cytokines compared to stiff matrix, while CD34À
cells appear unaffected (Figure 4Dii). This response to stiff matrix
is blocked with the phosphomimetic, deactivating S1943D-MIIA
(Figure 4Diii). Increased cell spreading as part of matrix engagement
on stiff substrate thus requires MIIB in CD34+
cells (Figure
2C), whereas differentiated cells use nonphosphorylated
MIIA. At the same FN density as above, the number of CD34+
CD38À
is 4-fold higher on soft matrix relative to stiff matrix (but
Blebb eliminates the difference), whereas the number of
CD34+
CD38+
remains constant (Figure 4E). CD34+
CD38À
cells
are thus sensitive to matrix elasticity, with sensitivity modulated
by MII.
In Vivo Roles in HSC/Ps: MIIB Contributes to
Differentiation, whereas MIIA Confers Survival
Based on our in vitro results, a major knockdown of MIIB in human
cells grafted into BM should repress asymmetric division
and lead to (1) an accumulation of human cells in marrow
and (2) a suppression of circulating human blood cells. To test
this hypothesis, fresh human CD34+
BM cells were transduced
with shRNA-carrying lentivirus to knock down MIIB, which
was followed by puromycin selection of transduced cells. Cells
were injected directly into bone of NOD/SCID/IL-2RgÀ/À
(NSG)
mice to study BM retention both at 16 hr and at 20 weeks
(Figure 5Ai); this same duration has been described by others
(Notta et al., 2011) as providing ‘‘a stringent test of long-term
A
B
D
E
C
Figure 3. MIIB Polarizes in and Promotes
Asymmetric Division of CD34+
to Differentiated
Cells
(A) Partial knockdown of MIIB decreases protein
by $40% in CD34+
CD38À
and CD34+
CD38+
. (Ai)
Mean Fluorescence Intensity (MFI) of MIIB protein
was measured by flow cytometry. (Aii) Hypothetical
model for asymmetric segregation of CD34 and
MIIB in division shown for WT versus MIIB
knockdown.
(B) MIIB segregates asymmetrically in dividing
CD34+
-derived cells unless MIIB is knocked down
(top). Bar = 5 mm. Intensities of MIIB and CD34
were measured (bottom) along the membrane
contour of dividing cells from ‘‘0’’ through the
cleavage furrow to the antipole, with distance
normalized by total length. A green arrow indicates
the difference in MIIB intensity between periphery
and cleavage furrow.
(C) MIIB membrane intensity bifurcates to CD34hi
and CD34lo
daughters in dividing cell pairs (Ci),
unless MIIB is knocked down. Percentage of
asymmetrically dividing CD34+
cells is suppressed
with MIIB siRNA versus control (Cii). Greater than
forty cells per group.
(D) Colony forming assays after 3 days in methylcellulose
medium supplemented with cytokines.
(Di) A relationship among different progenitors in
terms of CD34 and MIIB expression. (Dii) CFU-GM
increases with MIIB knockdown.
(E) Absolute CD34+
cell numbers expand after
MIIB knockdown, with normalization to an initial
total of 10,000 cells (left), and CFSE tracking
shows an increase in mean division number per
time (right). Slopes for si-MIIB are as follows:
control, (0.02: 0.015) for CD34+
CD38À
(Ei)
and CD34+
CD38+
(Eii), and (0.02: 0.02) for CD34À
(Eiii). For all graphs, *p < 0.05 between Control
( = Scrambled) versus MIIB siRNA for each data
point, mean ± SEM, and n R 3 donors.
See also Figure S2 and Table S3.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc. 85
repopulation’’ of human xenografts injected into the femurs of
NSG mice for which ‘‘HSCs were operationally defined by lymphomyeloid
engraftment that persisted for at least 20 weeks
after transplant.’’ Standards for mouse HSCs differ from those
of human (Doulatov et al., 2012), but 12–16 weeks is currently
considered as ‘‘long-term engraftment’’ (Oguro et al., 2013).
We injected directly into marrow rather than into blood to avoid
any potential effect of knockdown on trafficking from blood to
marrow. Human cells in mice were identified by dual immunostaining
for hCD45 and hCD47 (Figure S3A), since human
RBC and platelets do not express hCD45 while hCD47 confers
immunocompatability to all human cells within NSG mice
(Rodriguez et al., 2013; Takenaka et al., 2007). Partial permanent
MIIB knockdown (by $40%) (Figure 5Aii) leads to 3-fold
greater retention of cells in marrow when they are assayed
just 16 hr after marrow injection (Figure 5Bi); this could reflect
the fact that knockdown impairs migration through constraining
micropores by 20% (p < 0.05; Figure S2D). Despite this initial
retention advantage, the percentage of human peripheral blood
(PB) cells in circulation at 6 weeks after transplantation is 7-fold
lower for the MIIB knockdown cells (Figure 5Bii), and the difference
is maintained after 20 weeks (Figures 5Ci and 5Cii).
Sustained engraftment is evident in control mice with significant
human cell numbers in marrow and four of five mice showing
human cells in PB. In contrast, MIIB knockdown cells were
6-fold more abundant in marrow, but only one of five mice
had human cells in circulation. MIIB is thus required to generate
PB cells.
Because MIIA is the dominant isoform in hematopoietic cells
and is phosphoregulated distinctly in marrow cells versus PB
cells, we characterized MIIA contributions to hematopoiesis
by performing competitive transplants of BM from tamoxifeninducible
cre-Myh9 knockout mice. These conditional knockout
cells (with surface marker CD45.2) were mixed 1:1 with cells from
A
B D
C E
Figure 4. Phosphorylation of MIIA Regulates
the Biophysics of CD34+
Differen-
tiation
(A) Dephosphorylation of MIIA at S1943 promotes
assembly and function.
(B) CD34+
differentiation with soluble factors
decreases pS1943. (Bi) Representative images
showing pS1943 expression in fresh CD34+
cells.
