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HEMATOPOIESIS
Symmetry of Initial Cell Divisions Among Primitive Hematopoietic Progenitors
Is Independent of Ontogenic Age and Regulatory Molecules
By Shiang Huang, Ping Law, Karl Francis, Bernhard O. Palsson, and Anthony D. Ho
We have developed a time-lapse camera system to follow
the replication history and the fate of hematopoietic stem
cells (HSC) at a single-cell level. Combined with single-cell
culture, we correlated the early replication behavior with
colony development after 14 days. The membrane dye
PKH26 was used to monitor cell division. In addition to
multiple, synchronous, and symmetric divisions, singlesorted CD341/CD382 cells derived from fetal liver (FLV) also
gave rise to a daughter cell that remained quiescent for up to
8 days, whereas the other daughter cell proliferated exponentially. Upon separation and replating as single cells onto
medium containing a cytokine cocktail, 60.6% 6 9.8% of the
initially quiescent cells (PKH26 bright) gave rise again to
colonies and 15.8% 6 7.8% to blast colonies that could be
replated. We have then determined the effects of various
regulatory molecules on symmetry of initial cell divisions.
After single-cell sorting, the CD341/CD382 cells derived from
FLV were exposed to flt3-ligand, thrombopoietin, stem cell
factor (SCF), or medium containing a cytokine cocktail (with
SCF, interleukin-3, interleukin-6, granulocyte-macrophage
colony-stimulating factor, and erythropoietin). Whereas mitotic rate, colony efficiency, and asymmetric divisions could
be altered using various regulatory molecules, the asymmetric division index, defined as the number of asymmetric
divisions versus the number of dividing cells, was not altered
significantly. This observation suggests that, although lineage commitment and cell proliferation can be skewed by
extrinsic signaling, symmetry of early divisions is probably
under the control of intrinsic factors.
r 1999 by The American Society of Hematology.
H
mother cells. (2) In the peripheral nervous system, a sensory
organ precursor (SOP) responsible for forming external sensory
organs (ie, sensory bristles) generates an organ by dividing
asymmetrically to form precursor cells to produce 2 outer
support cells (a hair and a socket cell), a sensory neuron, and a
sheath cell.6 (3) In the central nervous system, MP2 precursors
are a pair of embryonic neural precursors that divides only once
to produce 2 different postmitotic neurons.
By analyzing the colony-forming potentials of individual
daughter cells in the hematopoietic system, Leary et al8,9 have
demonstrated that approximately 10% of the cell divisions of
multipotent progenitors are asymmetric. Mayani et al10 also
described asymmetry of cell division of hematopoietic progenitors. Recently, Brummendorf et al,11 from the same group,
reported a linkage between cell division rate and asymmetric
cell divisions. They showed that the proliferative potential and
cell cycle properties were unevenly distributed among daughter
cells derived from single-sorted HSC from fetal liver (FLV) and
that expansion potential is associated with asymmetric division.
According to this evidence asymmetric cell divisions seemed to
occur in 3% to 20% of the cultured cells and lineage commitment did not seem to be influenced by cytokines. Based on all
these observations, it has been suggested that stem cell differentiation is a stochastic event.
EMATOPOIETIC STEM CELLS (HSC) are characterized by the dual abilities to self-renew and to differentiate into progenitors of all the mature blood cell lineages. These
2 features are evident after bone marrow transplantation and
require that the HSC undergo rounds of asymmetric divisions to
generate mature cells of the distinct blood lineages as well as
cells to sustain long-term hematopoiesis.1 The 2 daughter cells
from a HSC may be initially equivalent, but subsequent cell
divisions must result in different fates of the progeny cells.2 It
has been suggested that the hallmark of a stem cell might be its
ability to divide asymmetrically to produce a daughter cell
identical to the mother and another cell committed to differentiation.3,4 Alternatively, a balance between symmetrical cell divisions that result in self-renewal versus that which result in
differentiation might be able to maintain the stem cell pool and
provide a source of multipotent progenitors. Even if the latter
were true, asymmetric division must have occurred during
ontogenesis of these 2 populations, and further asymmetric
divisions must occur during their multilineage differentiation.
A central question in developmental biology is how a single
cell can divide to produce 2 daughter cells that adopt distinct
fates.4 Theoretically, daughter cells with different fates can arise
by means of the following mechanisms. First, they may be
different from each other at the time of cell division, ie, due to
intrinsic factors. A parental factor, such as a transcription factor,
may be distributed unevenly to the daughter cells. Second, the
daughter cells may be similar at the time of cell division, but
become different upon subsequent exposure to environmental
signals, such as a cytokine, ie, due to extrinsic factors.2,4
Thus far, remarkably little is known if and how hematopoietic
stem cells divide in a self-renewing, asymmetric fashion.
Recently, studies of asymmetric division of neural stem cells in
Drosophila and mammals have provided exciting and new
insights into the mechanism of stem cell division and might
serve as a model for hematopoietic reconstitution.3,5-7 These
studies demonstrated that at least 3 types of asymmetric
divisions can be found in neural progenitors. (1) In Drosophila,
neuroblasts (NB) undergo a series of oriented asymmetric
divisions to renew themselves and produce smaller ganglion
Blood, Vol 94, No 8 (October 15), 1999: pp 2595-2604
From the Departments of Medicine and Bioengineering, University
of California, San Diego, CA; and the Department of Medicine V,
University of Heidelberg, Heidelberg, Germany.
