High-resolution video monitoring of hematopoietic
stem cells cultured in single-cell arrays identifies
new features of self-renewal
Brad Dykstra*†‡, John Ramunas‡§, David Kent*†, Lindsay McCaffrey*, Erin Szumsky§, Liam Kelly§, Kristen Farn§,
April Blaylock§, Connie Eaves*†, and Eric Jervis§¶
*Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada V5Z 4E6;†Department of Medical Genetics, University of British Columbia,
Vancouver, BC, Canada V5Z 1L3; and§Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada N2L 3G1
Communicated by Irving M. London, Massachusetts Institute of Technology, Cambridge, MA, March 30, 2006 (received for review November 9, 2005)
To search for new indicators of self-renewing hematopoietic stem
cells (HSCs), highly purified populations were isolated from adult
mouse marrow, micromanipulated into a specially designed mi-
croscopic array, and cultured for 4 days in 300 ng?ml Steel factor,
and its progeny were imaged at 3-min intervals by using digital
time-lapse photography. Individual clones were then harvested
and assayed for HSCs in mice by using a 4-month multilineage
repopulation endpoint (>1% contribution to lymphoid and my-
eloid lineages). In a first experiment, 6 of 14 initial cells (43%) and
17 of 61 clones (28%) had HSC activity, demonstrating that HSC
self-renewal divisions had occurred in vitro. Characteristics asso-
ciated with HSC activity included longer cell-cycle times and the
absence of uropodia on a majority of cells within the clone during
the final 12 h of culture. Combining these criteria maximized the
distinction of clones with HSC activity from those without and
identified a subset of 27 of the 61 clones. These 27 clones included
all 17 clones that had HSC activity; a detection efficiency of 63%
(2.26 times more frequently than in the original group). The utility
of these characteristics for discriminating HSC-containing clones
was confirmed in two independent experiments where all HSC-
containing clones were identified at a similar 2- to 3-fold-greater
efficiency. These studies illustrate the potential of this monitoring
system to detect new features of proliferating HSCs that are
predictive of self-renewal divisions.
video microscopy ? time-lapse imaging ? cell-cycle kinetics ? cell behavior ?
change with respect to one another over time and under different
conditions. Time-lapse micrography has been used for more than
half a century (1–4) to study cell morphology during attachment
and migration (5, 6), cell lifetimes (7, 8), growth (9), death (2, 10),
contact inhibition (11), clonal heterogeneity (12), and mitosis (13).
Software for extracting and analyzing cell lineage (14) and mor-
phology (15) data from videos of cells also has an extensive history.
Time-lapse studies of primitive hematopoietic cells have provided
information about their cell membrane dynamics when cocultured
with stromal cells (16, 17) or fibronectin (18), their kinetics of
division (19), their morphology and migration (20), their localiza-
tion in vivo (21), and their simultaneous expression of different
fluorescent proteins (22).
Here, we asked whether time-lapse video imaging could be
used to identify previously unidentified behavioral traits of
hematopoietic stem cells (HSCs) with functionally validated
long-term multilineage repopulating activity in vivo. A number
of groups have reported methods for obtaining highly purified
(?20% pure) populations of HSCs from normal adult mouse
bone marrow (23–28). One of these methods involves isolating
cells lacking surface markers characteristic of mature blood cells
(i.e., lineage marker-negative, or lin?cells) and able to efflux the
ime-lapse video imaging offers unique opportunities to deter-
mine how specific physical properties of individual living cells
fluorescent dyes, Rhodamine-123 (Rho?cells) and Hoechst
33342 (25). Efflux of Hoechst 33342 results in the appearance of
a side population of cells (SP cells) in two-dimensional plots of
fluorescent events (29). In mouse bone marrow (BM), the subset
of lin?Rho?SP cells represents ?0.004% of all of the cells.
