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HEMATOPOIESIS
From the Department of Molecular Biology, Umeå
University, Sweden.
Hematopoietic stem cells (HSCs) are unique in their capacity to
maintain blood formation following transplantation into
immunocompromised hosts. Expansion of HSCs in vitro is therefore
important for many clinical applications but has met with limited
success because the mechanisms regulating the self-renewal process are
poorly defined. We have previously shown that expression of the
LIM-homeobox gene Lhx2 in hematopoietic progenitor
cells derived from embryonic stem cells differentiated in vitro
generates immortalized multipotent hematopoietic progenitor cell lines.
However, HSCs of early embryonic origin, including those derived from
differentiated embryonic stem cells, are inefficient in engrafting
adult recipients upon transplantation. To address whether
Lhx2 can immortalize hematopoietic progenitor/stem cells
that can engraft adult recipients, we expressed Lhx2 in
hematopoietic progenitor/stem cells derived from adult bone marrow.
This approach allowed for the generation of immortalized growth
factor-dependent hematopoietic progenitor/stem cell lines that can
generate erythroid, myeloid, and lymphoid cells upon transplantation
into lethally irradiated mice. When transplanted into stem
cell-deficient mice, these cell lines can generate a significant
proportion of circulating erythrocytes in primary, secondary, and
tertiary recipients for at least 18 months. Thus, Lhx2
immortalizes multipotent hematopoietic progenitor/stem cells that can
generate functional progeny following transplantation into lethally
irradiated hosts and can long-term repopulate stem cell-deficient hosts.
(Blood. 2002;99:3939-3946) Hematopoietic stem cells (HSCs) can be isolated on
the basis of cell surface markers and have the unique capacity to
regenerate and maintain a functional hematopoietic system following
transplantation into immunocompromised mice.1 The ability
to isolate HSCs and their importance in clinical applications have led
to the development of a variety of different culture systems for
expanding HSCs in vitro.2-5 Expansion of HSCs using these
different culture systems has met with modest success because the
molecular and cellular mechanisms regulating the self-renewal process
in HSCs are largely unknown.6
An alternative approach for expanding HSCs in vitro is to immortalize
them by genetic manipulation, and stem cell-like cell lines have been
established using such methods. Most of these cell lines either show a
limited ability to generate functional hematopoietic cells after
transplantation or have thus far not been thoroughly analyzed in this
respect.7-10 One cell line was reported to generate
functional cells in vivo, but the genetic event that led to the
immortalization was unknown.11 Hence, generation of this
cell line cannot be easily reproduced. We have previously generated
HSC-like cell lines by expressing the LIM-homeobox gene Lhx2
in hematopoietic progenitor cells (HPCs) derived from embryonic stem
(ES) cells differentiated in vitro.12 However, HSCs of
early fetal origin, including those derived from ES cells differentiated in vitro, are for unknown reasons inefficient in engrafting in an adult environment.13-17
To address whether Lhx2 can reproducibly immortalize
hematopoietic progenitor/stem cells that have the capacity to engraft adult recipients, we expressed the Lhx2 gene in
hematopoietic progenitor/stem cells derived from adult bone marrow
(BM). This approach allowed for the generation of clonal and
cytokine-dependent stem cell-like cell lines. These cell lines
can generate different functional hematopoietic cells in vivo,
including a high proportion of circulating erythrocytes in stem
cell-deficient hosts for an extended time.
Mice
Retrovirus production and infection of BM cells
Clonal assays in vitro and spleen colony-forming unit assays Methylcellulose-based clonal assays were carried out in IMDM containing 1% methyl cellulose (Fluka, Neu-Ulm, Switzerland) supplemented with L-glutamine, 300 µg/mL iron-saturated transferrin (Boehringer, Ingelheim, Germany), 5% protein-free hybridoma medium II (Gibco-BRL), 1.5 × 10 4 M MTG, and
10% plasma-derived serum (Antech, Tyler, TX). The cells were plated in
final volume of 1.25 mL in 35-mm Petri dishes (Falcon 3008) in
triplicates. The following factors were used at a predetermined optimal
concentration: erythropoietin (Epo) (Eprex Cilag, Sollentuna, Sweden) 4 U/mL, macrophage colony-stimulating factor (M-CSF) 20 ng/mL,
granulocyte-CSF (G-CSF) 20 ng/mL, GM-CSF 10 ng/mL, thrombopoietin (Tpo)
20 ng/mL, and Flt3L 10 ng/mL. SF, IL-6, and IL-3 were used as
previously described. All growth factors, except where indicated, were
obtained from R&D Systems. In clonal assays of BM cells G418 was used
at 1 mg/mL. For spleen colony-forming unit (CFU-S) assays different
numbers of BM-HPCs, ranging between 300 to 106,
were intravenously injected into lethally irradiated (10 Gy in a single
dose from a 60Co source) B6-SJL mice. Ten days later the
spleens of the animals receiving transplants were removed and fixed in
Bouin solution (SIGMA), and the number of visible colonies on the
spleen were counted to determine the frequency of CFU-Ss for each
cell line.