Submitted December 1, 1998; accepted May 17, 1999.
Supported by National Institutes of Health Grants No. R01
DK49619-01 and U19 AI36612-01 and by the Pete Lopiccola Memorial
Foundation.
Address reprint requests to Anthony D. Ho, MD, Department of
Medicine V, Hospitalstr. 3, 69115 Heidelberg, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1999 by The American Society of Hematology.
0006-4971/99/9408-0037$3.00/0
2595
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2596
HUANG ET AL
The discoveries in neural stem cells have been made possible
by imaging studies of neuroblast divisions and development.4
Visualization of cell divisions with time-lapse camera systems
thus represents a powerful tool to study cell replication and
symmetry of division. In this study, we have monitored the
replication history of human candidate HSC with a time-lapse
camera system and demonstrated that asynchronous or asymmetric divisions of CD341 cells indeed occur. This asymmetry is
shown by different replication behavior of the 2 daughter cells.
One daughter cell remained quiescent, whereas the other
multiplied to yield hundreds to thousands of cells after 10 days.
Mitotic activity as well as asymmetric divisions decrease with
ontogenic age, ie, are more frequent among CD341/CD382
cells derived from FLV than those from adult bone marrow,
whereas the fraction of asymmetric divisions stays unchanged.
Monitoring of asymmetrical divisions represents a measurable
and reproducible measure of candidate HSC populations.
MATERIALS AND METHODS
Human hematopoietic progenitor cell preparations. FLV samples
were obtained from legal abortions at 17 to 24 weeks of gestational age
and were supplied by Advanced Bioscience Resources, Inc (Alameda,
CA). FLV cells were prepared by homogenizing the tissue through a
Cell Strainer (Becton Dickinson Labware, Lincoln Park, NJ) and were
washed once in RPMI 1640 containing 5% fetal calf serum (FCS;
Germini, Calabasas, CA). Umbilical cord blood (UCB) specimens were
collected at the University of California, San Diego delivery room as
well as supplied by Advanced Bioscience Resources Inc, using heparin
(Marsam, Cherry Hill, NJ) as anticoagulant. Healthy subjects were
recruited for adult bone marrow samples (ABM). Approximately 40 to
50 mL of bone marrow was drawn from multiple sites (10 to 15 mL each
time) from the posterior iliac crest. Subjects gave informed consent to
donate marrow for research. Low density mononuclear cells (MNC)
from FLV, UCB, and ABM were obtained by Ficoll-Hypaque (Histopaque 1077; Sigma Chemical Co, St Louis, MO) separation and
washing. All projects involving human subjects and use of human
tissues have been reviewed and approved by the Human Subjects
Committee of the University of California, San Diego.
Labeling with PKH-26. The procedure for staining cells using
PKH-26 (in a kit from Sigma) was provided by the manufacturer.
Briefly, CD341 cells (from different hematopoietic tissues) were washed
using PRMI 1640 medium without serum at room temperature and resuspended in 1 mL of Diluent C as supplied in the kit. Then, 1 mL of Diluent C
containing 8 3 106 molar PKH-26 dye was mixed with the cells and
incubated at room temperature for 5 minutes. The staining reaction was
halted by adding 2 mL of phosphate-buffered saline (PBS) containing 1%
FCS and incubating for 1 additional minute. The cells were washed with 4
mL of 10% FCS/RPMI 1640 medium. It has been demonstrated that the
PKH dye binds tightly to the lipid layer of cell membrane and is distributed
equally between the daughter cells after each division.12,13 Light and
phase contrast microscopy in preliminary experiments did not show
differences in the morphology of the cells after staining.
Flow cytometry and index sorting. The hematopoietic progenitor
cell preparations marked with PKH-26 were stained with CD34
(HPCA-2 fluorescein isothiocyanate [FITC]; Becton Dickinson Immunocytometry Systems [BDIS], San Jose, CA) and CD38 (Cy-chrome;
Pharmingen, San Diego, CA). Cells were stained with monoclonal
antibodies for 30 minutes on ice and washed 23 with 2% FCS/RPMI
1640 medium. Flow cytometric sorting was performed on a FACStarPlus
equipped with an Argon ion laser tuned at 488 nm. Single cells of
CD341/CD382 subsets were deposited singly onto a 72-well Terasaki
plate (Robinson Scientific, Sunnyvale, CA) with the use of an Automated Cell Deposition Unit (ACDU) and Index Sorting Device
according to preset sort-gates. Data acquisition was performed using
Lysys II (BDIS) software.13 The pulse processor module index sorting
device permitted the linkage of list-mode data of each cell to the
location of the well in the microtiter dish. Three-color, 5-parameter
multidimensional analysis was performed on a FACScan using PAINTA-GATEPLUS software (BDIS). This program allows the log-transformation of side scatter that permits easier delineation of different hematopoietic cell populations.