Assessment of the blood cells generated in mice after injection
of single lin?Rho?SP cells has shown that 40% of these cells can
produce all blood cell types for many (?4) months (25). Inter-
estingly, most of the markers used to isolate HSC-enriched
populations from steady-state mouse BM are not directly asso-
ciated with HSC functional potential, because these phenotypes
are altered when HSC are activated or stimulated to divide
(30–34). In fact, very few stable properties of HSCs, apart from
their defining developmental potential, have been identified. To
search for previously unidentified properties of HSCs that
remain relevant even while they are proliferating, we developed
a microwell-array imaging system to visualize clones derived
from individual HSCs over a 4-day period under conditions that
support HSC self-renewal divisions (25, 35, 36). Each clone was
then recovered and assayed for the presence of HSCs with
long-term multilineage in vivo repopulating activity. Video im-
ages of these assayed clones were then used to correlate visible
characteristics of the cultured cells with those that had produced
functionally defined daughter HSCs.
Cell-Division Kinetics of CD45midlin?Rho?SP Cells Determined by
High-Resolution Video Tracking.Invivotransplantationassaysof83
freshly isolated CD45midlin?Rho?SP cells showed that 31% of
these were functionally detectable HSCs (Fig. 1 A and C).
Additional cells of this phenotype were shipped overnight from
Vancouver, BC, to Waterloo, ON, and then 67 of these were
loaded into the individual wells of three silicone microwell array
chambers containing serum-free medium and 300 ng?ml murine
Steel factor plus 20 ng?ml human IL-11 and 1 ng?ml human flt3
ligand. The arrays were incubated at 37°C for 4 days and imaged
by using a ?5 objective at 3-min intervals throughout this period
to allow the morphology and behavior of each cell and its
progeny to be recorded and tracked (Fig. 2 B and C; and see
Movies 1 and 2, which are published as supporting information
information about the timing of every cell division that occurred
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
‡B.D. and J.R. contributed equally to this work.
¶To whom correspondence should be addressed at: Department of Chemical Engineering,
University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada N2L 3G1.
© 2006 by The National Academy of Sciences of the USA
May 23, 2006 ?
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(n ? 679) and, hence, the duration of each intervening cell cycle.
From these data, we constructed pedigree diagrams for each of
the 67 clones generated (Fig. 2D). The average times to the first,
these cycles were 39.5 ? 7.6, 18.2 ? 5.2, and 15.8 ? 3.8 h,
respectively (Fig. 2E). Sister cells (i.e., paired progeny derived
from the same parental cell) divided with remarkable synchrony
throughout the culture period (Fig. 2F). Transplantation data
were obtained on 61 individually harvested clones, and the
results showed that 17 of the 61 clones (28%) contained HSCs.
This finding demonstrated that a high proportion of the input
HSCs had executed at least one self-renewal division during
the 4-day culture period (Fig. 1 B and D).
Association of Smaller Clone Sizes and Longer Cell-Cycle Times with
Retention of HSC Activity. Retrospective analysis showed that the
4-day clones containing HSCs were significantly smaller than those
in which HSCs were not detected (log2average size 8.8 ? 1.1 cells,
n ? 17 versus 17.6 ? 1.2 cells, n ? 44, P ? 0.005, Fig. 3C). This
fewer cell generation (3.1 versus 4.1) in the clones in which HSC
self-renewal divisions were subsequently shown to have occurred.
The average cell-cycle time of cells that completed one, two, and
three divisions was also significantly longer for all three cycles (P ?
0.005) in the HSC-containing clones as compared with those
without detectable HSCs (Fig. 3A). The best discrimination be-
cell-cycle times (Fig. 3B). Note that, for these calculations, we
excluded clones in which three divisions or more did not occur,
although there were only seven such clones in all. Interestingly,
neither of the two starting cells that remained viable but did not
divide during the 4-day imaging period displayed repopulating
activity when subsequently injected into mice. Also, clones con-
taining HSCs had significantly (P ? 0.05, one-tailed t test) greater
asymmetry between the cell-cycle times of the daughters of the
clone founder than clones in which HSCs were not detected (for
details, see Supporting Text, which is published as supporting
information on the PNAS web site).
