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Prepublished online as a Blood First Edition Paper on September 5, 2002; DOI 10.1182/blood-2002-06-1780.
PLENARY PAPER
From the Department of Cancer Immunology and AIDS,
Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA;
Stowers Institute for Medical Research, Kansas City, MO; and Department
of Mathematics and Statistics, University of Missouri, Kansas City.
Hematopoietic stem cells (HSCs) maintain hematopoiesis by giving
rise to all types of blood cells. Recent reports suggest that HSCs also
possess the potential to generate nonhematopoietic tissues. To evaluate
the underlying mechanisms in the commitment of HSCs into multitissue
and multihematopoietic lineages, we performed oligonucleotide array
analyses targeting for prospectively purified HSCs, multipotent
progenitors (MPPs), common lymphoid progenitors (CLPs), and common
myeloid progenitors (CMPs). Here we show that HSCs coexpress multiple
nonhematopoietic genes as well as hematopoietic genes; MPPs coexpress
myeloid and lymphoid genes; CMPs coexpress myeloerythroid, but not
lymphoid genes, whereas CLPs coexpress T-, B-, and natural
killer-lymphoid, but not myeloid, genes. Thus, the stepwise decrease
in transcriptional accessibility for multilineage-affiliated genes may
represent progressive restriction of developmental potentials in early
hematopoiesis. These data support the hypothesis that stem cells
possess a wide-open chromatin structure to maintain their
multipotentiality, which is progressively quenched as they go down a
particular pathway of differentiation.
(Blood. 2003;101:383-389) Hematopoietic stem cells (HSCs) are clonogenic cells that possess
properties of both self-renewal and multilineage potential, giving rise
to all types of mature blood cells.1 Recent reports suggest that murine bone marrow fractions that are enriched for HSCs
can give rise to nonhematopoietic tissues including neural cells,
hepatocytes, myocytes, muscle tissue, and multiple organ tissues (for
reviews, see Graf,2 Goodell et al,3 and
Lagasse et al4). Thus, it is suggested that HSCs possess
the potential for differentiating into nonhematopoietic tissues,
although the plasticity of somatic stem cells is still under question
according to recent reports.5,6 Transdifferentiation from
HSCs into nonhematopoietic tissues suggests that HSCs maintain
accessibility to multiple differentiation programs for nonhematopoietic
as well as hematopoietic systems. Therefore, systematic analyses of
gene expression profiles at various stages of physiologic hematopoiesis initiating from HSCs may provide insight into understanding
developmental potential and plasticity of hematopoietic stem and
progenitor cells.
Changes in chromatin structure, allowing access for RNA polymerase to
initiate transcription, are essential for genetic programs to be
transcribed.7,8 The activation of chromatin remodeling can
occur prior to significant expression of genes.9,10 It has
been hypothesized that a wide-open chromatin structure is maintained in
early hematopoietic progenitors, enabling access to
multilineage-affiliated programs.11 This may lead to
"promiscuous" expression of genes affiliated with multiple lineages
in stem or progenitor cells prior to their lineage determination. In
fact, the coexpression of myeloerythroid genes, including
myeloperoxidase (MPO) and In this report, we systematically profiled gene expression in
rigorously purified self-renewing HSCs,15,16
non-self-renewing multipotential progenitors (MPPs),15,16
and lineage-restricted CLPs17 and CMPs.18 We
found that HSCs possess transcriptional accessibility for multiple
differentiation programs for nonhematopoietic as well as hematopoietic
systems. Promiscuous expression of lineage-related genes decreases
progressively as cells lose their multipotentiality and become lineage
restricted. These data support the concept that HSCs maintain a
wide-open chromatin structure that may allow HSCs to access
nonhematopoietic as well as hematopoietic developmental programs at
least at the transcriptional level, and that lineage potential is
hierarchically controlled by stepwise-regulated epigenetic programs
that guide transcriptional accessibility specific for each
hematopoietic stage.