Single-cell suspension culture technique. In the single-cell suspension culture system,13,14 each well contained a mixture of myeloid
long-term culture medium (Stem Cell Technology, Vancouver, British
Columbia, Canada) containing 12.5% horse serum, 12.5% FCS, 1024
mol/L 2-mercaptoethanol, 2 mmol/L L-glutamine, 0.2 mmol/L Iinositol, 20 mol/L folic acid, and antibiotics and supplemented with 2.5
U/mL recombinant human erythropoietin (Epo; Amgen, Thousand
Oaks, CA), 10 ng/mL recombinant human interleukin-3 (IL-3), 500
U/mL recombinant human (rh) IL-6, 10 ng/mL recombinant human
granulocyte-macrophage colony-stimulating factor (GM-CSF), 2.5
ng/mL recombinant human basic fibroblast growth factor (bFGF), 10
ng/mL recombinant human insulin-like growth factor-1 (IGF-1; Collaborative Research, Bedford, MA), and 50 ng/mL recombinant human stem
cell factor (SCF; Genzyme, Boston, MA). This combination of cytokines is referred to as ‘‘cocktail’’ in subsequent experiments. For
96-well microtiter plates, the cells were cultured in 200 µL, and for the
72-well Terasaki plates, the culture volume is 20 µL. All cultures were
incubated in 5% CO2 in air at 37°C in a fully humidified incubator. Cell
growth was scored for the presence of dispersed cells, cell clusters, or a
mixture of both on days 10 to 14. Cells were scored as dispersed cells
when an expansion of a minimum of 40 cells was generated that
appeared as dispersed round translucent cells after 10 to 14 days of
culture. Others grew into clusters of typically erythroid colonies or
myeloid colonies and were scored according to the typical morphology.
Other single cells grew into a mixture of clusters among dispersed blast
cells.
With our single-cell suspension culture technique, we were able to
define precisely the colony efficiency (CE), the growth pattern, and the
replating potential of each phenotype of CD341 cells.15 CE is defined as
the percentage of wells each initially containing 1 single cell that
developed into colonies after 10 to 14 days. Blast colony efficiency
(BE) is defined as the percentage of colonies that showed dispersed
(blast colonies) and mixed growth pattern (blasts with clusters in
between) that have the potential to give rise to further colonies when
replated in our suspension culture system.14,15
Experiments were also performed to test the influence of serum-free
medium (QB60; kindly provided by Dr Ronald Brown, Quality
Biologics, Gaithersburg, MD) on kinetics and symmetry of cell
division, as well as on colony formation, as compared with the
above-described myeloid long-term culture medium. In subsequent
experiments, the impact of various regulatory molecules on symmetry
of cell division was also determined by the addition of specific
cytokines instead of the above-described cocktail. The concentrations of
regulatory molecules used were as follows: 50 ng/mL SCF, 100 ng/mL
thrombopoietin (TPO; kindly provided by Amgen, Thousand Oaks,
CA), and 100 ng/mL Flt3-ligand (FL3-L; kindly provided by Dr
Douglas Williams, Immunex Corp, Seattle, WA).
Time-lapse camera system. Time-lapse measurements of cells in
multiple microscope fields over long periods of time (hours to days)
require instrumentation that can operate in an automatic manner.
Briefly, images were acquired using an inverted fluorescent microscope
(Nikon Diaphot 300; Nikon Inc, Melville, NY) with a 43 objective
such that an entire well of a Terasaki plate can be observed in a single
image (1,938 3 1,523 µm) field of view. Illumination was provided by a
100-W Mercury arc lamp that passed through a 41003 filter set (Chroma
Technologies, Brattleboro, VT). Digitized images were acquired and
stored on a SGI O2 workstation (Silicon Graphics, Mountain View, CA).
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SYMMETRY OF STEM CELL DIVISION
A motorized X, Y, Z stage (Ludl, Hawthorn, NY) moved the stage
between wells so that multiple images could be rapidly collected. All
acquisition and processing functions were controlled by the Isee
software (Inovision Corp, Durham, NC), which allowed for the analysis
of multiples from the list to create a composite image that showed
changes in cell shape or position over time.16 Cells in a Terasaki plate
can be simultaneously tracked in a single experiment and revisited at
prescribed time intervals.
After the cells were deposited as single cells, the replication history
of HSC was monitored initially every 3 to 12 hours for 7 to 10 days. For
each experiment, the number of wells analyzed were 72 to 216. The
replication history of the HSC was measured using the PKH membrane
dyes (see above), which were available in both green (PKH2) and red
(PKH26) forms. These dyes consist of a fluorophore attached to an
aliphatic carbon backbone that binds irreversibly to the lipid bilayer.
With each cell division, the fluorescent intensity of the PKH dye is
2597
reduced by one half. Thus, one can determine, using the time-lapse
camera system, the replication history of the daughter cells. We
determined the kinetics of cell division (by measuring the doubling
times), whether both daughters divided symmetrically, and under
which conditions the cells underwent asymmetric division. Initially, we performed these experiments with CD341 cells derived from FLV. The
same plates were kept in culture for 10 to 14 days whenever possible. The
colony efficiency and growth patterns were determined at the end of this
period. The relationship between short-term kinetics and symmetry of
division and the outcome of long-term culture was studied.