Association of a Late Prevalence of Cells with Uropodia with Loss of
HSC Activity. We also looked for other features of cell behavior in
activity, including the acquisition and loss of different types of
cellular projections. During the first 14–18 h, only 6 of the 67 wells
(?9%) contained cells with lagging posterior projections (uropo-
dia) although 45 (?67%) contained cells with other cytoplasmic
extensions. At later times, uropodia became more prevalent, par-
ticularly in some clones (Fig. 2 G and H). Filopodia (long, thin
projections) were observed with high-resolution imaging (?20 and
?40 objectives) on most cells at the start and end of the period of
monitoring, but these filopodia were not consistently visible in the
lower-resolution images collected every 3 min (by using the ?5
objective) and were therefore not included in this analysis. When
final 12 h of the 4-day culture period, the majority of the cells in 25
(P ? 0.05) with the presence or absence of HSC activity was found
only in the latter case, where none of the clones with a late
predominance of cells with uropodia were found to contain HSCs.
Identification of a Combination of Monitored Parameters That Are
Predictive of HSC Self-Renewal Divisions. We then asked whether
combining two different parameters of cell behavior in the
clones (time to third division and lack of uropodia on the 4th day
of culture) would identify HSC-containing clones more effi-
with a single CD45midlin?Rho?SP cell. (B) Representative FACS profiles from a mouse repopulated with the in vitro progeny of a single CD45midlin?Rho?SP cell
cultured for 4 days in an array chamber. (C) Proportion of peripheral blood (PB) leukocytes produced from a single freshly isolated CD45midlin?Rho?SP cell
transplanted 16 weeks previously. Filled circles identify mice in which the level of donor-type leukocytes indicated that at least one HSC was present in the clone
injected (?1% donor-type leukocytes at 16 weeks and ?1% of both lymphoid and myeloid cells present at some point during the period the mice were serially
had not shown all lineages to have been included in the cells produced. Mice showing no (?0.1%) repopulation by donor-type cells are not shown. Horizontal
bars show the geometric-mean size of the clones produced in vivo from the injected HSCs of HSC-containing clones. (D–F) Proportion of donor-type leukocytes
seen in the PB of mice injected 16 weeks previously with a 4-day clone derived from a single CD45midlin?Rho?SP cell in the first imaging experiment (D) and in
the second two experiments (E and F).
www.pnas.org?cgi?doi?10.1073?pnas.0602548103Dykstra et al.
ciently than either of these parameters on its own. To apply the
first parameter, we chose a minimal cell-cycle time that included
all HSC-containing clones and excluded a maximum number of
could be applied to other data sets, we set it equal to the mean
time to the third division measured on the entire data set minus
0.5 SD. For the data set shown in Fig. 3, this value was 67.23 h.
This value was then used as a gate to subdivide clones into two
groups; those clones in which the first cell to reach a third mitosis
did so in ?67.23 h and those in which the first cell to reach a third
clone sizes and longer cell-cycle times. (A) Du-
ration of the first, second, and third cell cycles
was significantly longer in clones containing
not complete a first, second, or third cell cycle
were excluded from this analysis. (B) The cu-
mulative time to a third division of cells in
than the corresponding value for clones with-
out HSCs. Clones in which there were fewer
time. Error bars represent SEM, n ? 67. (C)
Comparison of 4-day clone size distributions
for those that contained HSCs and those that
did not. Horizontal bars indicate the geomet-
(P ? 0.005). On average, clones with HSCs ex-
clones that did not contain HSCs.
microwells, each capable of holding up to ?150 cells that can be tracked simultaneously. (B) Higher-power view of a representative well containing one
CD45midlin?Rho?SP cell suspended in serum-free medium plus 300 ng?ml Steel factor, 20 ng?ml IL-11, and 1 ng?ml Flt-3 ligand. (C) Close-up of the well shown
could be timed. (E) Cell-cycle time histogram of 67 individually cultured CD45midlin?Rho?SP cells. A delayed initial cell cycle was observed, followed by
synchronously maintained subsequent divisions. Cells that did not complete the corresponding cell cycle were excluded from this histogram. (F) Comparison of
the cell-cycle times of individual progeny pairs, demonstrating the pronounced synchrony retained between such ‘‘sister’’ cells, despite the wide range of cycle
times observed. Cells whose sisters did not complete the corresponding cell cycle were not included in the plot. (G) Example of part of a clone in which many
cells have large trailing projections (uropodia). Arrows indicate cells with uropodia. (H) Example of part of a clone in which very few cells have uropodia.
