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HEMATOPOIESIS
From the University of Michigan, Department of Internal
Medicine, Ann Arbor; the Stowers Institute for Medical Research, Kansas
City, MO; the University of Washington, Departments of Pediatrics,
Microbiology, Surgery, and Molecular Biotechnology, Seattle; the
University of Bergen, Department of Pathology, Norway; the University
of Alabama at Birmingham, Department of Microbiology; Stanford
University, Department of Pathology, Stanford, CA; and the Institute
for Systems Biology, Seattle, WA.
Hematopoietic stem cells (HSCs) have self-renewal capacity and
multilineage developmental potentials. The molecular mechanisms that
control the self-renewal of HSCs are still largely unknown. Here, a
systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To
enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs
and a mixed population of stem and early multipotent progenitor (MPP)
cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA
clones with very low hybridization signals were selected for sequencing
and further analysis using microarrays on glass slides. Two populations
of cells, HSCs and MPP cells, were compared for differential gene
expression using microarray analysis. HSCs have the ability to
self-renew, while MPP cells have lost the capacity for self-renewal. A
large number of genes that were differentially expressed by enriched
populations of HSCs and MPP cells were identified. These included
transcription factors, signaling molecules, and previously unknown genes.
(Blood. 2002;99:488-498) Hematopoiesis is a dynamic process with significant
complexity in which a subset of hematopoietic stem cells (HSCs) gives rise to cells of both the myeloid and lymphoid lineages.1
In addition, HSCs have the ability to self-renew to produce more HSCs.
This property allows HSCs to repopulate the bone marrow of lethally
irradiated congenic hosts.
Mouse HSCs isolated with
Thy1.1loc-kit+Sca-1hiLin The rarity of the HSCs, coupled with an inability to maintain these
cells in culture for a significant period, has greatly hindered the
biochemical and molecular characterization of these cells. Although
various growth factors can induce stem cells to proliferate in vitro,
no known combination of growth factors has been shown to promote
sustained self-renewal of the HSCs. Rather, stem cells induced to
proliferate in vitro invariably undergo concomitant differentiation.
HSC functions are thought to be regulated by interactions of stem cells
with stromal cells.14 The dynamic process of HSC
development, including self-renewal, expansion, and maturation,
involves interactions between the intrinsic genetic program and
extrinsic signals from stromal cells. This dynamic process is
accompanied by global changes in gene expression profiles involving
molecular events such as signal transduction, transcriptional and
translational regulation, and chromatin modification. In the present
study, we intend to systematically look into these molecular events by
analyzing the gene expression profiles of self-renewing HSCs and
non-self-renewing MPP cells. Therefore, any changes in the components
of these aforementioned molecular events may provide insight into
understanding the molecular mechanisms in controlling self-renewal of HSCs.
Isolation of HSCs and generation of cDNA libraries
The cDNA library A was constructed from 1.3 × 106
Lin The cDNA library B was made from 2.8 × 104 twice-sorted
Lin Probe preparation for macroarray and microarray analyses
For preparation of cDNA probes used for microarray hybridization,
c-kit-phycoerythrin (PE) (2B8) and Sca-1-biotin
(D7)/Streptavidin-Cy-Chrome (Pharmingen) were used to stain
Lin Macroarray preparation, hybridization, and analysis To prepare for macroarray, the 2 HSC cDNA libraries, A and B, described above were plated onto LB agarose/ampicillin plates in 24 × 24-cm Q-trays (Genetix, Christchurch, England) with densities of 500 to 2000 colonies per tray, and incubated overnight at 37°C. The bacterial colonies were then picked up by a Q-bot robotic system (Genetix) and inoculated into 384-well plates. Thus, each cDNA clone was addressed by its position in the plate. The macroarrays were generated by spotting bacterial colonies from 384-well plates onto 24 × 24-cm nylon membranes using Q-bot (Genetix). Thirty-six thousand cDNA clones from library A and 18 000 clones from library B were spotted in duplicate, and the position of each clone on the membrane was recorded according to its address in the 384-well plate. These macroarray membranes were incubated on the top of LB-agarose plus antibiotics at 30°C overnight in Q-trays. DNAs were