Northern and Southern blot analysis Total RNA was prepared using the RNAgents system (Promega, Madison, WI). Ten micrograms of total RNA was separated on a 1% formaldehyde agarose gel, blotted onto a Zeta-Probe GT blotting membrane (Bio-Rad, Hercules, CA), and hybridized to radioactively labeled probes according to standard procedures.21 Genomic DNA was prepared by standard procedures and subsequently digested with BamHI, separated on 1% agarose gel, blotted onto Zeta-Probe GT blotting membrane, and hybridized to radioactively labeled Neo-probe. All membranes were analyzed in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).Transplantation of BM-HPC lines and Gpi assays Different numbers of BM-HPCs were transplanted by intravenous injection into lethally irradiated B6-SJL mice or sublethally irradiated (4-5 Gy) B6W mice. Mice were bled at various times after transplantation, and the blood was collected in microhematocrit tubes (Drummond Scientific, Broomall, PA). Erythrocytes were isolated from the erythrocyte fraction after centrifugation, lysed in a 7 mg/mL ethylenediaminetetraacetic acid solution, and subjected to glucose phosphate isomera (Gpi) analysis. Similarly, hemato/lymphoid organs were removed, single-cell suspensions were prepared, and following osmotic shock to eliminate erythrocytes, nucleated cells were lysed in ethylenediaminetetraacetic acid solution and subjected to Gpi analysis. B6-SJL and B6W mice have the Gpi1b allele at the Gpi locus. The Gpi1b type forms a more rapidly migrating band from positive to negative on electrophoresis than does the Gpi1a type. Electrophoresis and detection of the Gpi types was adapted from Eppig et al and Harrison et al.22,23 Gpi assays were scanned in a flatbed scanner (ScanMaker III, MICROTEK, Redondo Beach, CA) and analyzed using the image analyzing program Scion Image (Scion, Frederick, MD). Donor contribution (percent donor) was calculated as % donor = 100 × (intensity Gpi1a band/[intensity Gpi1a band + intensity Gpi1b band]).Serial transplantation Total BM cells were harvested from primary B6W recipients at 4, 7, 9, and 12 months after transplantation, and erythrocytes were eliminated by osmotic shock. One million or 2 million unfractionated nucleated cells were injected intravenously into sublethally irradiated B6W mice or lethally irradiated B6-SJL mice. For transplantation into tertiary recipients, total BM cells were harvested from a secondary B6W recipient 6 months after transplantation. Two million unfractionated nucleated cells from the secondary recipient were injected intravenously into sublethally irradiated B6W mice. The secondary recipient used as a source of cells for serial transplantation into the tertiary recipients had received BM cells harvested from a primary recipient 9 months after transplantation. Gpi analyses of erythrocytes and hematopoietic organs were carried out as described above.Flow cytometry The monoclonal antibodies used in this study were direct conjugates with phycoerythrin (PE), fluorescein isothiocyanate, or biotin. The following antibodies were purchased from Pharmingen (San Diego, CA): anti-CD45.1 (A20), anti-CD45.2 (104), anti-Sca-1 (E13-161.7), anti-Gr-1 (RB6-8C5), anti-c-kit (2B6), anti-Mac-1 (M1/70), PE-TER119, F4/80, anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (1044D), anti-CD19 (1D3), anti-Thy1.2 (53-2.1), anti-H-2Kb (AF-88.5), and anti-CD34 (RAM34). Unspecific antibody binding was prevented by incubating cells on ice in supernatant from the 2.4G2 hybridoma 15 minutes prior to all antibody labeling. The cells were incubated with specific antibodies on ice for 20 minutes, washed twice, and subsequently incubated with PE-conjugated streptavidin (Southern Biotechnology, Birmingham, AL). Labeled cells were washed twice before being analyzed in a FACScan (Becton Dickinson, Silicon Valley, CA).