Mitosis index is defined as the number of single-sorted cells that have
shown cell division after 8 days versus the total number of cells of the
same phenotype deposited. Asymmetric division is defined as the
number of cells that demonstrated at least 1 asymmetric division during
the course of 8 days versus the total number of cells deposited. The
asymmetric division index (ADI) is defined as the number of cells that
Fig 1. This figure demonstrates a typical symmetric division of one CD341/CD382 cell derived from FLV. We found that the first cell division
occurred consistently at 36 to 38 hours after culturing. Thereafter, the cell doubling time was 12 hours. Previous and subsequent experiments
with shorter observation intervals (eg, every 3 hours) have demonstrated that cells might migrate, partly due to movements of the culture plates
and partly due to the migratory property of the progenitor cells themselves.
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2598
HUANG ET AL
Fig 2. Divisional history of 1 single CD341/CD382 cell stained with
PKH26, derived from an FLV sample. At 0 hour, a cell showing bright
fluorescence is depicted. At 38 hours, the cell has divided, yielding 2
daughter cells with bright PHK26 fluorescence. At 98 hours, 1 cell has
maintained bright fluorescence among 32 cells with dim fluorescence. On day 8 (at 194 hours), 1 cell with bright fluorescence has
remained among several hundreds of other cells. The upper 4 pictures
were taken with fluorescence microscopy. The bottom picture shows
the same cell culture on day 8 in both phase contrast and fluorescence microscopy.
demonstrated at least 1 asymmetric division during the course of 8 days
divided by the number of dividing cells.
Statistical analysis. For statistical analysis, a personal computer
program, Testimate (supplied by IDV Daten analyse, Gauting-Munich,
Fig 3.
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SYMMETRY OF STEM CELL DIVISION
2599
Germany), was used. Data reported were given as the mean 6 standard
deviations or as the median and range, wherever applicable. The
Student’s t-test was applied to verify the differences in mitotic rate,
colony efficiency, percentage of asymmetric divisions, and ADI of
CD341/CD382 cells between 2 subgroups. The Kruskal-Wallis Analysis was applied to validate the differences in CD341/CD382 cells from
the MNC samples from various sources, eg, FLV, or UCB compared
with ABM. Wherever possible, the paired t-test was applied to verify the
difference between matched observations, eg, control and treated
preparations handled in parallel, or cells with symmetric divisions and
those with asymmetric divisions from the same sample.
RESULTS
In the first series of experiments, we have monitored the
replication history of hematopoietic progenitors that were
CD341. CD341 cells, without further fractionation derived
from FLV, UCB, or ABM, were used for the initial experiments.
The cells were stained in bulk with PKH26 for visualization in
fluorescence light, followed by resorting as single cells in
medium containing the above-described cytokine cocktail, and
were deposited onto a Terasaki plate. At least 72 wells and up to
216 wells were analyzed for each experiment. Preliminary
experiments using light and phase-contrast microscopy demonstrated that there was no significant difference in morphology
between cells before and after PKH26 staining or between cells
before and after the first divisions. Image analysis included
simultaneous assessment of cell number and fluorescence
intensity in each of 72 wells at defined intervals, eg, every 3 to
12 hours for 10 days. This enabled us to define precisely the
replication history of each single CD341 cell. We found that the
first division typically occurred at 36 to 38 hours after being
seeded. After the first division, the subsequent doubling times
were 12 hours, irrespective of the cell source, ie, from different
ontogenic ages. The majority (,65% to 75%) of the CD341
cells showed multiple synchronous symmetric divisions within
a single well. However, approximately 30% of the single-sorted
CD341 cells derived from FLV gave rise to a daughter cell that
remained quiescent for up to 8 days, whereas the other daughter
cell multiplied exponentially. Such asynchronous divisions
represented asymmetric divisions with respect to the replication
behavior of the 2 daughter cells.
We have then focused our studies on CD341/CD382 cells
derived from FLV, because our previous experiments indicated
that this subset contained significantly higher frequencies of
candidate stem cells with self-renewal capacity.14,15 Preliminary
experiments also demonstrated that 39.7% 6 10.3% (mean 6
SD) of CD341/CD382 cells derived from FLV underwent
asymmetric divisions and were consistently and significantly
higher than that of CD341/CD381 cells (30.7% 6 6.9%, n 5 5,
P 5 .0325, paired t-test).
Figure 1 demonstrates a typical symmetric division of 1
CD341/CD382 cell derived from FLV. We confirmed that the
first division of this cell type also occurred consistently at 36 to
38 hours after culturing; thereafter, the cell doubling time was
Fig 4. Percentages of asymmetric divisions found after the first,
second, third, and the fifth cell division. After the fifth cycle, it became
difficult to discern symmetry of divisions. Forty-two percent and 25%
of all asymmetric divisions occurred during the first and second
mitosis, respectively.
every 12 hours. The daughter cells continued to divide every 12
hours, giving rise to 4 cells at 48 to 50 hours, 8 cells at 60 to 62
hours, 16 cells at 72 to 74 hours, 32 cells with dim fluorescence
at 84 to 86 hours, and so forth.
Figure 2 demonstrates the divisional history of 1 single
CD341/CD382 cell that has divided asymmetrically, as monitored by time-lapse camera system over a period of 8 days. In
the first 36 to 38 hours, the image confirmed that 1 single cell
with very bright fluorescence was deposited in the well. After
36 to 38 hours, 2 cells with bright PKH26 fluorescence were
observed. Seventy-two to 74 hours after culturing, 1 bright cell
was observed among 8 other cells with dim fluorescence.