Bar = 5 mm. (Bii) pS1943 (normalized to MIIA) was
measured by flow cytometry [n = 3 donors, ± SEM;
fit to Log Y = aX + b (a,b): (CD34+
: À0.05, 5.74; in
red), (CD34À
: À0.10, 6.74; in green)]. Minimum
pS1943 per MIIA was measured for THP-1 cells
(0.07 ± 0.01). All cells were treated with SCF and
indicated cytokines. pS1943% values were
normalized as described in Figure S1D (n = 3, ±
SEM). pS1943 percentage for SCF only = $40%.
(C) MIIA S1943D phosphomimetic decreases
both cortical stiffness and cytoskeletal stability.
Aspiration length L, normalized by pipette radius,
Rp (L/Rp), versus time for various cells with
(slope, intercept, effective viscosity h) were as
follows: (Ci) CD34+
CD38À
(5.7/min, 8.3, 3.2 Pa/s)
and CD34+
CD38+
(2.2/min, 4.7, 8.5 Pa/s); (Cii)
MIIA-WT: (0.02/min, 0.5, 1,400 Pa/s), MIIAS1943D
(0.70/min, 1.6, 40 Pa/s). n = 5, ±SEM. The
inset bar graph in (Cii) shows the fraction of
transfected COS-1 cells after MIIB knockdown
that undergo cell division (2n and 4n cells) as
calculated by subtracting the fraction of polyploid
cells (n = 3, ±SEM, *p < 0.05). (Ciii) Representative
images of aspiration of transfected COS-1 cells
(bar = 10 mm).
(D) CD34+
CD38À
cells sense matrix elasticity with
changes in pS1943-MIIA similar to cytokines (Di).
(Dii) Soft matrix maintains high pS1943 in CD34+
.
*p < 0.05 (three donors, ±SEM). (Diii) pS1943
limits matrix sensing: cell area was normalized to
DNA to correct for ploidy of COS-1. *p < 0.05 for
GFP-S1943D 34 kPa versus GFP or GFP-MIIA
34 kPa (n R 20, ±SEM).
(E) CD34+
numbers on soft matrix (0.3 kPa)
scaled by stiff matrix (34 kPa) increase with
fibronectin (FN) density unless MII is inhibited.
For CD34+
CD38À
, EC50 $22.4 mg/ml, *p < 0.05
(n R 3, ±SEM).
See also Table S4.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
86 Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc.
wild-type mice (CD45.1) and injected into sublethally irradiated
recipient mice (CD45.1) (Figure 5Di). This knockout strategy
with mouse cells instead of human cells proved necessary for
understanding MIIA because our in vitro results for proliferation
indicated no effect with partial knockdown of MIIA in contrast
to major defects with MIIB partial knockdown (Figure 3A). At
8 weeks after transplantation of the mixed cells, the total percentage
of donor and competitor blood cells was $50% each,
and upon tamoxifen treatment, MIIA decreased as expected
only in CD45.2 donor cells (Figure 5Dii). In PB, donor myeloid
cells decreased rapidly compared to lymphoid cells (t1/2 =
$30–40 hr versus $20–25 days) (Figure 5E), but these half-lives
are within 2-fold of those reported for both myeloid (Basu et al.,
2002; van Furth and Cohn, 1968) and lymphoid (Fulcher and Basten,
1997; Sprent and Basten, 1973) lineages in mouse blood.
MIIA loss therefore does not greatly affect viability of terminally
differentiated lymphoid cells, while blood cell production from
progenitors is clearly suppressed. Consistent with this, we find
in BM that LinÀ
Sca-1+
c-Kit+
(LSK) CD150+
cells (which include
progenitors or HSC/Ps; Kiel et al., 2005) are reduced 10-fold at
just 8 weeks after Myh9 deletion (16 weeks since transplant),
with similar results for LSK in spleen and LS in blood (Figures
5Fi, 5Fii, and 5Fiii, respectively). MIIA is thus required for sustained
engraftment in vivo and hematopoiesis. An early
apoptotic fraction (Annexin-V+
and 7-AADÀ
) of the LSK population
also increased just 3 days after Myh9 deletion (Figure 5G),
although the total LSK number remained unchanged at this
time point (Figure 5F). Irreversible loss of MIIA therefore suppresses
differentiated cell numbers in the long term as defective
HSC/Ps progressively apoptose.
A
D
F G
E
B
C
Figure 5. MII Isoforms Regulate Hematopoiesis
In Vivo
(A) Scheme for in vivo experiments to test MIIB
functions (Ai). (Aii) MIIB expression kinetics with
shRNA knockdown relative to control. t1/2 = 81 hr.
n = 5 mice for each group (±SEM for all graphs),
transplanted via an intratibial route with 5 3 103
BM CD34+
cells per sublethally irradiated mouse.
Transplantation occurred 4 days after lentiviral
transduction, with 2 days puromycin selection.
(B) MIIB knockdown increases short-term (16 hr)
retention in bone marrow (BM) (Bi), but decreases
short-term (6 week) generation of peripheral blood
(PB) (Bii). *p < 0.01 control (scrambled) versus MIIB
shRNA.
(C) MIIB knockdown increases long-term
(20 weeks) BM engraftment (Ci), but suppresses
PB generation (Cii). For PB generation, the number
of positively engrafted mice is shown (R0.1% total
nucleated cells).
(D) Scheme for in vivo experiment to test MIIA
functions (Di). BM cells from Cre:Myh9loxP/loxP
(CD45.2) and from WT competitor (CD45.1) were
transplanted at a 1:1 ratio into lethally irradiated
WT recipients. (Dii) Eight weeks after reconstitution,
mice were treated with tamoxifen to delete
Myh9 as assayed by protein expression (*p < 0.01,
control versus tamoxifen, n R 3, ±SEM for all
graphs). Deletion occurred with t1/2 = 9.6 days. n R
8 mice for each group from two independent experiments
(±SEM for all graphs).