denatured by soaking the membrane in 0.5 N NaOH/1.5 M NaCl for 10 minutes and neutralized with 0.5 M Tris-Cl, pH 8.0/1.5 M NaCl for 5 minutes. DNAs were fixed by ultraviolet (UV) crosslinking (UVP, Upland, CA). For hybridization, membranes were prehybridized for 2 hours at 60°C in 100 mL solution (12.5 mM PEG8000; 250 mM NaCl; 85 mM Na2HPO4; 7.5 mM H3PO4 [86%]; 243 mM sodium dodecyl sulfate [SDS]; 10 mM ethylenediaminetetraacetic acid [EDTA]; 32 mM NaOH, pH 7.2) containing 100 µg/mL of denatured salmon sperm DNA and 100 µg/mL denatured mouse Cot-1 DNA. Denatured 33P-labeled probes from mature cell lineages were added to the prehybridized solution and incubated overnight in roller bottle or shaking bath at 60°C. Membranes were washed twice with 2x sodium chloride sodium citrate (SSC)/1% SDS at room temperature for 15 minutes, and then once in 0.2x SSC/0.1% SDS at 60°C for 30 minutes. Membranes were exposed to a phosphor imager screen and scanned using a phosphoimage scanner (Molecular Dynamics, Sunnyvale, CA). The hybridization result was analyzed using Spotfinder for image processing (H. Hammersmark and R.B., University of Washington, Seattle). Since each cDNA clone was spotted in duplicate, an average signal intensity of the duplicate was used. Local background hybridization signals surrounding each spot were subtracted prior to comparing spot intensity. Quantitative comparison of signal intensities was analyzed using Microsoft Excel. In order to avoid false-negative signals generated from slow or nongrowing bacterial colonies, a probe derived from the vector sequence was hybridized to the macroarray in parallel. The negative selection process was performed by selecting cDNA clones with positive signals for vector hybridization and negative or very weak signals for the cDNA probes derived from lineage-positive cells. Using this process, 3000 of 36 000 cDNA clones and 2000 of 18 000 cDNA clones were selected from library A and B, respectively, for microarray analysis.Microarray preparation, hybridization, and analysis cDNA inserts from 5000 clones selected using macroarray analysis described above were PCR-amplified using primers derived from the vector. For library A, ZAP-F (5'-AGTGGATCCAAAGAATTC-3') and ZAP-R (5'-CTCTAGAAGTACTCTCGAG-3') were used, and for library B, SubA1 (5'-CTTCGAACCGCGGATATCAGATC-3') and SubS2 (5'-AAGGTTCCTTCACAAAGATCCCTCGAG-3') were used. Amplified PCR products were purified using Sephacryl S500 (Pharmacia, Peapack, NJ), mixed 1 to 1 with dimethyl sulfoxide (DMSO) (Amersham, Piscataway, NJ), and spotted in duplicate onto each 75 × 25-mm coated type 7 microarray slide (Amersham) using Genespoter II (Molecular Dynamics). The microarray slides were air-dried and DNA was fixed by UV crosslinking at 500 mJ. The microarray slides were hybridized with Cy3- and Cy5-labeled probes described above. Briefly, the purified probes were concentrated with a speed-vacuum in the dark and resuspended in 20 µL hybridization buffer (5x SSC, 5x Denhardt, 0.1% SDS, 50% formamide, 100 µg/mL denatured salmon sperm DNA, 20 µg/mL polyA60 RNA, 100 µg/mL mouse Cot I DNA). An equal amount of Cy3- and Cy5-labeled probes were combined, denatured, and added onto the microarray slides, and hybridized at 42°C in a hybridization oven or in a humidified chamber for 16 hours to 18 hours. Microarray glass slides were washed twice with 2x SSC/0.1% SDS, and 0.2x SSC/0.1% SDS at 55°C for 5 minutes, and then with 0.1x SSC at room temperature for 1 minute. Glass slides were immersed in water for 10 seconds and dried immediately with N2. The hybridized microarray was scanned with a confocal dual-laser scanner at 523 nm and 633 nm (Molecular Dynamics) and analyzed using the custom array analysis software developed at the University of Washington (H. Hammersmark and R.B.). This array analysis software includes image processing, data normalization, and error analysis as described previously.16Reverse transcriptase-polymerase chain reaction Reverse-transcription reactions were carried out using SuperScriptII following the manufacturer's manual (Invitrogen). The following primers were used for PCR: Activin C,
5'-GACACCTTACTCTGGAGCTG-3' and 5'-GGGAGGCAGA GTAGATTACA-3'; BA_RA6B66,
5'-TCATGGTATGCCCTCGTGTA-3' and 5'-AAATGTGTGGGCTTTTCAGG-3';
BA_RA5A82, 5'-AACATGGCTTGGAGAC AACC-3' and 5'-AAGCCTGGGATTCACTGCTA-3';
BA_RA4B58, 5'-ATGAGGGC CATTGTTACACC-3' and 5'-TTATGGCCAGCTTGGTTCAC-3';
MSC_RA15C76, 5'-TGATACCCTTGGCTCGAAAC-3' and 5'-CCAAGTGCTGGGATTAAAGG-3';
MSC_RA16D13, 5'-GGCTCGAAATTAACCCTCAC-3' and 5'-CCAGATCTCGT
TACGGATGG-3'. The PCR condition was 35 cycles consisting of 94°C for
30 seconds, 55°C for 40 seconds, and 72°C for 1 minute.