Whereas the 1 PKH26 bright cell maintained its fluorescence
intensity and remained quiescent, the other cells continued to
divide symmetrically to give rise to 16, 32, 64 cells, etc, every
12 hours, such that on day 8, the same bright cell was observed
among hundreds of fluorescence-negative cells, thus providing
evidence that asymmetric divisions occurred among CD341/
CD382 cells derived from FLV. Other CD341/CD382 cells
initially gave rise to 2 daughter cells that appeared equivalent
after the first mitosis, but then divided asymmetrically after the
second division. Figure 3 shows a typical example, ie, 1
parental cell gave rise to 2 daughter cells, which in turn gave
rise to 4 cells, with 1 of 4 cells then remaining quiescent,
whereas the other 3 multiplied symmetrically, giving rise to
altogether 7 (6 1 1) cells at 60 hours after culturing. We have
analyzed the percentages of asymmetric divisions found after
the first, second, third, and up to the fifth cell division. The
results of 4 experiments are summarized in Fig 4. Forty-two
percent and 25% of all the asymmetric divisions occurred
during the first and second mitosis, respectively. Asymmetric
;
Fig 3. Other CD341/CD382 cells initially gave rise to 2 daughter cells that appeared equivalent, but then divided asymmetrically after the
second or third division. This figure depicts a typical example, ie, 1 parent cell gave rise to 2 daughter cells (at 38 hours), which in turn gave rise to
4 cells, with 1 of them then remaining quiescent and the other 3 multiplying symmetrically in subsequent divisions, yielding 7 cells at 60 hours, ie,
on day 3. Thereafter, 1 cell maintained bright PKH26 fluorescence, whereas the other 6 multiplied to yield hundreds of cells after 9 days, which all
showed very dim to nondetectable fluorescence.
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2600
divisions are rarely found in the third (13%), fourth (13%), and
fifth (7%) waves of cellular divisions.
To correlate the replication behavior of the CD341/CD382
cells in the first 8 days with the growth pattern of the
corresponding single cell after culture, we have continued to
incubate the plates for 10 to 14 days, as previously described.15
Our hypothesis is that cells showing asymmetric divisions
would give rise to more blast colonies with dispersed and mixed
growth patterns, whereas cells showing symmetric divisions
gave rise to more clusters. Figure 5 depicts the results from 9
experiments. The median percentage of blast colonies (BC;
which included wells with dispersed and mixed growth pattern)
was 33.3% for cells showing asymmetric divisions and 29.4%
for cells showing symmetric divisions. The median percentage
of cluster colonies (CC) was 35.3% for cells showing asymmetric divisions and 43.5% for those with symmetric divisions.
Although the colony data varied widely among individual
samples, they remained fairly consistent for cells within the
same specimen. In 8 of the 9 experiments, the percentage of BC
was higher in cells showing asymmetric divisions (Asym) as
compared with those showing symmetric divisions (Sym).
Paired t-test also confirmed a decrease, albeit of marginal
significance, in BC in the cells with symmetric division, with
P 5 .0348.
Because our conventional culture system made use of FCS,
which might contain minute quantities of various regulatory
molecules that could induce differentiation or apoptosis and
hence affect symmetry of divisions, we have compared the use
of serum-free medium (QB-60) versus serum-containing medium. The results of 5 experiments are summarized in Table 1.
Whereas there was no significant difference in mitotic rate,
colony efficiency, asymmetric division, and ADI between cells
in sera-containing media and those without, cells cultured in
serum-free medium showed minimum interference by background fluorescence and were therefore used for subsequent
studies.
To test the viability of the PKH26 bright cells, we have, on
the one hand, applied propidium iodine to the wells with
quiescent cells at 3211 to 51211 cell stages (days 4 to 8). All of
the cells were examined in light and fluorescence microscopy
and 70.3% 6 4.7% of the PKH26 bright cells were shown to be
viable. To examine the functional integrity of the quiescent cells
with bright PKH26 fluorescence derived from asymmetrically
divided HSC, we separated the fluorescence bright cells from
PKH26 dim cells at 3211 to 6411 cells stage (after 96 to 108
hours). They were then replated as single cells in 96-well plates
with medium containing the cytokine cocktail. Culture of such
cells for an additional 10 to 14 days showed that 60.6% 6 9.8%
of the PKH26 bright cells (n 5 137 cells from 3 different
samples) gave rise to colonies, whereas only 15.9% 6 11.1% of
the PKH26 dim cells (n 5 121 cells from 3 different samples)
did so. A total of 15.8% 6 7.8% of the PKH26 bright cells gave
rise to colonies with dispersed growth pattern, which upon
replating gave rise to a third generation of colonies.13-15
Colonies with dispersed growth pattern were observed in
2.5% 6 2.5% of the PKH26 dim cells, none of which showed
replating potential.
After establishing that asymmetric divisions occurred among
CD341/CD382 cells, we have determined the percentages of
HUANG ET AL
asymmetric divisions among samples derived from different
ontogenic ages. CD341/CD382 cells derived from FLV, UCB,
or ABM were sorted, stained with PKH26, and deposited as
single-sorted cells. Divisions were monitored every 12 to 24
hours for up to 8 days. Mitotic rate, symmetry of the initial
divisions, colony efficiencies, and ADI were documented. The
data from at least 5 experiments (number of cells analyzed was
72 to 216 per measurement point) are summarized in Table 2.