Isolation and characterization of stem/progenitor cells
RNA purification, labeling, and hybridization
Single-cell RT-PCR Single-cell RT-PCR was carried out based on a published procedure12 with the following modifications. (1) Single cells of HSCs and MPPs were directly triple sorted into 96-well arrays of 0.2-mL microamp tubes. (2) The lysis buffer contained 0.5% Triton X-100 instead of 0.4% NP-40. Primer information used in this assay can be obtained on request.Data analysis Pearson correlation coefficient.
Let yjk represent the
expression level of the jth gene in kth sample,
here k = 1,...m, and
j = 1,...,n, with m = 4, and
n = 24 818 in our sample data. Let k = 1
correspond to the sample gene expression observed in HSCs,
k = 2 in MPPs, k = 3 in CLPs, and
k = 4 in CMPs. The Pearson correlation coefficient
between any 2 samples is given by
Cutoff line and basal levels. The analysis software (Affymetrix) that converts raw hybridization intensities into expression levels ("average difference" in Affymetrix terms) for each gene is based on the comparison between the hybridization signals of perfect match (PM) and mismatch (MM).22 Thus, many negative values were obtained if the MM value was higher than the PM value, making it difficult to compare the expression patterns between 2 or more conditions when one of the conditions is a negative value. Therefore, we converted all negative values to a positive 20, using 20 as the background level.23 To estimate how many genes were expressed in each population of cells, "expressed" was defined as the expression level of a given gene being more than 100.23 Prescreening using screening filter.
The genes in our microarray data were considered as differentially
expressed and were screened for clustering analysis if they passed the
filter given by
|yj(m) K-means clustering.
The K-means clustering method groups items together according to their
similarity. The similarity/dissimilarity of the ith and
jth genes is given by the euclidean distance between the 2 observations:
Gene expression in purified cells The target populations of this study include HSCs,15,16 MPPs,15,16 CLPs,17 and CMPs18 (Figure 1). MPPs can generate both lymphoid and myeloid cells but do not have self-renewal activity.15,16 CLPs give rise to T, B, and NK cells but not myeloid cells, and at least contain clonogenic progenitors for T and B cells.17 CMPs exclusively generate myeloid cells and more than 60% of single CMPs were demonstrated to give rise to both MegE and GM components.18 HSCs, MPPs, CLPs, and CMPs were purified by multicolor FACS, as reported.15-18 The purified HSCs with long-term multilineage hematopoietic reconstitution activity16 were in the G0/G1 phase (98%), whereas 30% of MPPs were in S/G2/M phases, indicating that a majority of MPPs are cycling (Figure 1B). These data are compatible with the notion that HSCs are slowly dividing, but the MPPs represent an expanding subset.
We analyzed the gene expression in these purified stem and
progenitor cells using the MG-U74 set of oligonucleotide arrays A
and B representing 6000 known genes and 18 818 ESTs according to
the Affymetrix database. We first analyzed how many genes were expressed in each population by picking up genes with expression levels
above the cutoff line defined by a compensation method recommended by
the manufacturer (see "Materials and methods"). As shown in Table
1, about 42% of genes on the chips were detectable in each population.