Competitive repopulation assay Two hundred fifty cells (either RhloLin /loSca-1+c-kit+
or
RhhiLin/loSca-1+c-kit+)
from Ly5.2 mice were mixed with 4 × 104
Lin/lo cells from congenic host Ly5.1 mice17
and were then transplanted into lethally irradiated (11 Gy [1100
rads]) Ly5.1 mice by intravenous (IV) injection. Each group included 8 mice. Peripheral blood cells were harvested at different time points
after transplantation, as indicated in Figure 2C, and flow cytometric
analyses were performed using different specific cell surface markers
(CD3, B220, Mac-1, and Gr-1) together with anti-Ly5.2 antibody.
Construction and normalization of HSC cDNA libraries In order to enrich for genes that are predominantly expressed in the uncommitted, or lineage-negative cell populations, and remove highly redundant and housekeeping genes for further analysis, high-density array18 (macroarray) analysis was used and cDNA clones that hybridized to the lineage-positive probes were removed. cDNA clones that had no or very weak hybridization signals to the lineage-positive probes were selected. These negatively selected cDNA clones were subsequently subject to analysis of differential gene expression using a microarray system.19 The strategy for using macroarrays and microarrays to analyze the gene expression patterns of the HSC is illustrated and described in Figure 1A. First, 2 populations of HSCs were isolated from mouse bone marrow cells by FACS based on their cell surface antigen expression: the Lin/loSca-1+
population (A) containing MPP cells, LT-HSCs, and
ST-HSCs20; and the
Lin/loc-kit+Thy-1loSca-1+
population (B) enriched with LT-HSCs.5 Correspondingly,
cDNA libraries A and B were generated from each of these 2 complementary populations of HSCs. Sequence analysis of 200 randomly
chosen clones from each library indicated that they are very
diverse. Bacterial colonies derived from these HSC cDNA libraries were arrayed onto nylon membranes using the Q-bot robotic system to generate
the macroarrays, or high-density cDNA arrays. These macroarrays were
hybridized with the cDNA probes generated from committed or mature
hematopoietic cell lineages of the thymus, bone marrow, and spleen
(lineage-positive probes). Clones that hybridized to the
lineage-positive probes were eliminated through the process of negative
selection (see "Materials and methods") and the remaining clones
were selected and spotted onto microarray type glass slides. These
microarrays were used to analyze the gene expression profiles of HSCs
and MPP cells by hybridizing with fluorescent-labeled (Cy3 and Cy5)
cDNA probes derived from the sorted cell populations of HSCs and MPP
cells. This allowed the identification of genes preferentially
expressed in the HSCs and MPP cells, respectively.
To generate the macroarrays of cDNA libraries A and B, 36 000 clones
from library A (made from Lin The sequence analysis of the 2000 negatively selected clones from
library B provides a glimpse of the gene expression profile of adult
bone marrow enriched with HSCs. A partial annotated list of genes
identified in cDNA library B is shown in Table
1. Numerous gene encoding transcription
factors, chromatin modifiers, transmembrane proteins, and signaling
molecules were identified. Based on their molecular and cellular roles
in other tissues, contexts, or species, these genes might be likely
candidates for playing an essential role in stem cell functions. For
example, the bmi-1 gene is a Polycomb-group gene that
represses expression of Hox genes during development, and
regulates cell proliferation and senescence by down-regulating the
ink4a locus.21 The ink4a locus
expresses 2 genes by alternative splicing: p16 is a G1 cyclin inhibitor and p19ARF stabilizes expression of p53 by binding to Mdm2.