(E) PB lineages with deleted Myh9 are lost from
circulation with kinetics similar to clearance of WT
cells. The donor (top) and competitor (bottom)derived
granulocyte (Gran, Gr-1+
Mac-1+
, larger
side scatter), monocyte (Mono, Gr-1+
Mac-1+
,
smaller side scatter), T cell (T, CD3+
), and B cell (B,
B220+
) lineages in PB were quantified at the indicated
time points after vehicle (left) or tamoxifen
(right) treatment. Decay half-lives for tamoxifentreated
donor Gran, Mono, T, and B are 1.3, 1.7,
24.2, and 18.8 days, respectively
(F) Myh9 deletion decreases HSC/P subpopulations
across different hematopoietic organs in
the long term (8 weeks), but not in the short term
(3 days). Donor and competitor HSC/P cells were
quantified in BM (LSKCD150+
, Fi), spleen (LSK, Fii),
and PB (LS, Fiii) (control versus treated, *p < 0.01).
(G) Myh9 deletion increases apoptosis of LSK.
Treatment was for 3 days (*p < 0.01).
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc. 87
Transient Inhibition of MII with Blebbistatin Spares
Only Long-Term Multilineage Reconstituting Cells
Blebbistatin is a reversible inhibitor of all MII isoforms, and
dose-response studies of CD34+
cultures show that it has a surprising
but understandable effect: the diploid ‘‘HSC-enriched’’
population (as phenotypically defined per Majeti et al., 2007
and Novershtern et al., 2011) proves relatively stable to a 3day
treatment, which is long relative to the cell cycle, while the
Blebb-treated MPP and CPP are depleted by 1.8-fold (±0.5)
and 31-fold (±16), respectively. By suppressing only the progenitors
and sparing the HSC-enriched population, the net effect is
an enrichment of the latter among total CD34+
cells by up to 16fold
(Figure 6Ai). Whole-genome transcript profiles indeed show
that Blebb cultures correlate well with fresh HSC-enriched cells
and MPP, but not CPP (Figure 6Aii, Table S4, and Table S5),
whereas control CD34+
cultures correlate with fresh CPPs.
Blebb treatment beyond 3 days showed a progressive decrease
in the HSC-enriched population, consistent with the conditional
knockout studies above that suggest that MIIA is essential for
hematopoiesis in vivo (Figures 5D–5F).
Functional tests of HSC enrichment by Blebb were conducted
after washing out the drug and involved measuring the frequency
of human cells in NSG mice after limiting dilution serial transplantations
into multiple primary and secondary recipients (Figure
6B). A total duration of 32 weeks in primary plus secondary
xenografts was chosen as sufficient to assess long-term
A
C D
B Figure 6. MII Inhibition Maintains HSCEnriched
Population with Long-Term
Multilineage Reconstitution Potential
(A) Scheme for in vitro experiments (top). (Ai)
Representative flow cytometry contour plots for
CD34+
subpopulations, with dose-dependence of
2n cells showing 15.6- ± 4.1-fold enrichment at
20 mM Blebb. Absolute cell numbers were scaled
to 104
initial cells and fit to dose-response curves:
CPP and MPP IC50 = 10.5 mM; HSC-enriched
numbers = 646 ± 77 (n R 5 donors, ±SEM). These
IC50 values are within $2-fold of the inhibition
constant Ki for pure MII (Kova´ cs et al., 2004). (Aii)
Blebb-treated CD34+
cells show a gene expression
profile similar to fresh CD34+
CD38À
for
hematopoietic genes (Table S4 and Table S5).
Values are derived from two experiments.
(B) Limiting dilution serial transplant analyses
show functional HSCs after myosin inhibition
after 16 weeks (long-term). (Top) Scheme for
in vivo experiments. (Bi) Limiting dilution primary
transplant. The number of transplanted CD34+
cells versus the percentage of unsuccessful
engraftment determines the frequency of repopulating
cells (n = 26 recipients per group from
three independent experiments; p < 0.0005). (Bii)
Secondary transplantation of BM from primary
transplant demonstrates the maintenance of
higher HSC frequency with Blebb compared
to control (n R 13 recipients per group, p < 0.01)
(See Figure S3B). Transplantation with Blebbexposed
CD34+
-derived cells shows similar
multilineage engraftment in the NSG mice
compared to control cells, including myeloid
(CD33+
), lymphoid (CD19+
) (Biii) (±SEM), and
erythroid (GPA+
) (Biv). Bar = 5 mm.
(C) Kinetics of human-CD41+
platelets in circulation
were measured after transplantation of
human CD34+
-derived cells and normalized by
the initial number of CD41+
cells transplanted.
Areas under curves show significant differences
between drug-treated and control. p < 0.05 in
both phase I and phase II from at least nine recipients
in three experiments (±SEM).
(D) Effects of Blebb on progenitors. (Di) Enrichment
of polyploid MKs by Blebb (n = 4). y axis represents
the ratios between polyploid MKs and 2n + 4n MKs. EC50 = 7.5 mM; Hill coefficient = 7.0. (Dii) Enrichment of BFU-E relative to CFU-GM in the absence of Epo,
evaluated by colony forming assays. The maximum ratio was observed at 12.5 mM. IC50 = 10 mM, Hillslope = 5.0 (n = 3, ±SEM). (Diii) Sensitivity of erythroid
progenitors to Blebb in the presence of Epo. BFU-E = CD34+
IL-3R+
CD36À
; CFU-E = CD34À
IL-3RÀ
CD36+
. Absolute values were normalized to 104
initial cell
input and fit to dose-response curves. IC50, Hill coefficient for CFU-E, 2n: 8.7 mM, À4.4 and 4n: 12.9 mM, À6.3; BFU-E, 2n: 10.9 mM, À9.7, 4n: 13 mM, 28, and
Poly R 8n: 0.2 mM, 2.0. (n = 2, ±SEM).
