Hematopoietic stem cells (HSCs) in adult marrow are believed to be derived from fetal liver
precursors. To study cell kinetics involved in long-term hematopoiesis, we studied single-sorted candidate HSCs from fetal liver that were cultured in the presence of a mixture of stimulatory cytokines. After 8-10 d, the number of cells in primary cultures varied from <100 to
>10,000 cells. Single cells in slow growing colonies were recloned upon reaching a 100-200
cell stage. Strikingly, the number of cells in subclones varied widely again. These results are indicative of asymmetric divisions in primitive hematopoietic cells in which proliferative potential and cell cycle properties are unevenly distributed among daughter cells. The continuous
generation of functional heterogeneity among the clonal progeny of HSCs is in support of intrinsic control of stem cell fate and provides a model for the long-term maintenance of hematopoiesis in vitro and in vivo.
Key words:
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Introduction |
Blood formation originates in a small population of hematopoietic stem cells (HSCs)1 that have been defined
as pluripotent cells with self-renewal capacity (1, 2). The
mechanisms underlying the proliferation and differentiation
of HSCs are incompletely understood (3, 4). Both extrinsic
(e.g., growth factors and cell-matrix interactions) and intrinsic factors (e.g., developmentally controlled transcription factors) are involved in the regulation of HSCs. Although hematopoietic growth factors and an adequate
microenvironment are crucial for the survival and proliferation of HSCs, self-renewal/differentiation decisions in HSCs
seem to be derived largely independently of cytokines and
are postulated to be determined intrinsically (4).
Studies aimed at dissecting the molecular mechanism involved in stem cell regulation have been hampered by difficulties in obtaining populations of HSCs devoid of more
differentiated progenitor cells. Apart from a paucity of distinguishing phenotypic features, these difficulties are also
related to available HSCs assays. Thus, despite intense efforts to establish determinants by which primitive HSCs
can be defined prospectively, available in vivo and in vitro
stem cell assays only allow retrospective identification of
HSCs. In mice, candidate HSCs are believed to be among
the cells expressing a Thy-1.1loSca1hiLin
/lo phenotype (9,
10). This cell population represents <0.1% of murine fetal
liver and is highly enriched for multipotent progenitors (11). However, cells with this phenotype display considerable heterogeneity with respect to "stem cell" properties
such as CFU-S formation (12), and only a minority has the
ability to reconstitute lympho-myelopoiesis in lethally irradiated recipients (10, 13). In the last two decades, a large
amount of effort has also been directed towards the development of in vitro assays for human HSCs (for review see
reference 14). One prominent example of such an assay is
the long-term culture-initiating cell (LTC-IC) assay (15).
LTC-ICs are hematopoietic cells that are capable of generating myeloid colony-forming cells after at least 5 wk of
culture in the presence of irradiated feeder cells. LTC-ICs are highly enriched among cells with a CD34+CD38
phenotype, and the yield of LTC-ICs in such cells from adult human marrow and umbilical cord blood is ~20 (16) and
~50% (17), respectively. Based on these considerations, human CD34+CD38
cells are expected to be highly enriched
for HSCs. However, the frequency of CD34+CD38
cord
blood cells capable of initiating hematopoiesis in immune deficient mice is only 0.1% (18). The heterogeneity
within the CD34+CD38
compartment of human cells is
reminiscent of the functional heterogeneity as outlined
above for murine cells with a Thy-1.1loSca1hiLin
/lo phenotype.
To study the functional heterogeneity of candidate HSCs,
we followed the fate of single-sorted fetal liver CD34+CD38
cells that were cultured in cytokine-supplemented serum-free medium. By combining observations on in vitro growth
with a detailed characterization of individual cells produced
in culture, we observed that functional heterogeneity is
continuously generated among the clonal progeny of HSCs.
Furthermore, the growth characteristics of cell clones allowed
us to more closely and, to a certain degree, semiprospectively define cells with the highest proliferative capacity.
Our observations provide an explanation for the extreme functional heterogeneity among highly purified candidate
HSCs. Several possible mechanisms for the asymmetric cell
divisions that best explain our findings are discussed.