Whereas the mitotic rate, colony efficiency, and percent of
asymmetric divisions all decreased with ontogenic age, ie, from
FLV, UCB, to ABM, the fraction of cells undergoing asymmetric division among dividing cells, ie, ADI, was consistently at
45%, irrespective of ontogenic age.
We have then compared the use of medium alone without
addition of any regulatory molecules versus our conventional
cytokine cocktail. In these series of 5 experiments, we found
that, with medium alone, the cells died after approximately 3 to
4 days and, hence, the mitotic rate, ADI, and colony efficiency
were low to nonmeasurable, with cell debris and background
fluorescence. We then determined the effects of various regulatory molecules on symmetry of initial cell divisions. After
single-cell sorting and deposition of CD341/CD382 cells
derived from FLV, the cells were exposed to regulatory
molecules such as FL3-L, TPO, rhSCF, or a combination of the
3 or to medium containing the cocktail. Cell divisions were
monitored every 6 to 12 hours for up to 8 days. The results
(mean 6 SD) from 6 experiments are summarized in Table 3
and Fig 6. The mitotic rate, colony efficiency, and cells
undergoing asymmetric divisions decreased significantly upon
exposure to FL3-L, TPO, or SCF as compared with the cocktail.
With the exception of FL3-L, which induced a marginal
decrease, ADI did not change significantly upon exposure to
different regulatory molecules or combinations thereof and has
remained at approximately 40%. This invariance was also
confirmed in 2 experiments using a combination of FL3L1TPO1SCF. The ADI was 43.9% and 45.7%, respectively.
On monitoring the replication history, we also observed that,
with FL3-L, TPO, or SCF, the time interval between exposure to
cytokines and first mitosis was 48 to 50 hours instead of 36 to 38
hours. Thus, although mitotic rate, cloning efficiency, divisional
kinetics, and asymmetric divisions could be altered when using
various regulatory molecules, the ADI was not altered significantly.
DISCUSSION
The fate of HSC during the first hours and days after
transplantation in vivo or after seeding onto culture plates in
vitro has been largely unknown. Conventional stem cell assays
have attempted to estimate their proliferative and differentiating
potential by making use of their ability to form colonies after
incubation for 14 days.17,18 Various modifications of long-term
cultures (eg, long-term culture initiating cells [LTC-IC]) based
on the use of stromal feeder layer derived from bone marrow
have been used to estimate repopulating potential of stem cells,
but such assays were not able to determine another dimension of
stem cell activity, which is self-renewal capacity.19-22 Recent
advances in the understanding of neural stem cell biology might
serve as a model for HSC development.3,4 Based on these
studies of neural stem cells, a fundamental property of stem cell
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SYMMETRY OF STEM CELL DIVISION
2601
Table 2. Changes in Mitosis, CE, Asymmetric Division, and ADI
of CD341/CD382 Cells With Ontogenic Age
FLV
UCB
ABM
Mitotic Rate
(%)
82.2 6 9.1
76.3 6 3.8
47.1 6 15.5†
CE (%)
Asymmetric
Division
(%)
ADI
56.8 6 17.2
30.9 6 7.3*
19.8 6 10.0‡
36.6 6 12.8
34.7 6 14.7
20.6 6 10.4*
45.6 6 17.9
45.7 6 17.2
44.6 6 19.7
The means 6 SD are provided.
*P # .05 compared with FLV.
†P , .001 compared with FLV.
‡P , .01 compared with FLV.
Fig 5. Correlation between replication behavior of the CD341/
CD382 cells in the first 4 days with the growth pattern of the
corresponding single cell after 14 days of culture. Wells containing
cells showing asymmetric divisions (Asym) were compared with
those showing only symmetric divisions (Sym) in their abilities to
form BC, reflected in dispersed and mixed growth patterns, and their
abilities to form CC after a total of 14 days of culture. The figure
summarizes the results of 9 experiments. The percentages of the cells
that gave rise to colonies versus those cells that have initially divided
are shown. Although the colony data varied widely among samples,
the paired t-test showed a significant decrease of BC among cells
showing symmetric divisions versus those showing asymmetric
divisions. The bars represent the corresponding medians.
development seems to be asymmetric division, during which
the generation of cell diversity requires daughter cells to adopt
different pathways. A central question in HSC biology is if and
how a single HSC can divide to produce 2 progeny cells that
adopt distinct fates.
To follow the precise replication history and the fate of HSC
at a single-cell level, we have applied a time-lapse camera
system to directly monitor early cell divisions. Combined with
index sorting and single-cell culture to measure the colony
formation of various CD341 subsets, we were able to correlate
the early replication behavior with colony development. The
following conclusions can be drawn from the use of this
technology. First, we have confirmed definitively that approximately 30% of the single-sorted CD341 cells derived from FLV
gave rise to a daughter cell that remained quiescent for up to 8
days, whereas the other daughter cell proliferated exponentially.