See also Figure S3, Table S4, and Table S5.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
88 Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc.
multilineage engraftment of human HSCs in NSG mice (Notta
et al., 2011). Our blood analyses 16 weeks after primary transplantation
showed that positive engraftment required fewer
CD34+
cells ($1 in 10,000) from Blebb-treated cultures
compared to control cultures (Figure 6Bi; Figure S3B). If longterm
multilineage engraftment were due solely to progenitors
(such as MPPs), then the fact that Blebb-treated cultures have
relatively fewer progenitors (Figure 6A) would have required
that more (not fewer) Blebb-treated CD34+
cells be injected for
reconstitution. Both treated and control cultures also showed a
similar percentage of human CD34+
CD38À
and CD34+
CD38+
populations in BM after transplantation (Figure S3D), indicative
of engraftment, and Blebb results also compare well to uncultured
CD34+
cells in previous studies (Nishino et al., 2011). Sustained
secondary engraftment provides an assay for cells with
appropriate stem cell properties (Doulatov et al., 2012; Notta
et al., 2011; Oguro et al., 2013) and our secondary transplantation
results show that Blebb maintains a higher fraction of the
HSC-enriched population compared to untreated cultures ($5fold
once again). Both treated and control human CD34+
transplants
produced a similar percentage of multilineage myeloid
and lymphoid cells (Figure 6Biii) and a minor fraction of enucleated
human RBCs in the NSG mice (Figure 6Biv). The latter
were enriched by flowing blood through a microfluidic channel
coated with anti-hCD47 and then staining for the erythroid-specific
marker hGPA (Figure S3C).
MKs are unique among blood cells in being naturally polyploid
and become more so in vitro with Blebb treatment, which also
increases in vitro proplatelet formation (Shin et al., 2011). In the
NSG mice, human platelets (CD41+
) showed two phases of
circulation up to 2 weeks after transplantation that also
seemed to benefit from Blebb treatments (Figure 6C). In phase
I, human platelets are released into the circulation almost
immediately and reach an initial peak between $1–20 hr, consistent
with intravenously infused MKs (Figure S3E) (Fuentes
et al., 2010). Phase II peaks at $20–90 hr and reflects a successful
lodging of MKs in the marrow. Importantly, human cells
treated with Blebb generate more human platelets per transplanted
CD41+
cell by about 4-fold in both phases, and shear
forces appear to be important in regulating the size of human
platelets derived from MKs (Figure S3F). Blebb indeed enriches
for mature polyploid MKs in culture by $10 fold (Figure 6Di).
For other lineages, the sensitivity of individual progenitor lineages
to Blebb proves cytokine dependent (Figure 5A; Figure
S3G). For SCF and Tpo CD34+
-derived cells, the IC50 for
CFU-GM is lower than that of BFU-E, with Blebb producing up
to a 2-fold higher ratio of BFU-E to CFU-GM (Figure 6Dii).
Erythroid lineages are thus preserved under non-Epo and
submaximal MII inhibition. In contrast, when cells are cultured
with Epo, both CFU-E and BFU-E numbers are reduced
(Figure 6Diii). Functional studies thus reveal that short-term
reversible MII inhibition in combination with specific cytokines
enriches for HSCs, mature MKs, and even erythroid progenitors.
Sustained Blebbistatin Induces Apoptosis of
Dividing CD34+
Cells via Aryl Hydrocarbon
Receptors and p53 Pathways
Control cultures show that CPP and MPP progenitors undergo
2- to 3-fold more divisions as expected compared to the
phenotypically marked HSC-enriched population based on
CFSE tracking (Figure 7A), whereas Blebb-treated cells (20 mM)
do not divide. Decay rates of CFSE for CD34+
subpopulations
in either the presence or the absence of G-CSF (Figures S4A
and S4B) indicate that Blebb accelerates decay, consistent
with enhancing cell death. With the phenotypical HSC-enriched
population, this is likely due to inhibition of cytokinesis of 4n
cells trying to divide at the point drug was added. Apoptosis
was measured by cleaved caspase-3 and increased 2-fold
with Blebb (Figure 7B; Figures S4C–S4F), although G-CSF
modulates survival (Figure 7B; Figure S4B). Suppression of
MPP and CPP cell numbers with Blebb is therefore explained
by cytokinesis-associated death, which seems consistent with
similar roles for MII in apoptosis in C. elegans (Ou et al., 2010).
Transcription profiles of viable fractions from drug-treated
and control cells implicate intersecting pathways that involve
the aryl hydrocarbon receptor (AHR) and p53 (Table S4). AHR
antagonists counteract apoptosis (Vaziri and Faller, 1997) and
expand human HSC/Ps in culture (Boitano et al., 2010) (Figure
S5A). The antagonist CH-223191 improves viability of both
control CD34+
and CD34À
cells, specifically the cycling cells
(4n) (Figure 7B). The number of apoptotic cells with Blebb
treatment is also systematically decreased by the more potent
StemRegenin-1 (SR1) (Figure 7C). Both CH-223191 and SR1
rescue CD34+
cells from cell death by Blebb, since cell numbers
approximate control conditions (Figure 7D; Figure S5B).
Total p53 protein was then assayed in the presence of Blebb
and/or SR1. Total p53 protein increased $2 fold with Blebb
but reversed by SR1 (Figure 7E). Cyclic-pifithrin-a tests whether
induction of apoptosis is dependent on p53-mediated transcription
activity (Zuco and Zunino, 2008). The phenotypic
‘‘HSC-enriched’’ population is increased $2-fold with cyclicpifithrin-a
in both control and Blebb-treated cells (Figure 7F)
with an EC50 close to previous reports (30 nM) (Pietrancosta
et al., 2005). AHR and p53 are thus implicated in Blebb-induced
apoptosis.
DISCUSSION
Asymmetric division provides a means to maintain stemness
while generating the many differentiated cells required for a tissue
with high turnoversuch as blood (105
nucleated cells/s). However,
it has been unclear as to how two interconnected daughter cells
physically sort components to become distinct. While asymmetry
of stem cell division in C. elegans is driven by its one isoform of MII
(Ou et al., 2010), the mammalian homolog, MIIA, is expressed in
many cells other than stem cells and unlike MIIB, MIIA polarizes
very weakly if at all (Vicente-Manzanares et al., 2008; Raab et
al., 2012). Compared to any other hematopoietic lineage, CD34+
cells express the most MIIB relative to MIIA. MIIB polarizes
strongly to regions of high cell tension or curvature where it can
physically break the symmetry of cytokinesis (Sedzinski et al.,
2011). It is therefore almost predictable that MIIB in CD34+
cells
will polarize near a cleavage furrow and define the MIIBhi
daughter
cell in asymmetric division (Figure 3). Since MIIB is localized near
the membrane and is known to link to membrane proteins (Clark
et al., 2006), MIIBhi
could also help sort cell surface proteins
such as CD34 and thereby correlate with CD34hi
as seen. Depletion
of MIIB from the CD34lo
daughter cell is also propagated as a
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc. 89
key aspect of the MII isoform switch that defines and delineates
hematopoiesis (Figure 7G).