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Materials and Methods |
Purification of Fetal Liver Stem Cell Candidates
Cells with CD34+CD38
CD71
CD45RA
phenotype were
isolated from previously frozen samples of fetal liver obtained
from elective, therapeutic abortions in wk 10-16 of gestation.
The use of human material was approved by local Institutional
Review Boards as well as the Ethical Screening Committee of the
University of British Columbia. Cells were separated using Ficoll-Hypaque and processed for flow cytometry cell sorting as
previously described (6, 21). In brief, cells were labeled with
OKT-9-FITC (anti-CD71), 8G12-Cy-5 (anti-CD34), 8d2-PE
(anti-CD45RA), and Leu-17-PE (anti-CD38; Becton Dickinson, San Jose, CA) and sorted using a dual laser FACStar® Plus
(Becton Dickinson) equipped with an argon and helium-neon laser (Fig. 1). Cells either were collected in serum-free medium or were individually sorted directly into round-bottomed tissue culture plates (Nunc, Roskilde, Denmark) containing serum-free medium supplemented with hematopoietic growth factors (as indicated below) using an automatic cell deposition unit (Becton
Dickinson). The experimental design of the culture experiments
is shown in Fig. 2.

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Fig. 1.
FACS® profile of the CD34+CD38 candidate HSCs sorted
from fetal liver to initiate single cell cultures in 96-well plates. Viable,
propidium iodide negative (PI ) CD34+CD38 cells with a low side
scatter (SSC) and low levels of CD45RA and CD71 expression were recovered from the indicated sort windows.
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Culture of Cells
Single-cell Cultures.
Single-sorted cells were cultured in 96-well round-bottomed plates (60 cells/plate) with each well containing 100 µl of serum-free medium (21) containing 100 ng/ml
each of Steel factor (or stem cell factor) and Flt-3 ligand and 20 ng/ml each of IL-3, IL-6, and G-CSF. After 5-7 d of culture, another 100 µl of growth factor-containing medium was added.
After 6-9 d, the number of cells per well varied over a wide range
from wells with >10,000 cells to wells with <100 cells. Slowly
growing colonies with 100-250 cells after 8-13 d in culture were
recloned and the number and morphology (shape, size, and refractive index) of cells in each subclone were scored at various
time intervals. Slowly growing colonies were recloned again between days 8 and 19. Such recloning of subclones was repeated until the sixth generation.
Expansion Culture.
For the evaluation of the proliferative potential slow growing colonies that had reached a level of 5 × 104 cells
were transferred in 1-ml cultures of 24-well plates. CD34+CD38
cells were resorted every 5-10 d as described above if cultures became confluent (>106 cells/well) to initiate subcultures until the
percentage of CD34+CD38
cells per well dropped below 0.1%
of viable cells.
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Results |
Characterization of Primary Cultures Derived from Single
CD34+CD38
Fetal Liver Cells.
Single CD34+CD38
fetal liver stem cell candidates were sorted in individual wells
of microtiter plates containing serum-free medium supplemented with cytokines (see Materials and Methods). The
plating efficiency (number of wells containing visible single cells after sorting) was >90%, of which >80% appeared viable after 24 h. The cell number, morphology, and the percentage of CD34+CD38
cells in primary clones was analyzed at different time intervals. After 6-9 d, the number of
cells in the cultures varied over a wide range, indicating extensive heterogeneity among fetal liver CD34+CD38
cells. Rapidly growing clones of >10,000 cells as well as
slow growing clones of <50 cells were observed. All wells
with growing cells (n = 121) were transferred to 1-ml cultures after 5 × 104 or more cells were present between 10 and 21 d of culture. Cells with a CD34+CD38
phenotype
were resorted from expanded 1-ml cultures when these cultures became confluent (0.5-2.0 × 106 cells) and used
for continuation of cultures using identical culture conditions. This procedure was repeated until no more CD34+
cells were produced. Three different categories (A-C) were
defined based on the ability to produce CD34+CD38
cells
in culture. Clones producing CD34+CD38
cells up to
day 16 were categorized as A, whereas colonies producing CD34+CD38
cells for up to day 59 or more were categorized as B and C, respectively.