Constitutive expression of both bmi-1 and telomerase results
in immortalization of epithelial cells.22 Telomerase is
activated in most immortal cell lines and cancers, and possibly is
involved in the regulation of HSC self-renewal.23 The
Notch family has been implicated in the determination of HSC
fate.24-26 Recently, constitutive expression of the
activated form of Notch1 was shown to be able to immortalize pluripotent, cytokine-dependent HSCs.27
HSCs express genes that are shared by other tissues such as brain, muscle, and germ line cells (Table 1). For example, male-enhanced antigen (Mea) specifically expressed in the testis is involved in spermatogenesis.28 Some of the genes we identified are specific to hematopoietic tissues; for example, RGS18 is expressed in LT-HSCs and ST-HSCs as well as monocytes.15 Among unknown genes, there are genes that code for a variety of important functional domains and several genes with no obvious homology to any known functional domains. Some of the other unknown genes were analyzed by Northern hybridization. Of 10 unknown clones tested, 2 were expressed in all tissues (no. 1), 2 were expressed in some tissues (no. 26), and 6 had no detectable signals in any of the tissues examined (no. 8, no. 11). These 6 clones are candidates for HSC-specific genes (Figure 1B). Previously, Philips and his coworkers described the gene expression
profile of
Sca-1+AA4.1+c-kit+Lin
Differential gene expression between HSCs and MPP cells Since Rh staining allows for separation of HSCs into populations enriched for ST-HSC/early progenitor cells and LT-HSCs,9-11 the Lin/loSca-1+c-kit+ cells were
further separated into Rhlo and Rhhi
populations using flow cytometry (Figure
2A-B). In order to avoid a potential
interference from the intermediate staining of Rh (Rhitm)
cells, we chose a symmetrical portion of cells containing either the
15% highest staining or 15% lowest staining for Rh. A competitive repopulation assay was performed to confirm the functional differences between these 2 populations of cells. As Figure 2C shows, the engraftment rate using Rhlo cells is much higher than that
using Rhhi cells after transplantation. In addition, the
Rhlo cells could support hematopoiesis for up to 6 months
after transplantation (Figure 2C) and were able to reconstitute the
bone marrow in the secondary transplantation (F. L. and L. L., data not shown). The Rhhi cells, which gave rise to
both myeloid and lymphoid lineages, only engrafted the bone marrow for
less than 4 weeks (Figure 2C). This result demonstrated that we had
obtained 2 distinct cell populations: the Rhlo population
of cells were enriched for HSCs and the Rhhi population of
cells were enriched for MPP cells.5
To study differential gene expression among Rhlo and
Rhhi cells, we used the microarray glass slides on which
the 5000 negatively selected cDNA clones from both libraries A and B
had been arrayed in duplicate. These microarrays were hybridized to
Cy3- and Cy5-labeled probes generated from Rhlo and
Rhhi cells, respectively. Figure
3A shows a typical hybridization result
showing differentially expressed genes between the Rhlo and
the Rhhi cells. Rhlo-Cy3 (green) represents
genes that are expressed higher in the enriched HSCs.
Rhhi-Cy5 (red) represents genes that are expressed higher
in the enriched MPP cells. Yellow represents genes that are expressed
in a relatively similar level in both cell populations. Figure 3B shows
the results of statistical analysis based on 2 experiments with 4 points (2 points on each slide) representing each cDNA clone, and
selection using SpotUnite and SpotSelection software (H. Hammersmark
and R.B., see "Materials and methods"). The vertical axis
represents the Cy3/Cy5 signal intensity ratio and the horizontal axis
was sorted by mean ± standard deviation (sd) (mean indicates
average signal intensity of 4 spots for each cDNA insert). We chose
cutoff lines that were either 2-fold higher or 2-fold lower for the
Cy3/Cy5 intensity ratio. Thus, genes highly expressed in the
Rhlo population fell into the upper (positive) region and
genes highly expressed in the Rhhi population fell into the
lower (negative) region. The spots that fell very close to the left
axis represent cDNAs with very weak hybridization signals and, along
with spots of significant variation, were ignored. Using this strategy
we have identified approximately 30 genes that were highly expressed in
the LT-HSCs and around 30 genes that were highly expressed in the MPP
cells. These clones were marked with error bars (yellow, Figure 3B). Of
these, some were not found in the nonredundant database and therefore
were defined as novel or functionally unknown genes (Table 2).