Among these, around 23% of genes were expressed at low levels in each
population of cells. The expression levels of surface markers used for
sorting each population (such as c-Kit, Sca-1, and IL-7R) determined by
the array analysis were consistent with the definition of each
population based on FACS,15,17,18 which verifies
the quantification of gene expression in this assay (Figure 1). In
addition, the result of analyzing representative genes using
single-cell RT-PCR also verified the microarray analysis (see website
cited in "Isolation and characterization of stem/progenitor cells"). The pair-wise relationship between HSCs and MPPs,
represented by the Pearson correlation coefficient
(
Genes related to multiple nonhematopoietic tissues are predominantly expressed in HSCs Transcripts of a variety of nonhematopoietic genes were detected in early hematopoiesis (Table 1). HSCs expressed 43 of 58 genes specific to nonhematopoietic tissues detected by chip hybridization. These nonhematopoietic tissues included brain, liver, heart, kidney, pancreas, muscle, and endothelium as listed in Figure 2. Expression of the majority of these nonhematopoietic genes was progressively attenuated in MPPs and downstream CMPs and CLPs. Thus, promiscuous expression of nonhematopoietic genes (nonhematopoietic promiscuity) is most pronounced in the HSC population (Table 1).To exclude the possibility that the nonhematopoietic gene transcripts
may be derived from bone marrow nonhematopoietic cells sharing
the phenotype with HSCs, we further purified HSCs using CD45, a
hematopoiesis-specific marker.24 cRNA amplified from highly purified long-term HSCs of
Lin
Expression of hematopoiesis-affiliated genes during early hematopoietic development Hematopoiesis-affiliated genes on the chip contained 160 lymphoid-, 117 myeloid-, and some stem/progenitor-related genes. A partial list of these genes is shown in Figure 4. HSCs expressed more than 40% of the hematopoiesis-related genes. Interestingly, HSCs expressed GM- and MegE-affiliated genes, including myeloid cytokine receptors and transcription factors, but only a limited number of lymphoid genes. In contrast, MPPs expressed about 30% of hematopoietic genes related to both lymphoid (T and B) and myeloid (GM and Meg E) lineages. CMPs expressed 26% of myeloid (GM- and MegE-affiliated) genes but not lymphoid genes, whereas CLPs expressed 45% of lymphoid (T-, B-, and NK-affiliated) genes but not myeloid genes. Hence, coexpression of myeloerythroid genes (myeloid promiscuity) exists in HSCs, MPPs, and CMPs, whereas coexpression of T/B/NK lymphoid genes (lymphoid promiscuity) exists mainly in MPPs and CLPs. These data strongly suggest that myeloid and lymphoid promiscuity is distributed in a hierarchical and asymmetrical fashion during hematopoietic development and, therefore, the expression of lineage-related genes can precede commitment12,14 (Figures 1, 4, and 5).
Differential expression of nonhematopoietic and hematopoietic genes during hematopoietic development Because groups of genes with similar expression behavior (up-regulation or down-regulation under the same condition) are likely to be functionally related,30 we next compared the relative expression patterns of genes within these populations. Among a variety of clustering methods, including self-organization maps (SOMs)31 and hierarchical clustering,32 we found K-means clustering, which uses genes with known functions as initial seeds for clusters,33 to be most appropriate34 (see "Materials and methods"). We picked 137 known genes, the biologic functions of which have been well characterized, as our initial seeds. A total of 5223 genes that passed our initial screening filter were subjected to further analysis. The expression levels of these genes were first standardized (or normalized) and then analyzed by K-means clustering using Minitab data analysis software. The final partition of the 5223 genes/ESTs resulted in 100 clusters, each containing a different number of genes (see "Materials and methods"). We focused on genes that were dominantly expressed in each population, grouping them into 4 categories (Figure 4; Table 1).The clustering analysis revealed again that the majority of nonhematopoiesis-affiliated genes fell into category A (Table 1). Category A also contained genes that might play a role in the regulation of stem cell properties such as self-renewal (Figure 4A). These include Wnt1, desert hedgehog (DHH), TCF3 (a target of Wnt signaling), and Smoothened (SMO; a coreceptor of DHH), which are potentially involved in maintaining stem cell compartments.35 Genes related to cell growth arrest (eg, gut-enriched Kruppel-like factor and ZFP36),36,37 immortalization of cells (eg, Bmi-1, a polycomb-group protein),38 leukemogenesis (eg, HoxA9 and Meis1)39 and commitment (eg, Manic Fringe [Notch activity regulator])40 were also found in this category. We found that 13.8% of the genes (n=5223) were significantly up-regulated in