Retrospective analysis revealed that colonies that gave
rise to the highest number of CD34+CD38
cells for the
longest time period corresponded to primary clones with
slow growth properties in the first 9 d of culture (Table 1).
This fraction represented 16% of the clones analyzed,
whereas the majority of clones (60%) were fast growing
(>103 cells at day 9) with a relatively low expansion potential. Furthermore, the number of cells in fast growing colonies already started to plateau at day 10, whereas the cell
number in slow growing clones was still expanding at day 12 (Fig. 3). These observations underscore the functional heterogeneity of the CD34+CD38
cell compartment in human fetal liver and suggest that categories A and B represent the property of more differentiated progenitor cells.
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Table 1
Production of CD34+CD38 Cells by Single Cells in Serum-free Medium Supplemented with Steel Factor, Flt-3, IL-3, IL-6,
and G-CSF
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Fig. 3.
Clonal heterogeneity in early proliferative response of single-sorted CD34+CD38 fetal liver cells cultured in growth factor-supplemented SFM. Single CD34+CD38 fetal liver cells were sorted into the
wells of round-bottomed microtiter plates containing serum-free medium
supplemented with Steel factor, Flt-3 ligand, IL-3, IL-6, and G-CSF at
37°C. At the indicated time intervals, the number of cells in each well was
scored.
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Expansion Potential of Single-sorted CD34+CD38
Fetal
Liver Cells.
To further characterize the expansion potential
of slow growing clones, the experiment described above
was repeated with CD34+CD38
cells from a different fetal liver. The CD34+CD38
expansion potential per clone
varied over a wide range, from 1.43 × 104 to 2.7 × 1012,
with an average expansion of 8.65 × 108 CD34+CD38
cells
in the 27 clones that were analyzed. The maximum continued production of CD34+CD38
cells was 129 d. Strikingly, a single clone produced 2.7 × 1012 CD34+CD38
cells over 106 d in culture (corresponding to at least 41 "self-renewal" population doublings). Interestingly, in this
clone (Fig. 4), the calculated average cell cycle time of
CD34+CD38
cells in culture was very long up to day 15 (70.2 h), decreased from day 29 to 57 (to 43.3 h), and finally gradually increased again up until day 117 (72.4 h).
These findings indicate that after an initial period of slow
growth, the CD34+CD38
cells divided more rapidly before decreased turnover and eventual loss of CD34+CD38
cell production. Similar growth kinetics were observed for
the other clones with lower proliferative potential examined in these experiments.

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Fig. 4.
Expansion potential and average cell cycle time of
CD34+CD38 cells resorted from expanded cultures initiated with a single-sorted CD34+38 fetal liver cell. Initial culture and subculture of the
CD34+CD38 cells was in serum-free media supplemented with cytokines. The average cell cycle time of CD34+CD38 was calculated as described in Table 1.
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In general, these results document that extensive heterogeneity exists in the ability to produce CD34+CD38
cells
among slow growing CD34+CD38
fetal liver cells. To
examine whether the early proliferative response of single
slow growing clones is a good predictor of maximum expansion potential, the cell numbers in clones at different time intervals were plotted against the total number of
CD34+CD38
cells produced by that clone. Linear regression analysis revealed a significant negative correlation (P = 0.0006) between the cell number at day 6 and the maximum CD34+38
expansion potential (Fig. 5). This result
suggests that the proliferative potential and cell cycle characteristics in primitive hematopoietic cells are linked.

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Fig. 5.
Correlation between early proliferative response at day 6 of
slow growing clones derived from single-sorted CD34+CD38 fetal liver
cells and maximum CD34+38 expansion potential of different clones.
The maximum expansion potential was determined by repeated subculture of CD34+CD38 cells sorted from confluent 1-ml cultures initially
seeded with the content of a single well. *Estimated cell numbers: amount
of CD34+38 was not determined due to low cell numbers; expansion
was calculated on total cells for these clones.
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Heterogeneity in Subclones Derived from Slow Growing
CD34+CD38
Fetal Liver Clones.