Description of the high-resolution time-lapse array system and representative culture results. (A) A digital image of an array showing 40 silicone
Dykstra et al.
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vol. 103 ?
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mitosis took longer than this threshold period (see Supporting
Text and Fig. 5, which is published as supporting information on
the PNAS web site, for additional details). We then further
subdivided the clones into two groups based on whether or not
the majority of the cells within the clone displayed uropodia at
any point during the final 12 h of culture. Selection of clones in
which the time to the third division was ?67.23 h and ?50% of
the cells exhibited uropodia in the last 12 h of culture identified
clones that contained HSCs at a 2.26-fold-higher frequency than
in the original 61 clones analyzed (Fig. 4, Table 1).
The robustness of these criteria to identify HSC-containing
clones was then tested by applying them to similar data acquired
from two independently executed experiments of the same
design. As in the first experiment, maintenance of HSC activity
was evident in the clones analyzed after culturing single
CD45midlin?Rho?SP cells for 4 days (Fig. 1 E and F). Impor-
tantly, application of the same criteria identified in the first
experiment to the data obtained from the two later experiments
allowed the HSC-containing clones to again be predicted with a
2 to 3-fold-increased efficiency (Table 1).
Here, we describe a time-lapse video monitoring system that allows
high-resolution real-time tracking of cells in multiple expanding
clones in vitro to be coupled with functional assays of the individ-
ually harvested clones at the end of the monitoring period. These
unique features have made it possible to address questions about
the biology of HSCs that have not been amenable to investigation.
The objective of our study was to identify parameters that might be
associated with HSC self-renewal divisions in vitro. From a survey
of numerous cell features (see Table 2, which is published as
supporting information on the PNAS web site, for details of other
features considered), we identified two that each showed a signif-
icant association with clones containing HSCs after 4 days of
culture: a prolonged cell-cycle time measured over thee divisions
and a reduced proportion of progeny with uropodia at any time
between 84 and 96 h of culture. In combination, these parameters
identified all of the HSC-containing clones in each of the three
experiments performed and consistently enhanced the identifica-
tion of HSC-containing clones 2- to 3-fold independent of the
these biomarkers are, indeed, robust features of mouse bone
These findings extend the results of previous studies that
correlated longer cell-cycle times of primitive hematopoietic
cells of both mouse (37) and human (38, 39) origin with the
retention of their primitive cell properties. The experiments
described here have taken this line of investigation a step further
through the use of a more highly purified HSC starting popu-
lation, a higher spatial–temporal-resolution monitoring system,
and functional assessment of the HSC activity retained (or not)
by each tracked clone. In this way, a link between HSC cell-cycle
time and their self-maintenance in culture could be definitively
established. Schroeder (40) has recently described a comple-
mentary computer-aided culture and time-lapse imaging system
that he has used to describe the generation of HSC-derived
clones on stromal cell feeder layers but without data for HSC
HSC-containing clones. Circles indicate the mean time to a third division in
each clone. Bars indicate the ranges of these times. Arrows indicate that one
or more of the cells did not complete a third division by the end of the culture
period. Asterisks indicate wells in which the original cell had not yet divided
contained a detectable HSC, and open circles represent clones that did not.
Gray symbols represent clones that were excluded by one or both of the two
criteria applied (i.e., the average time to a third division was ?67.23 h and?or
?50% of cells within the clone displayed uropodia during the final 12 h of
allowed the frequency of HSC-containing clones in the remainder to be
increased from 28% to 63%, a 2.26-fold increase.
Use of behavioral parameters defined by cell tracking to predict
Table 1. Application of selection criteria developed from the ‘‘training set’’ of data to results
from two additional experiments
Control NongatedGate 1*Gate 2†
Gates 1, 2
No. of HSC-containing clones
Test set #1
No. of HSC-containing clones
Test set #2
No. of HSC-containing clones
6 of 11
17 of 61
17 of 36
17 of 40
17 of 27
3 of 16
4 of 24
4 of 13
4 of 18
4 of 12
4 of 18
5 of 73 5 of 42
5 of 35
5 of 27
*Excluding clones containing one or more cells with a cumulative time to a third division faster than the mean
minus 0.5 SD.