Such asynchronous divisions probably represented asymmetric
divisions and were found more frequently among CD341/
CD382 cells than in CD341/CD381 cells. These divisions could
be observed during the first and subsequent rounds of mitosis
among CD341/CD382 cells. Second, the percentage of such
Table 1. Impact of Medium Containing Serum Versus Serum-Free
Medium on Mitosis, CE, Asymmetric Division, and ADI of
CD341/CD382 Cells Derived From FLV
Medium
Mitotic
Rate
(%)
CE
(%)
Asymmetric
Divisions
(%)
ADI
Serum-free
78.7 6 8.4 47.1 6 13.8 31.3 6 14.0 39.5 6 16.0
Serum-containing 75.4 6 11.9 50.0 6 14.0 36.5 6 10.7 51.2 6 10.8
The means 6 SD are shown. Both media contained the cytokine
cocktail described in text (SCF, Epo, IL-3, IL-6, GM-CSF, bFGF, and
IGF-1).
asymmetric divisions decreased with ontogenic age, ie, higher
in CD341/CD382 cells derived from FLV than in those from
UCB or ABM. However, despite the fact that asymmetric
divisions, along with mitotic rate and colony efficiency, decreased significantly with ontogenic age, the ADI, ie, the ratio
of cells undergoing asymmetric divisions versus dividing cells,
remained constant at approximately 40%. Third, we have
demonstrated that cells showing asynchronous or asymmetric
divisions gave rise to more blast colonies than those showing
symmetric divisions, a phenomenon consistent with the observations by Young et al.23 Upon replating as single cells onto fresh
medium, 15.8% 6 7.8% of the PHK bright cells gave rise to
colonies with dispersed growth pattern and demonstrated replating potential, whereas none of the PKH dim cells had replating
potential. Fourth, whereas significant changes in mitotic rate,
colony formation, and asymmetric divisions were dependent on
exposure to regulatory molecules, the ADI remained unchanged
at approximately 40%. This interesting finding supports the
notion that growth factors are not essential for determining the
symmetry of divisions and hence the fate of the daughter cells.
Thus, commitment decision to self-renewal versus to differentiation is probably controlled by intrinsic programming and not by
regulatory molecules.24,25 In a series of experiments, Ogawa et
al have reported disparate differentiation in paired hematopoietic progenitors.8,9,26,27 Initially in a murine stem cell model,26,27
later confirmed in the human HSC,8,9 they demonstrated that
approximately 20% of the progenitors divided asymmetrically,
giving rise to different differentiation pathways from paired
daughter cells from a single progenitor. When mouse-derived
primitive progenitors were cultured individually, asymmetric
divisions took place in almost 20% of the cases and always
involved multipotent progenitors.26,27 Symmetric divisions involved both multipotent and monopotent progenitors and ocTable 3. Regulatory Molecules on the Mitotic Rate, CE, Asymmetric
Division, and ADI of CD341/CD382 Cells Derived From FLV
Regulatory
Molecule
FL3-L
TPO
SCF
Cocktail
Mitotic Rate
(%)
48.9 6 10.1*
58.2 6 4.4*
64.5 6 13.2‡
84.3 6 3.5
CE (%)
Asymmetric
Divisions
(%)
ADI
9.2 6 9.4†
8.8 6 0.9†
29.6 6 19.8†
68.2 6 14.4
16.9 6 7.1*
22.0 6 5.1‡
29.5 6 7.6‡
39.5 6 7.6
33.3 6 9.6‡
34.0 6 7.4
46.1 6 9.2
46.9 6 8.8
The results are shown as the mean 6 SD.
*P , .01 compared with cytokine cocktail.
†P , .001 compared with cytokine cocktail.
‡P # .05 compared with cytokine cocktail.
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2602
HUANG ET AL
Fig 6. Mitotic rates, asymmetric divisions, colony efficiencies, and ADI of CD341/CD382 candidate hematopoietic stem cells upon exposure to
regulatory molecules: recombinant human TPO, FL3-L, and SCF. Cytokine cocktail (containing Epo, IL-3, IL-6, GM-CSF, SCF, bFGF, and IGF-1) was
used for comparison.
curred in the rest. The same group obtained similar results in
studies of human HSC.8,9 Given this evidence, they suggested
that stem cell differentiation is a stochastic process. Mayani et
al10 described asymmetry of cell division of hematopoietic
progenitors. In their studies, individually sorted human cord
blood-derived primitive hematopoietic cells were allowed to
undergo 1 division, after which the 2 daughter cells were
physically separated and cultured in either the same or different
cytokine combinations. These investigators used cytokine combinations favoring erythropoiesis (mast cell growth factor
[MGF]1IL-61IL-31Epo) or myelopoiesis (MGF1IL-61fusion protein of IL-3 and GM-CSF1macrophage colonystimulating factor [M-CSF]1granulocyte-colony-stimulating
factor [G-CSF]) in the culture media. Asymmetric division was
defined as a division that yields 2 daughter cells with distinct
functional properties, ie, 1 of the daughter cells gave rise to
erythroid and the other to myeloid or mixed colonies, corresponding to asymmetric division of peripheral sensory organ progenitors described in neural stem cells.3 According to these investigators, asymmetric divisions occurred in 3% to 17% of the
cultured cells and lineage commitment did not seem to be
influenced by cytokines. The fundamental question of whether
asymmetric divisions of HSC with 1 cell remaining quiescent
and maintaining self-renewal capacity occur was not addressed
by these studies. With our present technology, we were able to
visualize the behavior of dividing CD341/CD382 cells during
the first rounds of cell divisions and to correlate this behavior
with their corresponding fate in further cell culture. The focus of
our study was on an earlier level in the hierarchy of HSC
development, corresponding to the asymmetric cell divisions of
primitive neuroblasts.3
Denkers et al28 recently described a similar time-lapse
recording system of human hematopoietic progenitors in culture. With this present technology, we were able to define
retrospectively cells that gave rise to asymmetric divisions, with
1 daughter cell that remained PKH26 bright and hence quiescent after mitosis and another that gave rise to multilineage
progenitors, as shown in the formation of typical erythroid and
myeloid clusters. More blast colonies could be derived from
single-sorted cells with a quiescent daughter cell, whereas more
clusters could be derived from those with symmetric divisions.