How MYH10 is ultimately repressed in differentiation requires
further study: RUNX1 downregulates MYH10 during MK differentiation
(Lordier et al., 2012), but RUNX1 does not anticorrelate in
general with MYH10 and is not required for normal functions
once HSCs are formed from vascular endothelial cells during
embryonic development (Chen et al., 2009). Nevertheless, asymmetric
processes are hinted at by a number of polarizable proteins
in our early CD34+
cells, including Cdc42, which polarizes
in correlation with HSC aging (Florian et al., 2012). Generic polarization
of a protein in hematopoietic cells seems predictive of a
role in asymmetric division (Beckmann et al., 2007), but MIIB’s
role in physically breaking the symmetry of cytokinesis seems
unique and motivates deeper study of biophysical factors that
feed back into transcription programs and perhaps even regulate
cancer stem cell differentiation (Cicalese et al., 2009).
Human HSC/Ps expand when cultured on flexible tropoelastin
matrices plus serum and cytokines (Holst et al., 2010) and also
expand when cultured serum-free on endothelial cells that
secrete cytokines (Butler et al., 2012). However, endothelial cells
and secreted matrix on plastic could be locally soft or regionally
rigid. On FN-coated hydrogels that are marrow-mimetic soft
or else endosteal-like stiff, minimal cytokines in serum-free
media can for some conditions enrich for early CD34+
cells,
consistent with our finding that both soft gels and blebbistatin
suppress MIIB polarization and enhance pS1943-MIIA in early
CD34+
cells.
Blood Cells that Lack Myosin-II Become Polyploid
or Die Trying
Motile cells that are sufficiently adherent can generate enough
traction forces to pull themselves apart even in the absence of
MII, whereas cells in suspension or daughter cells that cannot
A B C
D
G
E F
Figure 7. Inhibition of MII Blocks Division
and Activates AHR-Dependent Apoptosis
(A) The mean division numbers for each HSPC
subpopulation were calculated by fitting Gaussians
to CFSE data, and Blebb blocks division
(n R 3 donors, ±SEM).
(B) Increased apoptosis by sustained MII inhibition.
CD34+
-derived cells cultured in SCF and Tpo
were treated with Blebb for 3 days and fixed,
followed by intracellular flow cytometry with the
anti-(cleaved caspase-3) and 7-AAD for DNA
(n = 3, ±SEM, p < 0.05 for all except CD34À
4n).
(C) SR1 decreases the absolute number of
apoptotic cells generated by Blebb treatment.
Nucleated (Hoechst+
) Annexin-V+
7-AADÀ
cells
were quantified by flow cytometry calibrated by
APC-beads. Absolute values were normalized
to control (vehicle-treated cells). IC50 = $380–
1,200 nM, Hill coefficient = 0.62. *p < 0.05
Blebb +0 nM versus +750 nM SR1 (n = 3, ±SEM).
(D) Phenotypic HSC-enriched population is
maximized by synergy between myosin inhibition
and AHR antagonism. CD34+
-derived cells in SCF
and Tpo were treated with different doses of the
selective AHR antagonist SR1, with or without
20 mM Blebb for 3 days. Absolute values were
normalized to 104
initial cell input and fit to
dose-response curves. EC50 and maximum cell
number for HSC-enriched, Control: 15.5 nM, 4000,
and Blebb: 10 nM, 1800; MPP, Control: 15.5 nM,
2200, and Blebb: 15.5nM, 1,100; CPP, Control:
41.5 nM, 7,500, and Blebb: 4.7 nM, 2,400. Hill coefficient
for all the graphs is $1–2. n R 3 (±SEM).
(E) SR1 reverses upregulation of p53 protein by
Blebb. Total p53 protein was quantified by intracellular
flow cytometry. *ANOVA p < 0.05, Tukey’s HSD
Test p < 0.05 for DMSO versus Blebb (n = 3, ±SEM).
(F) Pharmacological inhibition of p53 transcription
by cyclic-pifithrin-a increases the absolute cell
number of phenotypic HSC-enriched. EC50 =
63.1 nM for both control and Blebb, Hill coefficient
= 2.6, 1.8 for control and Blebb, respectively
(n = 3, ±SEM).
(G) Summary of results for biological functions of
MII in adult hematopoiesis with perturbations of MII
pathways.
See also Figure S4, Figure S5, and Table S4.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
90 Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc.
crawl away with sufficient force (to break the intercellular bridge)
tend to become polyploid (Zang et al., 1997). HSC/Ps grow well
as suspension cells that do not adhere and spread strongly on
substrates compared to other solid tissue cell types, and they
only possess a thin cortical cytoskeleton; cytokinesis defects
are thus likely to favor polyploidy in these cell types. In a blood
cancer line that only expresses MIIA, partial knockdown of
MIIA indeed increases polyploidy in vitro as does blebbistatin
and the cancer cells survive (Shin et al., 2011). In healthy human
and mouse primary cells, however, such a process of endomitosis
is usually seen only for MKs (among blood cells at least),
which implies that other cell types are either never tetraploid or
apoptose if they become so. Irreversible ablation in vivo of
MIIA in primary blood cells indeed enhances apoptosis and
depletes most dividing blood cell types (Figures 5D–5G). This
seems consistent with cell death in blebbistatin treatments
being downstream of MII inhibition. Although the specificity of
this drug has been questioned (Shu et al., 2005), the reversible
3-day treatment here with blebbistatin of primary CD34+ cells
in vitro increases ploidy of viable MKs and enhances apoptosis
of progenitors with slower dividing stem/progenitor cells dying
only with more sustained drug treatments. Our pharmacological
results with AHR inhibitors provide some functional evidence
of a transcriptome-implicated link between a failure of these
normal primary, nonadherent cells to divide and AHR upstream
of p53 in apoptosis (Figure 7G, right), but since AHR is primarily
nuclear, any interaction with MII is likely to be indirect.