To further analyze the
functional heterogeneity within the most primitive, slow
growing CD34+CD38
fetal liver cells, we recloned cells
recovered from category C at days 8-19, when cell numbers had reached between 100 and 250 cells. Individual
subclones were analyzed with respect to cell number and
morphology at different time intervals and, in some cases, also
analyzed for total CD34+CD38
cell production as described
above for primary clones. Surprisingly, the clonal heterogeneity observed in the primary single-sorted CD34+CD38
fetal liver cells was preserved. This is shown for five consecutive generations of subclones of a single slow growing
colony of CD34+CD38
cells in Fig. 6. Furthermore, as
shown in Fig. 7, the distribution pattern of the three categories remained more or less constant through multiple
generations of recloning, with fast growing clones representing the majority. Slowly growing clones that did not show morphological features of terminal (macrophage) differentiation represented 20% of primary clones and between 20 and 30% of second to fourth generation subclones, and decreased to <10% by the sixth generation (Fig.
7). Our results indicate that the number of CD34+CD38
cells with extensive replating potential in slow growing
clones decreases upon multiple rounds of recloning.

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Fig. 6.
Clonal heterogeneity of subclones is preserved
through at least five consecutive
generations of recloning of slow
growing clones derived from single-sorted CD34+CD38 fetal
liver cells. The average number
of cells in multiple clones of categories A, B, and C (see text) is
plotted. A single "category C"
clone (dotted line and *) was recloned when 100-200 cells were
present and this was repeated for
subsequent category C subclones that were produced from
a single initial clone.
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Fig. 7.
Percentage of slow, intermediate, and fast growing clones in
the progeny of single-sorted CD34+CD38 fetal liver cells shown in Fig.
6. n = number of clones/generation. Figure represents pooled data from
two experiments.
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Compared with the findings in slow growing clones, recloning of intermediate or fast growing clones revealed a
more homogeneous growth pattern in subclones. In such
subclones, the vast majority (>90%) either developed into
mature macrophages or produced up to a few thousand
CD34+CD38
cells over 2-3 wk before terminal myeloid
differentiation.
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Discussion |
The data presented in this paper provide insight into
three major aspects of stem cell biology. First, the data
show that CD34+CD38
candidate HSCs from fetal liver
display extensive functional heterogeneity when cultured as
single cells in defined culture conditions. Secondly, this
heterogeneity is generated in the slow growing progeny of
single-sorted fetal liver cells. Thirdly, cells with the highest
overall proliferative potential could be recognized by their
slow growth kinetics, allowing early identification of colonies containing cells with a very high proliferative potential.
The majority of subclones derived from slow growing
CD34+CD38
cells were fast growing clones with a low
proliferative potential. However, a minority of subclones
showed similar growth kinetics to the parental clones, and
this heterogeneity was preserved through four to six generations of recloning. Because culture conditions were kept
constant in all these experiments, our findings support the
conclusion that differences in the fate of individual stem cells are continuously and intrinsically generated. What
could be the mechanisms involved in the heritable functional heterogeneity within the clonal progeny of slow
growing candidate HSCs? The most simple hypothesis is
that daughter cells are endowed with different cell fates via
asymmetric cell divisions (Fig. 8). Such asymmetric cell divisions would result in one daughter cell being similar to
the mother cell and the other daughter cell more committed to terminal differentiation. Alternatively, differences in
cell fate could be acquired after mitosis by unknown mechanisms. Direct evidence for asymmetric divisions in early
hematopoiesis was previously reported using time lapse video
recordings of CD34+CD71loCD45RAlo bone marrow progenitor cells in culture (22). In our study, we also observed
a distinct polarity among slow growing CD34+CD38
cells:
such cells were small, motile, and appeared as "commas" with most of the cellular volume preceding a cytoplasmic tail of one to two cell diameter (5-10 µm). In previous studies,
it was reported that ~20% of the cell divisions in early hematopoiesis would qualify as asymmetric (5, 6), whereas
extensive amplification of cell numbers during proliferation
and expansion of more committed hematopoietic progenitors would involve primarily symmetric divisions (3).

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Fig. 8.