To confirm the differential expression of selected genes using
microarray analysis, RT-PCR was performed and 15% of the clones were
confirmed to be expressed preferentially in HSCs. Activin
The differentially expressed genes fall into a variety of gene families (Table 2). Among the genes that are highly expressed in HSCs are several clones identified as transcription factors. Nfix belongs to the conserved nuclear factor I (NFI) family of transcription/replication proteins. Loss of function of Nfia, one of 4 Nfix family members, causes severe developmental defects, suggesting this family may have distinct roles in vertebrate development.31 Two novel genes that encode a PI3 kinase and a PKA kinase were also identified. The homologue of PKA-like kinase in Schizosaccharomyces pombe, Kin, was found to be important for growth polarity.32 PI3 kinase can be activated by c-kit/stem cell factor receptor signaling and is essential for male fertility.33 Furthermore, PI3 kinase has been known to be essential for cell survival.34 RBM3, an RNA-binding protein, is up-regulated by granulocyte macrophage-colony-stimulating factor (GM-CSF)35 and is closely related to the Y chromosome ribonucleic acid recognition motif (YRRM).36 YRRM has been implicated in azoospermia, a male infertility disease, and spermatogenesis.36 This suggests that RBM3 might be involved in the regulation of HSC development. Histone acetyltransferase 1 (HAT1) is also highly expressed in the Rhlo cells. HAT1 belongs to the GCN5-related N-acetyltansferase superfamily and functions to acetylate histone H4, thereby participating in chromatin-related transcriptional regulation.37 Recently HAT1 was found to be involved with telomeric silencing by the transcriptional repression of telomere-proximal genes.38 In contrast, Set oncoprotein, a component of a recently identified protein complex, INHAT, that inhibits the activity of HAT,39 was highly expressed in the Rhhi cell population. Among the genes that are highly expressed in Rhhi cells is
the growth factor Activin We also identified approximately 70 genes that are expressed in both LT-HSCs and MPP cells (Table 3). Among these is the transcription factor GCN5, a histone acetyltransferase, which interacts with Notch 1 and plays an important role in the recombination recognition sequence binding protein J (RBP-J)-mediated transactivation by Notch 1.47 Loss of GCN5 leads to increased apoptosis and mesodermal defects during mouse development.48 Fiz1, a zinc finger protein with 11 C2H2-type zinc fingers, interacts with receptor tyrosine kinase Flt3.49 Flt3 has been shown to play a role in proliferation and survival of hematopoietic progenitor cells as well as differentiation of early B-lymphoid progenitors, dendritic cells, and natural killer cells.50 TOK-1, a POU-domain-containing transcription factor, is a p21 C-terminal-binding protein and preferentially binds to an active form of cyclin-dependent kinase 2 (CDK2) via p21.51 p21 is a negative cell-cycle regulator and is involved in maintaining the quiescent state of HSCs.52 One of the identified apoptosis-related molecules is survivin. Survivin is an inhibitor of apoptosis and is overexpressed in various human cancers but undetectable in normal tissues.53 Overexpression of survivin resulted in an accelerated S phase shift, resistance to G1 arrest, and activated Cdk2/Cyclin E complex leading to Rb phosphorylation.54
The mechanisms that regulate the self-renewal of HSCs are largely unknown. The deterministic model of hematopoiesis suggests that HSC self-renewal is autonomous; thus, expression of specific sets of genes could determine the stem cell fate. Therefore, a systematic approach was used to study differential gene expression profiles in adult HSCs. The ability to isolate a cell population enriched with HSCs allowed us to study the gene expression profile of HSCs. Using macroarray and microarray techniques, we were able to obtain a snapshot of genes expressed by enriched populations of HSCs as well as MPP cells. The identification of multiple genes that regulate proliferation, cell survival, immortalization, and differentiation gives new insights into HSC functions. This is important not only to study their biologic behaviors but also for potential clinical applications. For example, the efficacy of treatment modalities for cancer, such as radiation therapy and many chemotherapy agents, is constrained by dose-limiting bone marrow toxicity. Therefore, the ability to isolate HSCs with self-renewal and repopulating potentials, in