Translation
Mouse knockouts of MIIB are embryonic lethal (Ma et al., 2010),
and since MIIA is at least weakly polarizable (Figure 2D), any
deficiencies or mutations in MIIB might be partially compensated
by MIIA. Inhibiting both isoforms transiently, as shown here,
might be exploited to further maintain and perhaps expand
HSCs and maybe other stem cells in suitably designed microenvironments.
Importantly, given the successful long-term
engraftment of drug treated cells, genes that are truly essential
for hematopoiesis (profile in Figure 5Aii) might be clearly identified.
Enrichment of highly adherent stem cells by the methods
here might require optimization of adhesion to both suppress
motility and provide sufficient anchorage signals for viability.
Our findings ultimately reveal not only a biophysical hierarchy
of actomyosin forces in adult hematopoiesis but also some utility
in controlling those forces to enrich for stem cells.
EXPERIMENTAL PROCEDURES
MS-IF Cytometry
For intracellular flow cytometry, cells were fixed with 4% paraformaldehyde in
PBS for 10 min, washed with PBS, and resuspended in 0.1% saponin in HBSS.
The samples were then stained with antibodies against MIIA and MIIB for
30 min at room temperature, along with hematopoietic surface markers and
Hoechst 33342, followed by secondary antibody staining conjugated with
Alexa 488 and 647 (Invitrogen). The samples were analyzed on an LSR II
(BD) to obtain the mean fluorescent intensity (MFI) values of MIIB and MIIA
across different samples and subpopulations. Each value was normalized
by a standard cell line (MFI from COS cells) to correct for differences in
fluorescence intensities caused by laser fluctuations. Normalized MFI values
from flow cytometry were then calibrated by MS results for MSCs (Raab
et al., 2012) that served as a standard to calculate the stoichiometric ratio between
MIIB and MIIA of each sample.
Standard methods can be found in Supplemental Experimental Procedures
for MS, microarray, gene correlation analysis, cell culture, confocal microscopy,
micropipette analysis, construction of matrix-coated gels, and
xenotransplantation, among other techniques.
Statistical Analyses
All statistical analyses were performed using GraphPad Prism 5. Unless
otherwise noted, all statistical comparisons were made by unpaired two-tailed
Student t test and were considered significant if p < 0.05. All dose-response
data were fitted to sigmoidal dose-response with variable slope with x axis
in a log scale.
SUPPLEMENTAL INFORMATION
Supplemental Information for this article includes Supplemental Experimental
Procedures, five figures, and five tables and can be found with this article
online at http://dx.doi.org/10.1016/j.stem.2013.10.009.
AUTHOR CONTRIBUTIONS
J.W.S. and D.E.D. designed research; J.W.S., A.B., K.R.S., D.A.C., I.L.I., and
F.R. performed research; J.S. and P.C.D. contributed to new analytic tools;
C.L. and C.G. engineered, and C.A.H. supplied, the MYH9 floxed mutant
mice; and J.W.S., K.R.S., J.S., J.A.C., and D.E.D. wrote the paper.
ACKNOWLEDGMENTS
We thank Dr. Robert Adelstein, Dr. Mary Anne Conti (NIH-NHLBI), and
Dr. Leonard Zon (Harvard) for invaluable comments. We gratefully acknowledge
Arielle Glatman Zaretsky for technical assistance in mouse BM transplantation
and the Stem Cell Xenograft Core at the University of Pennsylvania,
A. Secreto, J. Glover, and Dr. G. Danet-Desnoyers for cells and engraftment
studies. This study was supported by the National Institutes of Health
(P01DK032094; R01HL062352; R01-EB007049; P30-DK090969; NCATS-
8UL1TR000003), the Human Frontier Science Program (I.I. and D.E.D.), the
National Science Foundation (D.E.D.), the Nano Science and Engineering
Center-Nano Bio Interface Center (D.E.D.), and the American Heart
Association (J.-W.S.).
Received: July 29, 2012
Revised: July 31, 2013
Accepted: October 17, 2013
Published: November 21, 2013
REFERENCES
Basu, S., Hodgson, G., Katz, M., and Dunn, A.R. (2002). Evaluation of role of
G-CSF in the production, survival, and release of neutrophils from bone
marrow into circulation. Blood 100, 854–861.
Beckmann, J., Scheitza, S., Wernet, P., Fischer, J.C., and Giebel, B. (2007).
Asymmetric cell division within the human hematopoietic stem and progenitor
cell compartment: identification of asymmetrically segregating proteins. Blood
109, 5494–5501.
Boitano, A.E., Wang, J., Romeo, R., Bouchez, L.C., Parker, A.E., Sutton, S.E.,
Walker, J.R., Flaveny, C.A., Perdew, G.H., Denison, M.S., et al. (2010). Aryl
hydrocarbon receptor antagonists promote the expansion of human hematopoietic
stem cells. Science 329, 1345–1348.
Butler, J.M., Gars, E.J., James, D.J., Nolan, D.J., Scandura, J.M., and
Rafii, S. (2012). Development of a vascular niche platform for expansion
of repopulating human cord blood stem and progenitor cells. Blood 120,
1344–1347.
Canman, J.C., Cameron, L.A., Maddox, P.S., Straight, A., Tirnauer, J.S.,
Mitchison, T.J., Fang, G., Kapoor, T.M., and Salmon, E.D. (2003).
Determining the position of the cell division plane. Nature 424, 1074–1078.
Chen, M.J., Yokomizo, T., Zeigler, B.M., Dzierzak, E., and Speck, N.A. (2009).
Runx1 is required for the endothelial to haematopoietic cell transition but
not thereafter. Nature 457, 887–891.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc. 91
Chen, G., Hou, Z., Gulbranson, D.R., and Thomson, J.A. (2010). Actin-myosin
contractility is responsible for the reduced viability of dissociated human
embryonic stem cells. Cell Stem Cell 7, 240–248.
Cicalese, A., Bonizzi, G., Pasi, C.E., Faretta, M., Ronzoni, S., Giulini, B.,
Brisken, C., Minucci, S., Di Fiore, P.P., and Pelicci, P.G. (2009). The tumor
suppressor p53 regulates polarity of self-renewing divisions in mammary
stem cells. Cell 138, 1083–1095.