MPPs but maintained at various levels in CLPs and CMPs (category B, Figure 4B). These included 26% of hematopoietic (both myeloid and lymphoid) genes, which were elevated at the MPP stage. Thus, MPPs coexpress genes related to multiple myeloid and lymphoid lineages (Figure 4C-D), suggesting that both myeloid and lymphoid promiscuity may operate at this stage. Other known genes in this category include regulatory molecules of cell cycling such as cyclins, CDC molecules, and cell cycle checkpoint molecules (BRCA, MAD2, etc). Several kinases related to cell proliferation such as Nek2, Sak-b (a homolog to Drosophila Polo) and Esk41 were also found in this category. These data are compatible with the fact that MPPs are highly proliferative cells (Figure 1B) and suggest that MPPs are at a priming stage for both myeloid and lymphoid differentiation. The majority of genes preferentially expressed in CLPs (41% of
hematopoietic-related genes, category C) and CMPs (25% of
hematopoietic-related genes, category D) were lymphoid and myeloid
genes, respectively (Table 1; Figure 4). Genes in category C included
B, T, and NK lymphoid-associated genes (ie, E2A,
Ikaros, HES-1, Notch1,42 GATA-3, BLNK, TCR In addition to mutually exclusive regulation in the expression of
lymphoid-versus myeloid-related genes, a number of genes were
up-regulated at the CLP (Figure 4C) or CMP stage (Figure 4D) as a
result of transition from the MPP stage. These genes encode molecules
related to cell differentiation and functions, such as lymphoid-related
Lck,
Gene expression profiling by microarray in murine HSCs has been reported by us and others previously.16,43,44 These studies identified a large number of genes that are predominantly expressed in HSCs. However, these were performed by subtraction of total mRNA in HSCs from those in mature cells (such as unfractionated bone marrow cells), resulting in exclusion of lineage-affiliated genes from the survey. In the present study, we systematically profiled gene expression without pre-excluding multilineage-affiliated genes. Our data demonstrate that both nonhematopoietic and hematopoietic lineage-affiliated genes are transcribed at a low level in HSCs, and the size of the "functional genome" (defined by transcriptional accessibility for lineage-affiliated programs) is progressively decreased as HSCs undergo differentiation (Figure 5). The expression of lineage-specific genes can occur prior to the lineage decision in the hematopoietic system.45 This notion has been obtained by previous studies that demonstrated the coexpression of representative myeloid or lymphoid genes in hematopoietic progenitors.12-14 Here, we significantly extend this view by using oligonucleotide microarray analysis. HSCs coexpress myeloid (GM- and MegE-affiliated) but not lymphoid genes. MPPs coexpress myeloid and lymphoid genes. CMPs and CLPs coexpress a vast majority of GM- and MegE-affiliated genes, and T-, B-, and NK-lymphoid genes, respectively. Thus, our genome-wide gene profiling reveals that HSCs predominantly exhibit myeloid promiscuity, MPPs exhibit both lymphoid and myeloid promiscuity, and CLPs and CMPs exclusively possess lymphoid and myeloid promiscuity, respectively (Figures 4-5). The distribution of hematopoietic promiscuity shown here is compatible with our single-cell RT-PCR study that demonstrates the coexpression of GM- and MegE-affiliated genes in single HSCs and CMPs, and of T- and B-lymphoid genes in single CLPs.14 Accordingly, lineage promiscuity might be a common transcriptional feature in uncommitted stem or progenitor cells, which may represent their immediate lineage potential.14 In this context, lineage commitment might require inactivation of programs for unselected lineages (lineage exclusion) as well as activation or stabilization of programs for committed lineages (lineage specification).46 It is of interest that the most primitive HSCs expressed myeloid but not lymphoid genes.13 Our data strongly suggest that in normal hematopoietic development, priming of myeloid genes precedes that of lymphoid genes. This phenomenon may reflect both evolution and ontogeny. For example, primitive hematopoietic cells appearing during embryonic development can produce primitive erythroid cells and macrophages, but fail to form lymphoid cells,47 and the appearance of macrophage/erythroid cells precedes lymphoid cell formation during evolution.48 One of the most striking results in this study is that primitive HSCs
positive for CD45, a hematopoietic cell-specific marker, express almost
70% of genes affiliated to nonhematopoietic tissues. The expression of
nonhematopoietic genes is down-regulated progressively in HSC
descendants. Recent reports demonstrate that bone marrow contains cells
capable of differentiation into multiple organs, including endothelial
cells, skeletal and cardiac muscles,49 neuronal and glia
cells,50 parenchymal liver cells24 or
epithelial cells,51 as well as hematopoietic cells.