Generation of cytokine-dependent cell lines from adult BM We previously showed that Lhx2 could immortalize HPCs derived from ES cells differentiated in vitro.12 To elucidate whether Lhx2 also can immortalize hematopoietic progenitor/stem cells derived from adult mice, BM cells from 5-FU-treated B6-cast mice were transduced with virus containing the Lhx2 gene or control vector. Transduced cells were subsequently cultured in the presence of SF or SF/IL-6. After 4 to 5 weeks in culture, a high proportion (> 99%) of the cells transduced with Lhx2 showed blastlike morphology with a large nuclei and a small rim of cytoplasm (Figure 1A). These cultures of expanded blastlike cells are referred to as BM-derived HPC (BM-HPC) lines. The cells transduced with control vector differentiated into neutrophils and mast cells after 3 to 4 weeks of culture, and no viable cells were present after an additional 2 to 3 weeks. The doubling time of BM-HPC lines established in SF or in SF/IL-6 was 40 hours and 22 hours, respectively. However, the lines established in SF/IL-6 did not proliferate in IL-6 alone because after 24 hours in culture in the presence of IL-6 alone, more than 80% of the cells were not viable based on trypan exclusion. Thus, BM-HPC lines are strictly dependent on SF for growth, whereas IL-6 only promotes proliferation in combination with SF. Northern blot analysis revealed that the BM-HPC lines express Lhx2 from the retroviral vector at a similar level as a cell line generated from ES cells differentiated in vitro (Figure 1B). The 2 BM-HPC lines we chose to study further were clonal because they both harbor one unique insertion site of the retroviral vector in the genome (Figure 1C). Cell surface marker analysis of the BM-HPC lines showed that those generated in SF/IL-6 expressed high levels of the mouse HSC marker Sca-1 (Figure 1D, top), whereas the cell line generated in SF alone displayed lower expression of this marker (Figure 1D, bottom). Other cell surface markers analyzed did not reveal any major differences between these cell lines because both were c-kit+, H-2Kb+ (major histocompatibility complex class I), CD45.2+, CD34, Gr-1 (marker for
neutrophils), Mac-1low/ (monocyte/macrophage),
F4/80 (macrophage), TER119 (erythroid),
CD19 (B cell), CD3 (T cell) (Figure 1E).
Both the doubling time and cell surface marker phenotype for the BM-HPC
lines have been stable for at least 10 weeks in vitro. Thus, expression
of Lhx2 in hematopoietic progenitor/stem cells derived from
BM of adult mice generates immortalized and cytokine-dependent
cell lines.
A broad combination of growth factors efficiently promotes proliferation of the BM-HPC lines at low cell density, whereas no significant proliferation was observed in SF or SF/IL-6 Normal hematopoietic progenitor/stem cells only proliferate when cultured in a broad combination of early-acting growth factors.24-26 We have also previously shown that SF-dependent HPCs immortalized by Lhx2 do not respond to SF at low cell density because the SF-dependent proliferation is due to an additional and cell nonautonomous mechanism.20 This prompted us to analyze the response of the BM-HPC lines to different growth factors/growth factor combinations in clonal assays. The total number of colonies generated by the BM-HPCs in clonal assays in the presence SF/Mix (SF/Tpo/IL-3/IL-6/GM-CSF/G-CSF/M-CSF/Epo) was arbitrarily set as 100% and served as reference for all other combinations (Figure 2A). The frequency of colony-forming cells (CFCs) using this factor combination did not differ between the 2 cell lines and ranged between 30 and 40 CFCs per 100 cells plated. The main difference between line nos. 5 and 9 is that the latter is highly responsive to IL-3 because the plating efficiency in SF/IL-3 was 77% for line no. 9 and 5% for line no. 5 (Figure 2A). Thus, line no. 5 required a more complex mix of growth factors for efficient proliferation. None of the cell lines responded to Tpo, Flt3L, or Epo in combination with SF, and M-CSF in combination with SF promoted a low response of line no. 9, where the plating efficiency was low (6%) and the colonies generated were small (< 100 cells). The BM-HPCs reveal a limited potential in vitro because the major cell type in the colonies, independent of factor combination, was large macrophagelike cells expressing Mac-1 (data not shown). Neither line no. 5 nor line no. 9 showed significant proliferation in response to SF or SF/IL-6 at these cell densities (< 104/mL) (Figure 2A). Thus, the BM-HPC lines respond efficiently to a broad combination of growth factors, whereas their response to SF and SF/IL-6 appear to be cell density-dependent similar to the cell lines we generated from Lhx2-transduced ES cells differentiated in vitro.20