vitro amplification of these cells, and transplantation without tumor cells would greatly increase the success rate of current cancer therapies. Ex vivo-expanded stem cells have been extensively tested for possible use in transplantation. Soluble jagged-1, a Notch ligand, induced the survival and expansion of human stem cells with multipotent repopulating capacity.55 The presence of Notch 1 in the HSC library suggests that these proteins may play a role in self-renewal or maintaining identity of HSCs. Although various growth factors can induce stem cells to proliferate in vitro, they result in concomitant differentiation. The function of stem cell factor (SCF) has been known as a hematopoietic stem cell survival factor.56 Recently, it has also been shown that the in vitro culture of the HSCs, in the presence of both SCF and thrombopoietin (TPO), induced self-renewal cell division in which only one of the daughter cells had self-renewal potential.57 Asymmetric cell division requires unequal segregation of cell-fate determinants, such as mRNAs and proteins, which are important to maintain self-renewal and repopulating potentials, during mitosis. This type of asymmetric cell division is also observed in germline cell division. During Drosophila oogenesis, a germline stem cell divides asymmetrically to produce a daughter stem cell and a cytoblast, which further divides to eventually produce oocytes. This process is controlled by several factors including decapentaplegic (DPP)58 as well as by intrinsic mechanisms involving pumilio, nanos, arrest, and bag-of-marbles. Expression of BMP4, the mammalian homologue of DPP, in HSCs (Table 1) suggests that BMP4 may also play a similar role. The differential gene expression pattern between Rhlo
and Rhhi indicated that some of the genes were either HSC-
or MPP-specific (Table 2), and many of them were expressed in both
populations of cells (Table 3). Some of the MPP highly expressed genes
included Activin In summary, cDNA libraries were generated from mouse HSCs with long-term reconstituting potential and from progenitor cells, and the differential gene expression patterns were studied using bioinformatics and array technologies. Although the microarray system has been known for its tendency to lose less abundant mRNAs due to limited resolution, our approach overcomes this tendency by removing cDNAs that show high hybridization signals using macroarray and thereby enriching for cDNAs derived from less-abundant mRNAs. On the other hand, our subtraction technique using cDNA probes derived from mature populations of cells may lead to a loss of molecules that are expressed in multiple developmental stages. The systematic approach used in this study provides an effective and efficient method for identification of genes that are specifically expressed in HSCs with self-renewal capability. This study will therefore provide a fundamental tool for identifying important candidate genes involved in the regulation of self-renewal and expansion of HSCs.
We thank G. van den Engh, K. Allen, and D. Corden for the assistance of cell sorting. We thank E. Hammersmark for the assistance on microarray analysis software. We appreciate V. Ng and X. Tao for their assistance on analysis of Est sequences. We thank A. Banta for the involvement of macroarray work. We are grateful to Drs G. Vassilopoulous, D. Russel, and N. Wolf for the consultation on the bone marrow transplantation and repopulation assay. We appreciate the help of R. Krumlauf, B. Steenhard, and D. Stenger on the manuscript proofreading. We are particularly grateful to X. He, J. Zhang, and C. Niu for their work on the reanalysis of cDNA sequences listed in the tables.
Submitted June 8, 2001; accepted September 14, 2001.
Supported in part by National Institutes of Health grant 1P01 DK53074-02 and Stowers Institute for Medical Research.
I.-K.P. and Y.H. contributed equally to this article.
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: Linheng Li, Stowers Institute for Medical Research, 1000 E 50th St, Kansas City, MO 64110; e-mail: lil{at}stowers-institute.org.
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Top
Abstract
Introduction
chain
(B12-1); lineage negative (Lin
ZAPExp
vector. The plasmids (pBK; Stratagene, La Jolla, CA) bearing the cDNA
inserts were excised from their parent vector 
indicates
Rhlo lymphoid lineage; 
, Rhlo myeloid
lineage; 
, Rhhi lymphoid lineage; 
,
Rhhi myeloid lineage.
A Developmental Approach. Oxford United Kingdom: Oxford University Press; 2001:35-47.