The intrinsic timetable model of stem cell biology. In this
model, the functional properties of stem cells are (i) linked to the number
of preceding cell divisions or generations (g) and (ii) unevenly distributed
among daughter cells upon each cell division. It is postulated that each
stem cell division is asymmetric and results in daughter cells that differ in
cell cycle properties. As a result, the time interval between successive generations (t 1, t 2, etc.) is variable between clones of the same generation
and the interval between divisions is subject to both intrinsic (developmental) and extrinsic (microenvironment and growth factors) control.
Clonal variations in turn-over time result in an extreme hierarchy of stem
cells varying in replicative history and related functional properties that is
difficult to reconcile with the concept of stem cells as a homogeneous
population of cells.
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To date, limited data on the mechanism involved in asymmetric division of HSCs are available. Investigations of asymmetric division in plants (for review see reference 23), Caenorhabditis elegans (24), Drosophila and other species (for review
see reference 25), as well as in mammalian neural stem cells
(26) have elucidated several distinct possibilities, and several working models based on these better defined systems
are shown in Fig. 9. Similar mechanisms are implicated in
asymmetric cell divisions in Drosophila and mammalian neural progenitor cells (for review see references 29, 30). Many
different proteins involved in these processes have been
identified, some of which, i.e., the notch- (27) and numb- (28) families of proteins, may also be involved in early
hemopoiesis (Fig. 9 a). Indeed, Drosophila neuroblast stem
cells have been shown to undergo self-renewing asymmetric
divisions in culture (31), very similar to the data on fetal liver
HSCs reported here. Recently, Milner et al. showed that
constitutive expression of the intracellular domain of notch-1
in the 32D myeloid progenitor cell line leads to inhibition of
differentiation and to expansion of undifferentiated cells in
response to G-CSF (32). These effects have recently been
shown to be transmitted by the Notch-1-ligand Jagged-1
(33). The pros gene, which encodes for the homeodomain-containing nuclear protein prospero, is involved in asymmetric divisions of Drosophila neuroblasts (Fig. 9 c and references 34, 35). This might be of special interest in light of
recent data on the role of homeobox transcription factors
HOXA10 (36), HOXB4 (37), and others in hematopoiesis
(38, 39). Studies on oligodendrocyte precursor cells by Gao
and Raff (26) also resemble the observations reported here.
Gao and Raff concluded that the differentiation and maturation of oligodendrocyte precursor cells is an intrinsic property of the cell and that asymmetric divisions of precursors contribute to differences in cell fate. Interestingly, the cell cycle time of different precursors was also found to be related to cell fate in their studies. Finally, it also seems important to
exclude asymmetric inheritance of extrachromosomal rDNA
circles (40) as an explanation for the divergence in the fate of
HSCs (Fig. 9 d).
It is tempting to speculate that our observations provide
an explanation for the recent observation that committed
progenitor cells do not mediate early hematopoietic reconstitution after blood cell transplantation in mice (41). According to this scenario, slow growing clones, capable of
engrafting lethally irradiated recipients, would produce rapidly growing and differentiating subclones while simultaneously producing more primitive precursors. Data on ontogeny-related changes in the functional properties and
telomere length of fetal liver compared with cord blood
and adult bone marrow cells (42, 43) have led to the speculation that the replicative life span of phenotypically identical HSCs may decrease with age and could be limited to
<100 cell divisions (4). A model of early stem cell biology
that is compatible with both asymmetric divisions of the
HSCs and ontogeny-related changes in HSC function is
shown in Fig. 8.
In conclusion, the data shown here provide further evidence that the fate of the most primitive HSC is primarily
determined intrinsically and regulated only in a permissive
way by extrinsic factors in agreement with previous studies
in model systems (8, for review see reference 44). The
mechanisms underlying the asymmetric divisions of HSCs
documented here appear to be a fruitful area for further
studies.
Address correspondence to Peter Lansdorp, Terry Fox Laboratory, BC Cancer Research Centre, 601 West
10th Ave., Vancouver, BC, V5Z 1L3, Canada. Phone: 604-877-6070, ext. 3026; Fax: 604-877-0712;
E-mail: peter{at}terryfox.ubc.ca
This work was supported by National Institutes of Health grant AI-29524 and by a grant from the National
Cancer Institute of Canada with funds from the Terry Fox Run as well as a grant from the Deutsche Forschungsgemeinschaft (to T.H. Brummendorf ).
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