The BM-HPC lines have CFU-S activity Immature hematopoietic progenitor/stem cells can generate colonies on the spleen upon transplantation into lethally irradiated recipients.1,27 To test whether the BM-HPC lines have this potential, we transplanted different numbers of the respective cell line into lethally irradiated B6-SJL. Control mice did not show any visible colonies on the spleen after 10 days (Figure 2B), whereas all mice injected with BM-HPCs showed numerous colonies on the spleen (Figure 2C). The estimated frequency of CFU-Ss in the respective cell line was 2 ± 1 per 300 cells for line no. 5 and 4 ± 2 per 103 cells for line no 9.BM-HPC lines are multipotent and generate functional cells in vivo An important characteristic of HSCs is that they should be able to generate mature and functional hematopoietic cells in vivo. One of the most stringent assays to test for function in vivo is to analyze whether the cells can protect mice from radiation-induced death.1,28 Therefore, we transplanted the BM-HPC lines (Gpi1a, CD45.2) into lethally irradiated B6-SJL mice (Gpi1b, CD45.1). Sixty-four percent of the mice that received 3 × 106 BM-HPCs survived the acute effects of irradiation (Figure 3A). All recipients transplanted with less than 3 × 106 cells died within 21 days, and the control mice receiving no cells died within 16 days (Figure 3A). Gpi analysis of erythrocytes in peripheral blood of the surviving mice 1 month after transplantation confirmed the presence of significant numbers of donor erythrocytes in the circulation (Figure 3B). Donor contribution to circulating erythrocytes decreased over time and was undetectable 3 months after transplantation (Figure 3B), at which time endogenous erythropoiesis was completely restored. Flow cytometry analysis of different hematopoietic organs from animals receiving transplants revealed the presence of donor-derived (CD45.2+) leukocytes. Leukocytes generated by the BM-HPC lines were myeloid cells (Mac-1+ and Gr-1+) (Figure 3D,E,K,L), B cells (CD19+) (Figure 3F,I,M), and T cells (CD3+, CD4+/CD8+) (Figure 3G,H,J,N). The Sca-1low line appears to be less efficient in generating lymphoid cells as compared with the Sca-1+ line (compare Figure 3M,N with Figure 3F,G). These findings clearly demonstrate that the BM-HPC lines are multipotent able to generate myeloid cells and lymphoid cells in addition to erythrocytes. The
decrease in donor cells in peripheral blood was followed by a similar
decrease of donor cells in all hematopoietic organs analyzed (BM,
spleen, thymus, lymph nodes), and donor cells were undetectable 3 to 4 months after transplantation (data not shown). Together, these
observations indicate that the BM-HPC lines are multipotent, can
provide some radioprotection, and can short-term repopulate lethally
irradiated wild-type B6 hosts.
BM-HPC lines long-term repopulate stem cell-deficient mice To further investigate the long-term repopulating potential of the BM-HPC lines, they were transplanted into the stem cell-deficient B6W mouse strain, which provides a less competitive environment for engraftment.29 BM-HPC lines were transplanted at different cell doses (3 × 104-3 × 106 cells) into sublethally irradiated B6W recipients, and donor contribution to circulating erythrocytes was monitored regularly. All recipients showed significant donor contribution to erythrocytes in peripheral blood 1 month after transplantation. The proportion of donor-derived erythrocytes ranged from 9% to 85%, roughly correlating to the number of cells transplanted (Figure 4A). All mice receiving 2 × 106 or fewer BM-HPCs lost donor contribution in all hematopoietic organs analyzed within 4 to 5 months after transplantation (data not shown). However, most of the animals receiving 3 × 106 cells had a significant proportion of donor-derived cells in the erythrocyte fraction in peripheral blood for an extended time (Figure 4B). Donor contribution decreased in all recipient mice 2 to 3 months after transplantation, and thereafter it stabilized, increased, or fluctuated. Six of 13 recipients had a donor contribution above 20% during the whole time period, and 7 of 13 recipients had undetectable levels of donor erythrocytes at one or more time points. Six mice analyzed 1 year after transplantation showed significant donor contribution, ranging from 23% to 72% (Figure 4B). Mice killed at various time points, including those showing no donor contribution to peripheral blood (Figure 4C), revealed no significant difference in the donor contribution to nucleated cells in the BM (average 54% ± 8%). These data reveal that all mice receiving 3 × 106 BM-HPCs showed sustained engraftment, suggesting that lack of donor contribution to circulating erythrocytes reflects fluctuations in erythropoiesis that are not due to progenitor/stem cell exhaustion.