†Excluding clones containing ?50% of cells with uropodia during the final 12 hours of culture.
www.pnas.org?cgi?doi?10.1073?pnas.0602548103 Dykstra et al.
activity in the clones produced. We anticipate that further use of
both systems will provide valuable insights into how primitive
hematopoietic cells interact with external cues to regulate their
self-renewal and differentiation potential.
Multiple studies have associated a variety of cell projections with
primitive hematopoietic cells (18, 21, 41–43). In particular, Frim-
berger et al. (16) observed several types of projections on the
leading edge and periphery of cells in HSC-enriched populations
using high-speed optical-sectioning microscopy and inverted fluo-
rescent video microscopy. Giebel and colleagues (43) have de-
scribed the appearance of uropodia at the rear pole of human
negatively associated with retained HSC activity. Clearly, attention
to the criteria used to define different categories of projections as
at which they are assessed will be important to future investigations
of whether these projections play a role in HSC biology.
Although our approach is potentially applicable to any HSC-
containing population, all candidate biomarkers would need to be
screened again if a different isolation strategy were used, because
the non-HSC component of such populations would likely be
different. A strength of the approach used here is that it can be
adapted to any source of HSCs or HSC isolation strategy because
it makes no assumptions about the biological homogeneity of the
cells being monitored. The most useful biomarkers are, however,
those that can be directly linked to the defining developmental
properties of HSCs. The technology and experimental design
described here, thus, represent an important advance in the defin-
itive identification of such features. In addition, the system we have
described has the flexibility of allowing specific cells with tracked
surface or internal components will further broaden the scope of
cellular events that can be monitored. We, thus, anticipate increas-
ing application of this powerful technology to many areas of cell
or -Ly5.2 mice. Transplant recipients were Ly5-congenic C57BL?
cGy?min. Peripheral blood (PB) was collected at 4, 8, 12, and 16
weeks after transplant, and the leukocytes were then stained with
antibodies for donor and recipient CD45 allotypes plus lymphoid-
as the detection of donor-derived leukocytes at ?1% levels in the
PB for at least 16 weeks. Multilineage repopulation was defined as
the detection of ?1% of both donor type lymphoid and myeloid
cells at 4, 8, 12, and?or 16 weeks after transplantation. Evidence of
was used to infer that ?1 HSC had been injected. For further
details, see Supporting Text and Fig. 6, which is published as
supporting information on the PNAS web site.
Purification, Culture, and Shipment of CD45midlin?Rho?SP Cells. Cell
purification was performed as described in ref. 25, with minor
modifications. See Supporting Text and Fig. 7, which are pub-
lished as supporting information on the PNAS web site, for
details. For controls, single CD45midlin?Rho?SP cells were
sorted into the individual wells of a round-bottom 96-well plate
containing 100–200 ?l of serum-free medium (SFM) (see Sup-
porting Text), visually confirmed, and then injected individually
directly into sublethally irradiated C57BL?6J-W41?W41recipi-
ents. The, CD45midlin?Rho?SP cells to be imaged were sorted
and collected into a 1.4-ml Eppendorf tube prefilled with SFM
in Vancouver, BC, and then shipped via overnight courier
(18–22 h) at 4°C to the University of Waterloo in Ontario. Upon
arrival, the cells were warmed to 25°C, and 300 ng?ml murine
Steel factor (StemCell Technologies, Vancouver), 20 ng?ml
human IL-11 (Genetics Institute, Cambridge, MA), and 1 ng?ml
human Flt-3 ligand (Immunex, Seattle, WA) added to the
medium. Single CD45midlin?Rho?SP cells were then microma-
nipulated into the individual microwells of an array chamber
(Fig. 2A, prepared as described below), which was then placed
at 37°C in a humidified, 5% CO2atmosphere and imaged every
3 min by using phase contrast optics. The time of cytokine
addition was set as 0 hours of culture time for all experiments.
At the end of the 4 days of culture, the clones in the arrays were
harvested individually, placed into separate 0.65-ml microcen-
trifuge tubes, and shipped via overnight courier at 4°C to
Vancouver, where the cells in each tube were resuspended and
injected into individual sublethally irradiated C57BL?6J-W41?