Clark, K., Langeslag, M., van Leeuwen, B., Ran, L., Ryazanov, A.G., Figdor,
C.G., Moolenaar, W.H., Jalink, K., and van Leeuwen, F.N. (2006). TRPM7, a
novel regulator of actomyosin contractility and cell adhesion. EMBO J. 25,
290–301.
Conti, M.A., Even-Ram, S., Liu, C., Yamada, K.M., and Adelstein, R.S. (2004).
Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle
myosin heavy chain II-A in mice. J. Biol. Chem. 279, 41263–41266.
Doulatov, S., Notta, F., Laurenti, E., and Dick, J.E. (2012). Hematopoiesis:
a human perspective. Cell Stem Cell 10, 120–136.
Dulyaninova, N.G., House, R.P., Betapudi, V., and Bresnick, A.R. (2007).
Myosin-IIA heavy-chain phosphorylation regulates the motility of MDA-MB-
231 carcinoma cells. Mol. Biol. Cell 18, 3144–3155.
Engler, A.J., Sen, S., Sweeney, H.L., and Discher, D.E. (2006). Matrix elasticity
directs stem cell lineage specification. Cell 126, 677–689.
Florian, M.C., Do¨ rr, K., Niebel, A., Daria, D., Schrezenmeier, H., Rojewski, M.,
Filippi, M.D., Hasenberg, A., Gunzer, M., Scharffetter-Kochanek, K., et al.
(2012). Cdc42 activity regulates hematopoietic stem cell aging and rejuvenation.
Cell Stem Cell 10, 520–530.
Fuentes, R., Wang, Y., Hirsch, J., Wang, C., Rauova, L., Worthen, G.S.,
Kowalska, M.A., and Poncz, M. (2010). Infusion of mature megakaryocytes
into mice yields functional platelets. J. Clin. Invest. 120, 3917–3922.
Fulcher, D.A., and Basten, A. (1997). B cell life span: a review. Immunol. Cell
Biol. 75, 446–455.
Giebel, B., Corbeil, D., Beckmann, J., Ho¨ hn, J., Freund, D., Giesen, K., Fischer,
J., Ko¨ gler, G., and Wernet, P. (2004). Segregation of lipid raft markers
including CD133 in polarized human hematopoietic stem and progenitor cells.
Blood 104, 2332–2338.
Hawkins, E.D., Hommel, M., Turner, M.L., Battye, F.L., Markham, J.F., and
Hodgkin, P.D. (2007). Measuring lymphocyte proliferation, survival and
differentiation using CFSE time-series data. Nat. Protoc. 2, 2057–2067.
Holst, J., Watson, S., Lord, M.S., Eamegdool, S.S., Bax, D.V., Nivison-Smith,
L.B., Kondyurin, A., Ma, L., Oberhauser, A.F., Weiss, A.S., and Rasko, J.E.
(2010). Substrate elasticity provides mechanical signals for the expansion of
hemopoietic stem and progenitor cells. Nat. Biotechnol. 28, 1123–1128.
Kiel, M.J., Yilmaz, O.H., Iwashita, T., Yilmaz, O.H., Terhorst, C., and Morrison,
S.J. (2005). SLAM family receptors distinguish hematopoietic stem and progenitor
cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121.
Knoblich, J.A. (2010). Asymmetric cell division: recent developments and
their implications for tumour biology. Nat. Rev. Mol. Cell Biol. 11, 849–860.
Kova´ cs, M., To´ th, J., Hete´ nyi, C., Ma´ lna´ si-Csizmadia, A., and Sellers, J.R.
(2004). Mechanism of blebbistatin inhibition of myosin II. J. Biol. Chem. 279,
35557–35563.
Lichtman, M.A. (1970). Cellular deformability during maturation of the myeloblast.
Possible role in marrow egress. N. Engl. J. Med. 283, 943–948.
Lordier, L., Bluteau, D., Jalil, A., Legrand, C., Pan, J., Rameau, P., Jouni, D.,
Bluteau, O., Mercher, T., Leon, C., et al. (2012). RUNX1-induced silencing
of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization.
Nat Commun 3, 717.
Ma, X., Jana, S.S., Conti, M.A., Kawamoto, S., Claycomb, W.C., and Adelstein,
R.S. (2010). Ablation of nonmuscle myosin II-B and II-C reveals a role for
nonmuscle myosin II in cardiac myocyte karyokinesis. Mol. Biol. Cell 21,
3952–3962.
Majeti, R., Park, C.Y., and Weissman, I.L. (2007). Identification of a hierarchy
of multipotent hematopoietic progenitors in human cord blood. Cell Stem
Cell 1, 635–645.
Maupin, P., Phillips, C.L., Adelstein, R.S., and Pollard, T.D. (1994). Differential
localization of myosin-II isozymes in human cultured cells and blood cells.
J. Cell Sci. 107, 3077–3090.
Merkel, R., Simson, R., Simson, D.A., Hohenadl, M., Boulbitch, A., Wallraff, E.,
and Sackmann, E. (2000). A micromechanic study of cell polarity and plasma
membrane cell body coupling in Dictyostelium. Biophys. J. 79, 707–719.
Nilsson, S.K., Debatis, M.E., Dooner, M.S., Madri, J.A., Quesenberry, P.J., and
Becker, P.S. (1998). Immunofluorescence characterization of key extracellular
matrix proteins in murine bone marrow in situ. J. Histochem. Cytochem. 46,
371–377.
Nishino, T., Wang, C., Mochizuki-Kashio, M., Osawa, M., Nakauchi, H., and
Iwama, A. (2011). Ex vivo expansion of human hematopoietic stem cells by
garcinol, a potent inhibitor of histone acetyltransferase. PLoS ONE 6, e24298.
Notta, F., Doulatov, S., Laurenti, E., Poeppl, A., Jurisica, I., and Dick, J.E.
(2011). Isolation of single human hematopoietic stem cells capable of longterm
multilineage engraftment. Science 333, 218–221.