BM-HPCs efficiently engraft secondary recipients A unique characteristic of HSCs is their capacity to generate repopulating cells that can be detected following transplantation into secondary recipients.30,31 To test if the BM-HPC lines display this potential, we transplanted 1 × 106 to 2 × 106 BM cells from primary B6W recipients into sublethally irradiated secondary B6W recipients and lethally irradiated B6-SJL recipients. Eight secondary B6W recipients showed a significant fraction of donor-derived erythrocytes in peripheral blood at 1 month after transplantation, and all mice in this group maintained a high donor contribution thereafter (> 70%) throughout the analysis period (Figure 5A). One mouse in this group had 100% donor contribution to circulating erythrocytes 3 months after transplantation, and 3 mice in this group had 100% donor-derived erythrocytes in peripheral blood 6 months after transplantation (Figure 5A). Six secondary B6W recipients did not generate detectable levels of donor erythrocytes in peripheral blood until 4 months after transplantation, and mice in this group analyzed at 7 months after transplantation maintained a significant donor contribution in peripheral blood. In one mouse we did not detect donor-derived erythrocytes until 6 months after transplantation, and this mouse maintained a significant donor contribution at 8 months after transplantation (Figure 5A). Six of 13 secondary wild-type B6-SJL recipient animals showed a significant fraction of donor-derived erythrocytes in peripheral blood 3 months after transplantation, ranging from 36% to 58% (Figure 5B). Four of 5 secondary wild-type B6-SJL mice analyzed 4 months after transplantation showed significant donor contribution to circulating erythrocytes, ranging from 30% to 51% (Figure 5B). This is in contrast to the primary B6-SJL recipient animals where no donor-derived erythrocytes could be detected beyond 2 months after transplantation (compare Figure 5B with Figure 3B). Secondary B6W and B6-SJL recipients lacking donor-derived erythrocytes in peripheral blood had at least 50% donor contribution to nucleated cells in BM (Figure 5C). This observation supports the idea that lack of donor contribution to erythrocytes reflects fluctuations in erythropoiesis and is not due to progenitor/stem cell exhaustion.
BM-HPCs engraft tertiary recipients An additional property that has been used to characterize HSCs is their ability to generate repopulating cells in the secondary recipient that can be detected following transplantation into tertiary recipient.32,33 To test whether the BM-HPCs also have this property, 2 million BM cells from a secondary B6W recipient were serially transplanted into sublethally irradiated B6W tertiary recipients. Analysis of the tertiary recipients revealed that all 7 mice analyzed had significant donor contribution to circulating erythrocytes at 2 months after transplantation, ranging from 24% to 44% (Figure 6A). Five of the tertiary recipients analyzed at 3 months after transplantation had donor-derived erythrocytes in peripheral blood, ranging from 49% to 72% (Figure 6A). Morphologic analysis of the nucleated cells in the BM of tertiary recipients showed numerous myeloid cells (Figure 6B). Gpi assays of the nucleated cells in the BM of the tertiary recipients revealed a high proportion (> 90%) of donor cells (Figure 6C), indicating that other hematopoietic lineages in addition to the erythroid lineage were generated in the tertiary recipients. Thus, similar to normal HSCs, the BM-HPC lines can generate repopulating cells in the secondary recipient that can be detected by serial transplantation into tertiary recipients.