However, most of these reports lack clonal evidences for hematopoietic
and nonhematopoietic differentiation. The plasticity of somatic stem
cells has also been challenged recently by several reports,
particularly as to the conversion from nonhematopoietic stem cells to
hematopoietic tissues. McKinney-Freeman and coworkers found that only
CD45+, but not CD45 Thus, stage-specific distribution of lineage promiscuity (Figure 5) may reflect the selective closing and opening of chromatin domains specific for each progenitor or stem cell type. We have also observed that a variety of chromatin-related genes (such as histone, chromobox, Hdac, and Dnmt) display stage-specific expression patterns that are potentially related to accessibility of each type of cell for lineage-affiliated genes or programs (X.H. and L.L., unpublished results, December 2001). Further studies are required to understand the epigenetic programs controlling the stage-specific transcriptional accessibility in early hematopoiesis.
We thank Dr I. L. Weissman for scientific discussion. We thank Drs R. Krumlauf, E. Rothenberg, and C. J. Sherr for critically reviewing the manuscript. We are grateful to Drs L. Wiedemann and P. Nelson for scientific discussion and to D. di Natale and D. Stenger for assistance on manuscript editing. We are grateful to Dr R. Perera and his coworkers D. Stark and A. McKee for assistance on Affymetrix technique and analysis, and M. A. Handley for technical assistance in FACS operation. We thank Drs A. Mushegian, M. Coleman, and E. Glynn for bioinformatics assistance, and W. Walker and her coworkers for animal care. We are grateful to summer interns R. Dalal for help on data analysis and S. Young for blast search.
Submitted June 17, 2002; accepted August 14, 2002.
Prepublished online as Blood First Edition Paper, September 5, 2002; DOI 10.1182/blood-2002-06-1780.
Supported in part by the Leukemia Research Foundation and Damon Runyon Cancer Research Foundation (K.A.); and by Stowers Institute for Medical Research (L.L.).
K.A. and X.H. contributed equally to this work.
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
-globin, has been demonstrated in a
fraction of a multipotential hematopoietic cell line and in immature
progenitors in human bone marrow cells and mouse intraembryonic
aorta-gonad-mesonephros (AGM) regions by single-cell multiplex reverse
transcription-polymerase chain reaction (RT-PCR)
analysis.
/lo) bone marrow cells, obtained
through Lin+ depletion by sequentially using Dynabeads and
fluorescence-activated cell sorter (FACS), were stained with
APC-c-Kit-, PE-Sca-1-, and biotin/SA-PerCPCy5.5-conjugated Thy-1.
The
c-Kit+Thy1loLin
-positive (IL-7R
RII/III+ cells. Cell cycle analysis
was performed by using Hoechst 33342 (10 µM/L).
yj(1)|> 100 and
yj(m)/yj(1) > 2 for
j = 1,...,n, where
yj(m) and yj(1) are the order
statistics with yj(1) 
...
,
LMO4, FOG, and IL-11R,
G-CSFR, GM-CSFR). Interestingly, the
majority of the genes categorized in categories C and D are likely to
be reciprocally regulated between CLPs and CMPs, representing the
myeloid-versus-lymphoid branch point. This result suggests that
transcriptional regulation of lymphoid-affiliated (T, B, and NK
lineages) or myeloid-affiliated (MegE and GM lineages) genes is a
mutually exclusive event in the progression from MPPs to either CLPs or CMPs.
5, TdT, RAG-1 and
myeloid-related LIM and SH3 protein 1, LMO4,
SDR1, macrophage inflammatory protein (MIP), and
small inducible cytokine A9. This indicates that up-regulation of
lineage-affiliated genes is also required for lineage specification.
evidence of the significant induction by a highly toxic coplanar polychlorinated biphenyl [in Japanese].
Fukuoka Igaku Zasshi.
1997;88:135-143