Videotracking System. Cells were cultured in custom-designed mi-
crowell chambers. Briefly, these microwell arrays were constructed
by applying silicone gel to a glass coverslip to form a film ?20 ?m
thick, and a 100-?m-wide glass scraper was then used to machine
a reservoir to contain the culture medium. To deposit the cells
containing ?50 cells that were then allowed to settle. Each of the
settled cells using a glass micropipette guided by a 3-axis motorized
micromanipulator. The micropipettes were made from capillary
tubes (3-000-203-G?X; Drummond) by using a vertical pipette
puller (Model 720; Kopf) and cut with a single-crystal diamond-
tipped glass etcher to give an opening 15–30 ?m wide. Images were
obtained on a Zeiss Axiovert 200 microscope equipped with
phase-contrast optics and a Sony XCD-SX900 digital camera. Cells
were exposed to light only during imaging. Each cell in each image
of the ?1850-image time courses was scored for morphological
characteristics, location, and parentage by using human-assisted
custom cell-tracking software that generated pedigree diagrams
(EXCEL, MATLAB, and PRISM) to test correlations between candi-
date biomarkers and HSC activity (details of the human-assisted
tracking techniques and a list of candidate biomarkers that were
tested are given in the Supporting Text and Table 2).
We acknowledge gifts of reagents from StemCell Technologies, Genetics
Institute, and Immunex; technical assistance from the Terry Fox Flow
Cytometry Facility and staff of the Animal Resource Centre of the
British Columbia Cancer Agency; and secretarial assistance from Ad-
rienne Wanhill. This work was supported by grants from the Stem Cell
Network (to C.E. and E.J.) and the National Cancer Institute of Canada
(with funds from the Terry Fox Foundation) and National Heart, Lung,
and Blood Institute?National Institutes of Health Grant P01 HL-55435
(to C.E.). B.D. held a Stem Cell Network Studentship and a Terry Fox
Foundation Research Studentship. D.K. held a Stem Cell Network
Studentship and a Studentship funded jointly by the Canadian Institutes
of Health Research and the Michael Smith Foundation for Health
1. Schwobel, W. (1952) Mikroskopie 7, 115–120.
2. Schrek, R. & Ott, J. N., Jr. (1952) Am. Med. Assoc. Arch. Pathol. 53, 363–378.
3. Kramis, N. J. (1956) J. Biol. Photogr. Assoc. 24, 27–29.
4. Siskin, J. (1963) in Cinemicrography Cell Biology, ed. Rose, G. G. (Academic,
New York), pp. 143–168.
5. Allen, R. D., Zacharski, L. R., Widirstky, S. T., Rosenstein, R., Zaitlin, L. M.
& Burgess, D. R. (1979) J. Cell Biol. 83, 126–142.
6. DiMilla, P. A., Stone, J. A., Quinn, J. A., Albelda, S. M. & Lauffenburger, D. A.
(1993) J. Cell Biol. 122, 729–737.
7. Hsu, T. C. (1960) Tex. Rep. Biol. Med. 18, 31–33.
Dykstra et al.
May 23, 2006 ?
vol. 103 ?
no. 21 ?
8. Froese, G. (1964) Exp. Cell Res. 35, 415–419. Download full-text
9. Zetterberg, A. & Killander, D. (1965) Exp. Cell Res. 39, 22–32.
10. Marin, G. & Bender, M. A. (1966) Exp. Cell Res. 43, 413–423.
11. Martz, E. & Steinberg, M. S. (1972) J. Cell Physiol. 79, 189–210.
12. Absher, P. M. & Absher, R. G. (1976) Exp. Cell Res. 103, 247–255.
13. Concha, M. L. & Adams, R. J. (1998) Development (Cambridge, U.K.) 125,
14. Sylwester, D., Dennis, S. M. & Absher, M. (1980) Comput. Biol. Med. 10,
15. Potel, M. J., Sayre, R. E. & Robertson, A. (1979) Comput. Biol. Med. 9,
16. Frimberger, A. E., McAuliffe, C. I., Werme, K. A., Tuft, R. A., Fogarty, K. E.,
Benoit, B. O., Dooner, M. S. & Quesenberry, P. J. (2001) Br. J. Haematol. 112,
17. Wagner, W., Saffrich, R., Wirkner, U., Eckstein, V., Blake, J., Ansorge, A.,
18. Fruehauf, S., Srbic, K., Seggewiss, R., Topaly, J. & Ho, A. D. (2002) J. Leukoc.
Biol. 71, 425–432.