Using a similar technique to identify quiescent HSC, Young et
al23 also reported that cell production capacity was largely
attributed to cells exhibiting quiescent behavior, which is
consistent with our observations. When the PKH bright cells
were replated onto fresh medium containing cytokine cocktail,
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SYMMETRY OF STEM CELL DIVISION
each single-picked cell gave rise to colonies with dispersed and
cluster growth patterns. Under the present conditions, we have
not yet been able to establish that these cells would undergo
further rounds of asymmetric divisions and, hence, represent
precise replication of the mother cells.
Two mechanisms may be responsible for the adoption of
different fates by the daughter cells. (1) The intracellular or
intrinsic mechanism involves an inherited determinant that is
asymmetrically segregated into 1 daughter cell at the time of
division.4,6,7 (2) The extracellular or extrinsic mechanism may
result from communication of the daughter cells with each other
or with surrounding cells.4 Current research indicates a stereotypic mechanism for the asymmetric division of stem cells.
Evidence from neural stem cell research supports the idea that
asymmetric divisions are defined mostly by cell-autonomous
information, whereas extrinsic signal might also be involved
initially in instructing the asymmetric fates of daughter cells.
Our observation of a fairly consistent ADI, irrespective of
ontogenic age or of exposure to regulatory molecules, supports
this hypothesis. The results indicate that, although the pattern of
commitment can be skewed by extrinsic signaling, the proportion of asymmetric divisions is probably under the control of
intrinsic factors.
Using a different approach, Brummendorf et al11 also drew
similar conclusions from studies of single-sorted candidate
HSC from FLV. They reported that the results from culturing
and replating of hematopoietic progeny cells from single-sorted
HSC were indicative of asymmetric divisions in primitive
hematopoietic cells. The proliferative potential and cell cycle
properties were shown to be unevenly distributed among
daughter cells derived from single-sorted HSC from FLV.
Judging from the continuous generation of functional heterogeneity among clonal progeny of HSC, they suggested that
intrinsic control of stem cell fate is more likely than extrinsic.
Interestingly, Reddy et al29 also demonstrated that HSC from
mouse bone marrow took 36 to 40 hours to complete the first
division and then only 12 hours to complete each of 5
subsequent divisions. Our present study in human HSC derived
from FLV, UCB, or ABM confirmed this inertia of the first
mitosis and that each of the subsequent divisions took only 12
hours. The inertia for the first division might represent just an
artifact caused by the trauma to the cells due to the preparation
from the primary tissue, subsequent sorting, and staining
procedures. However, subsequent divisions then took 12 hours
in our culture conditions with the cytokine cocktail. Divisions
resulting in 1 quiescent daughter cell as well as symmetric
divisions resulting in equivalent daughter cells also occurred in
intervals of 12 hours. This was a surprising finding, because the
sorted population probably represented a relative heterogenous
mixture of cells. We have as yet no satisfactory explanation for
this phenomenon. Our preliminary experiments with ‘‘early’’
regulatory molecules showed a slight prolongation of the cell
doubling times. However, this observation requires further
confirmation and analysis.
Our technology will permit precise definition of cytokine and
cellular determinants of replication behavior of primitive progenitors. In continuation of the present project, we will define if
cellular determinants such as the number and type of stroma
cells will have an impact on symmetry of HSC divisions.
2603
Furthermore, intracellular determinants have been shown to
define the symmetry of division of neural stem cells. Recent
genetic analysis has identified several proteins that differentially
segregate during division and may be involved in determining
the asymmetry of the division. These important cell fate
determinants range from transcription factors (such as PROS)30
to modulations of cell-cell interactions (such as Numb and
Notch)6 and are asymmetrically localized during division of
neuroblasts.6,7,31-33 Simultaneously, mounting evidence indicates that transcription factors may play a key role in the
differentiation of hematopoietic progenitors.34 Our method may
enable us to correlate the expression of such factors and
symmetry of cell division and to define the differential expression of such factors in the daughter cells of a single CD341/
CD382 cell that adopt different fates.
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1999 94: 2595-2604
Symmetry of Initial Cell Divisions Among Primitive Hematopoietic
Progenitors Is Independent of Ontogenic Age and Regulatory Molecules
Shiang Huang, Ping Law, Karl Francis, Bernhard O. Palsson and Anthony D. Ho
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