Transplanted mice are engrafted with transduced cells To confirm that the cells of donor Gpi type in engrafted mice were transduced with the retroviral vector, genomic DNA prepared from hemato/lymphoid organs of primary, secondary, and tertiary recipient animals was analyzed for the presence of proviral sequence. A proviral insertion could be detected in genomic DNA derived from spleen and BM of all mice receiving transplants showing donor cells based on the presence of Gpi1a marker in these organs (Figure 7 and data not shown). Mice with donor cells in the BM as determined Gpi assays also had a significant fraction of G418-resistant (G418R) CFCs in the BM (Table 1), further supporting that the donor cells contained retroviral DNA. Furthermore, the integration site of the provirus in the genome of cells in different hemato/lymphoid organs of engrafted primary, secondary, and tertiary recipients appears to be identical to that of the original cell line (Figure 7A-B). These results show that cells engrafting primary, secondary, and tertiary recipients are derived from the original hematopoietic progenitor/stem cell transduced with Lhx2 and expanded in vitro.
Expression of the LIM-homeobox gene Lhx2 in adult hematopoietic progenitor/stem cells reproducibly generates multipotent hematopoietic progenitor/stem cell lines immortalized by a similar mechanism as previously described for hematopoietic progenitor/stem cells derived from ES cells differentiated in vitro.12,20 The BM-HPC lines have some radioprotective properties and can generate erythroid, myeloid, and T and B lymphoid cells upon transplantation. The BM-HPC lines also demonstrate robust contribution to the mature erythrocyte population in stem cell-deficient primary, secondary, and tertiary recipients mice for an aggregate time of at least 18 months, revealing a remarkable potential for self-renewal and differentiation in vivo. Our results also indicate that recipients can survive when the erythrocytes are exclusively derived from the cell line, because in some instances 100% of the circulating erythrocytes in the stem cell-deficient animals receiving transplants were of donor origin. This strongly suggests that the BM-HPC lines produce functional erythrocytes in vivo. Collectively, these characteristics make the BM-HPC lines unique in comparison with the previously described immortalized stem cell-like cell lines.7-11 Although the data presented suggest that the BM-HPC lines are HSC-like, the BM-HPC lines are less efficient in engrafting immunocompromised mice as compared with normal HSCs. Because Lhx2 is most likely not expressed in HSCs,34 the ectopic expression of Lhx2 in HSCs may alter these cells to reduce their fitness in vivo. This idea is supported by the observations that they can long-term repopulate in a less competitive environment provided by the BM of a stem cell-deficient host. The dependence on a certain host environment for long-term engraftment is unexpected because the difference between so-called long-term and short-term repopulating stem cells has been suggested to be due to intrinsic differences in their ability to self-renew.35 Whether properties distinct from intrinsic capacity to self-renew between different subpopulations of HSCs can contribute to the difference between long-term and short-term repopulating stem cells remains to be elucidated. Normal HSCs show decreased efficiency in engrafting upon serial transplantation that is suggested to be caused by intrinsic changes and/or to dilution of HSCs.36-39 However, engraftment of BM-HPCs in the secondary and tertiary recipients appears to be more efficient than in the primary recipients because reproducible engraftment was observed in all secondary and tertiary recipients with fewer BM-HPCs as compared with the number injected into primary recipients. Furthermore, the BM-HPCs could also contribute to a significant proportion of circulating erythrocytes in secondary wild-type recipients 4 months after transplantation, whereas primary wild-type recipients never showed donor contribution in peripheral blood beyond 2 months after transplantation. These observations suggest that the BM-HPC lines acquire characteristics in the BM environment that increase their ability to compete with host cells. The nature of this change of phenotype is at present unknown, but it is further supported by the observation that we have thus far been unable to reestablish any type of cell line from animals receiving transplants, suggesting that the change is intrinsic to the BM-HPCs. Another striking observation is the homogeneity of the BM-HPC lines
despite being generated from such a heterogeneous cell population as BM
from 5-FU-treated mice. Also, the third independently generated BM-HPC
line (line no. 28, Figure 1A) shows the identical pattern of cell
surface marker expression as BM-HPC line no. 5 (eg,
Sca-1+/c-kit+/CD34
We thank Dr Sara Wilson for critical reading of the manuscript.
Submitted September 20, 2001; accepted January 23, 2002.
Supported by the Swedish Cancer Society and the Tobias Foundation. K.R. was supported by the Foundation for Strategic Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Leif Carlsson, Dept of Molecular Biology, Umeå University, 901 87 Umeå, Sweden; e-mail: leif.carlsson{at}micro.umu.se.
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Abstract
Introduction
50%) to nucleated cells in the BM. Bands
corresponding to the respective Gpi isoforms are indicated.