19. Punzel, M., Liu, D., Zhang, T., Eckstein, V., Miesala, K. & Ho, A. D. (2003)
Exp. Hematol. 31, 339–347.
20. Francis, K., Palsson, B., Donahue, J., Fong, S. & Carrier, E. (2002) Exp.
Hematol. 30, 460–463.
21. Suzuki, N., Ohneda, O., Minegishi, N., Nisikawa, M., Ohta, T., Takahashi, S.,
Engel, J. D. & Yamamoto, M. (2006) Proc. Natl. Acad. Sci. USA, 103, 2202–2207.
22. Stadtfeld, M., Varas, F. & Graf, T. (2005) Methods Mol. Med. 105, 395–412.
23. Osawa, M., Hanada, K. I., Hamada, H. & Nakauchi, H. (1996) Science 273,
24. Wagers, A. J., Sherwood, R. I., Christensen, J. L. & Weissman, I. L. (2002)
Science 297, 2256–2259.
25. Uchida, N., Dykstra, B., Lyons, K. J., Leung, F. Y. K. & Eaves, C. J. (2003) Exp.
Hematol. 31, 1338–1347.
26. Benveniste, P., Cantin, C., Hyam, D. & Iscove, N. N. (2003) Nat. Immunol. 4,
27. Chen, C.-Z., Li, L., Li, M. & Lodish, H. (2003) Immunity 19, 525–533.
28. Matsuzaki, Y., Kinjo, K., Mulligan, R. C. & Okano, H. (2004) Immunity 20,
29. Goodell, M. A., Brose, K., Paradis, G., Conner, A. S. & Mulligan, R. C. (1996)
J. Exp. Med. 183, 1797–1806.
30. Sato, T., Laver, J. H. & Ogawa, M. (1999) Blood 94, 2548–2554.
31. Huygen, S., Giet, O., Artisien, V., Di Stefano, I., Beguin, Y. & Gothot, A.
(2002) Blood 100, 2744–2752.
32. Uchida, N., Dykstra, B., Lyons, K., Leung, F., Kristiansen, M. & Eaves, C.
(2004) Blood 103, 4487–4495.
Reilly, J., Carlson, J. E., Frimberger, A. E., Stewart, F. M. & Quesenberry, P. J.
(1998) J. Exp. Med. 188, 393–398.
34. Zhang, C. C. & Lodish, H. F. (2005) Blood 105, 4314–4320.
35. Miller, C. L. & Eaves, C. J. (1997) Proc. Natl. Acad. Sci. USA 94, 13648–13653.
36. Audet, J., Miller, C. L., Eaves, C. J. & Piret, J. M. (2002) Biotechnol. Bioeng.
37. Suda, T., Suda, J. & Ogawa, M. (1983) J. Cell Physiol. 117, 308–318.
38. Brummendorf, T. H., Dragowska, W., Zijlmans, J. M., Thornbury, G. &
Lansdorp, P. M. (1998) J. Exp. Med. 188, 1117–1124.
C. T., Abonour, R. & Orschell, C. M. (2005) Blood 105, 3109–3116.
40. Schroeder, T. (2005) Ann. N.Y. Acad. Sci. 1044, 201–209.
41. Frimberger, A. E., Stering, A. I. & Quesenberry, P. J. (2001) Blood 98,
42. Wagner, W., Ansorge, A., Wirkner, U., Eckstein, V., Schwager, C., Blake, J.,
Miesala, K., Selig, J., Saffrich, R., Ansorge, W. & Ho, A. D. (2004) Blood 104,
J., Kogler, G. & Wernet, P. (2004) Blood 104, 2332–2338.
www.pnas.org?cgi?doi?10.1073?pnas.0602548103 Dykstra et al.