Novershtern, N., Subramanian, A., Lawton, L.N., Mak, R.H., Haining, W.N.,
McConkey, M.E., Habib, N., Yosef, N., Chang, C.Y., Shay, T., et al. (2011).
Densely interconnected transcriptional circuits control cell states in human
hematopoiesis. Cell 144, 296–309.
Oguro, H., Ding, L., and Morrison, S.J. (2013). SLAM family markers resolve
functionally distinct subpopulations of hematopoietic stem cells and multipotent
progenitors. Cell Stem Cell 13, 102–116.
Ou, G., Stuurman, N., D’Ambrosio, M., and Vale, R.D. (2010). Polarized myosin
produces unequal-size daughters during asymmetric cell division. Science
330, 677–680.
Pietrancosta, N., Maina, F., Dono, R., Moumen, A., Garino, C., Laras, Y.,
Burlet, S., Que´ le´ ver, G., and Kraus, J.L. (2005). Novel cyclized Pifithrin-alpha
p53 inactivators: synthesis and biological studies. Bioorg. Med. Chem. Lett.
15, 1561–1564.
Raab, M., Swift, J., Dingal, P.C., Shah, P., Shin, J.W., and Discher, D.E. (2012).
Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates
myosin-II heavy chain. J. Cell Biol. 199, 669–683.
Ren, Y., Effler, J.C., Norstrom, M., Luo, T., Firtel, R.A., Iglesias, P.A., Rock,
R.S., and Robinson, D.N. (2009). Mechanosensing through cooperative interactions
between myosin II and the actin crosslinker cortexillin I. Curr. Biol. 19,
1421–1428.
Rodriguez, P.L., Harada, T., Christian, D.A., Pantano, D.A., Tsai, R.K., and
Discher, D.E. (2013). Minimal ‘‘Self’’ peptides that inhibit phagocytic clearance
and enhance delivery of nanoparticles. Science 339, 971–975.
Sedzinski, J., Biro, M., Oswald, A., Tinevez, J.Y., Salbreux, G., and Paluch, E.
(2011). Polar actomyosin contractility destabilizes the position of the cytokinetic
furrow. Nature 476, 462–466.
Shin, J.-W., Swift, J., Spinler, K.R., and Discher, D.E. (2011). Myosin-II inhibition
and soft 2D matrix maximize multi-nucleation and cellular projections
typical of platelet-producing megakaryocytes. Proc. Nat. Acad. Sci. USA
108, 11458–11463.
Shu, S., Liu, X., and Korn, E.D. (2005). Blebbistatin and blebbistatin-inactivated
myosin II inhibit myosin II-independent processes in Dictyostelium.
Proc. Natl. Acad. Sci. USA 102, 1472–1477.
Sneddon, I.N. (1965). The relation between load and penetration in the
axisymmetric boussinesq problem for a punch of arbitary profile. Int. J. Eng.
Sci. 3, 47–57.
Sprent, J., and Basten, A. (1973). Circulating T and B lymphocytes of the
mouse. II. Lifespan. Cell. Immunol. 7, 40–59.
Takenaka, K., Prasolava, T.K., Wang, J.C., Mortin-Toth, S.M., Khalouei, S.,
Gan, O.I., Dick, J.E., and Danska, J.S. (2007). Polymorphism in Sirpa modulates
engraftment of human hematopoietic stem cells. Nat. Immunol. 8,
1313–1323.
Ting, S.B., Deneault, E., Hope, K., Cellot, S., Chagraoui, J., Mayotte, N., Dorn,
J.F., Laverdure, J.P., Harvey, M., Hawkins, E.D., et al. (2012). Asymmetric
segregation and self-renewal of hematopoietic stem and progenitor cells
with endocytic Ap2a2. Blood 119, 2510–2522.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
92 Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc.
Trumpp, A., Essers, M., and Wilson, A. (2010). Awakening dormant haematopoietic
stem cells. Nat. Rev. Immunol. 10, 201–209.
Ubukawa, K., Guo, Y.M., Takahashi, M., Hirokawa, M., Michishita, Y., Nara,
M., Tagawa, H., Takahashi, N., Komatsuda, A., Nunomura, W., et al. (2012).
Enucleation of human erythroblasts involves non-muscle myosin IIB. Blood
119, 1036–1044.
van Furth, R., and Cohn, Z.A. (1968). The origin and kinetics of mononuclear
phagocytes. J. Exp. Med. 128, 415–435.
Vaziri, C., and Faller, D.V. (1997). A benzo[a]pyrene-induced cell cycle
checkpoint resulting in p53-independent G1 arrest in 3T3 fibroblasts. J. Biol.
Chem. 272, 2762–2769.
Vicente-Manzanares, M., Koach, M.A., Whitmore, L., Lamers, M.L., and
Horwitz, A.F. (2008). Segregation and activation of myosin IIB creates a rear
in migrating cells. J. Cell Biol. 183, 543–554.
Wang, F., Kovacs, M., Hu, A., Limouze, J., Harvey, E.V., and Sellers, J.R.
(2003). Kinetic mechanism of non-muscle myosin IIB: functional adaptations
for tension generation and maintenance. J. Biol. Chem. 278, 27439–27448.
Yamazaki, S., Ema, H., Karlsson, G., Yamaguchi, T., Miyoshi, H., Shioda, S.,
Taketo, M.M., Karlsson, S., Iwama, A., and Nakauchi, H. (2011).
Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation
in the bone marrow niche. Cell 147, 1146–1158.
Zang, J.H., Cavet, G., Sabry, J.H., Wagner, P., Moores, S.L., and Spudich,
J.A. (1997). On the role of myosin-II in cytokinesis: division of
Dictyostelium cells under adhesive and nonadhesive conditions. Mol. Biol.
Cell 8, 2617–2629.
Zuco, V., and Zunino, F. (2008). Cyclic pifithrin-alpha sensitizes wild type p53
tumor cells to antimicrotubule agent-induced apoptosis. Neoplasia 10,
587–596.
Cell Stem Cell
Myosin-II Regulates Adult Hematopoiesis
Cell Stem Cell 14, 81–93, January 2, 2014 ª2014 Elsevier Inc. 93