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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2595-2604
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
From the Departments of Medicine and Bioengineering, University of
California, San Diego, CA; and the Department of Medicine V, University
of Heidelberg, Heidelberg, Germany.
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ABSTRACT |
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, single-sorted
CD34+/CD38 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% ± 9.8% of the initially quiescent cells
(PKH26 bright) gave rise again to colonies and 15.8% ± 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
CD34+/CD38 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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIETIC 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 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.
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 CD34+ 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
CD34+/CD38 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.
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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, CD34+ 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 × 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 2× 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
CD34+/CD38 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 PAINT-A-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, 104 mol/L
2-mercaptoethanol, 2 mmol/L L-glutamine, 0.2 mmol/L I-inositol, 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 CD34+
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 4× objective such that an entire well of a
Terasaki plate can be observed in a single image (1,938 × 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). 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 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.
Initial- ly, we performed these experiments with CD34+
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
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, Germany), was used.
Data reported were given as the mean ± 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
CD34+/CD38 cells between 2 subgroups.
The Kruskal-Wallis Analysis was applied to validate the differences in
CD34+/CD38 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.
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RESULTS |
In the first series of experiments, we have monitored the replication
history of hematopoietic progenitors that were CD34+.
CD34+ 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 CD34+ 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 CD34+ cells showed
multiple synchronous symmetric divisions within a single well. However,
approximately 30% of the single-sorted CD34+ 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
CD34+/CD38 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% ± 10.3% (mean ± SD) of
CD34+/CD38 cells derived from FLV
underwent asymmetric divisions and were consistently and significantly
higher than that of CD34+/CD38+ cells (30.7% ± 6.9%, n = 5, P = .0325, paired t-test).
Figure 1
demonstrates a typical symmetric division of 1 CD34+/CD38 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 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.
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| Fig 1.
This figure demonstrates a typical symmetric division of
one CD34+/CD38 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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| Fig 2.
Divisional history of 1 single
CD34+/CD38 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.
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Figure 2 demonstrates the divisional history of 1 single
CD34+/CD38 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
CD34+/CD38 cells derived from FLV. Other
CD34+/CD38 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) 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 divisions are rarely found in the third (13%), fourth (13%), and fifth (7%) waves of cellular divisions.
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| Fig 3.
Other CD34+/CD38
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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| 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.
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To correlate the replication behavior of the
CD34+/CD38 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 = .0348.
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| Fig 5.
Correlation between replication behavior of the
CD34+/CD38 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.
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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.
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Table 1.
Impact of Medium Containing Serum Versus Serum-Free
Medium on Mitosis, CE, Asymmetric Division, and ADI of
CD34+/CD38 Cells Derived From FLV
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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
32+1 to 512+1 cell stages (days 4 to 8). All of the cells were examined
in light and fluorescence microscopy and 70.3% ± 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 32+1 to 64+1 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% ± 9.8% of the PKH26
bright cells (n = 137 cells from 3 different samples) gave rise to
colonies, whereas only 15.9% ± 11.1% of the PKH26 dim cells (n = 121 cells from 3 different samples) did so. A total of
15.8% ± 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% ± 2.5% of the PKH26 dim
cells, none of which showed replating potential.
After establishing that asymmetric divisions occurred among
CD34+/CD38 cells, we have determined the
percentages of asymmetric divisions among samples derived from
different ontogenic ages. CD34+/CD38
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
CD34+/CD38 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 ± 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
FL3-L+TPO+SCF. 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.
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Table 3.
Regulatory Molecules on the Mitotic Rate, CE, Asymmetric
Division, and ADI of CD34+/CD38 Cells
Derived From FLV
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| Fig 6.
Mitotic rates, asymmetric divisions, colony efficiencies,
and ADI of CD34+/CD38 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.
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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 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
CD34+ 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
CD34+ 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
CD34+/CD38 cells than in
CD34+/CD38+ cells. These divisions could be
observed during the first and subsequent rounds of mitosis among
CD34+/CD38 cells. Second, the percentage
of such asymmetric divisions decreased with ontogenic age, ie, higher
in CD34+/CD38 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% ± 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 occurred 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]+IL-6+IL-3+Epo) or
myelopoiesis (MGF+IL-6+fusion protein of IL-3 and GM-CSF+macrophage
colony-stimulating factor [M-CSF]+granulocyte-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
CD34+/CD38 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, 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. 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
CD34+/CD38 cell that adopt different fates.
| |
FOOTNOTES |
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.
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.
Address reprint requests to Anthony D. Ho, MD, Department of Medicine
V, Hospitalstr. 3, 69115 Heidelberg, Germany.
| |
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ABSTRACT
INTRODUCTION