From The Beatson Institute for Cancer Research, Cancer Research Campaign Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United Kingdom
Received for publication, September 12, 2000, and in revised form, November 14, 2000
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ABSTRACT |
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The difficulties associated with
studying molecular mechanisms important in hemopoietic stem cell (HSC)
function such as the problems of purifying homogeneous stem cell
populations, have prompted us to adapt the murine ES cell system as an
in vitro model of HSC generation and function. We now
report that careful analysis of the time course of HSC generation in
differentiating ES cells allows them to be used as a source of known
and novel hemopoietic gene products. We have generated a subtracted
library using cDNA from ES cells collected just prior to and just
following the emergence of HSCs. Analysis of this library shows it to
be a rich source of known hemopoietic and hemopoietic related gene products with 44% of identifiable cDNAs falling into these camps. We have demonstrated the value of this system as a source of novel genes of relevance to HSC function by characterizing a novel membrane protein encoding cDNA that is preferentially expressed in primitive hemopoietic cells. Intriguingly, further analysis of the known components of the subtracted library is suggestive of erythroid preconditioning of the ES cell-derived HSC. We have used dot-blot and
in situ analysis to indicate that this erythroid
preconditioning is probably restricted to primitive but not definitive
HSC.
The hemopoietic stem cell
(HSC)1 occupies a pivotal
position within the hemopoietic hierarchy and it is at this cellular
level that all hemopoietic function is ultimately regulated (1). It is
also at this level that dysfunctions involved in the pathogenesis of
leukemias and myeloproliferative disorders frequently arise (2). For
these reasons, it is clear that a more complete understanding of the
molecular mechanisms regulating the generation and function of
hemopoietic stem cells is central to our appreciation of physiological and pathological stem cell function. Unfortunately, a number of practical limitations associated with studies on adult HSC have precluded in depth analyses of genes of relevance to stem cell function. Foremost among these difficulties is the problem of purifying
homogenous populations of stem cells for molecular analysis. To
alleviate the problems associated with studies on adult HSC, we have
recently turned our attention to the murine in vitro
embryonal stem (ES) cell system and have been attempting to develop
this as a tractable model of stem cell generation and function that may
be more amenable to in depth molecular analyses.
Embryonal stem cells are totipotent cells derived from the murine
blastocyst at 3.5 days post-coitum and are maintained in an
undifferentiated state by in vitro culture in the presence of the differentiation inhibiting agent leukemia inhibitory factor (LIF) (3-5). Upon removal of the LIF, ES cells when cultured as
aggregates, or embryoid bodies (EBs) will spontaneously commit in
vitro to a range of embryological tissues including epidermis (6),
neuronal and glial cells (7), muscle cells (8), and most notably
hemopoietic cells (9). Hemopoiesis in differentiated EBs is typically
represented by regions of hemoglobinization which are referred to as
blood islands and which are composed principally of erythrocytes with
small numbers of macrophages (9). This hemopoietic cell generation
occurs in the absence of exogenous growth factors, however, addition of
appropriate hemopoietic growth factors or use of supportive stromal
cells during the differentiation process can lead to the generation of
progenitor cells for all hemopoietic lineages in the developed embryoid
body (10-16). This therefore suggests that at some point between being
an ES cell with no direct hemopoietic potential and being a developed
EB with the capacity to contain progenitors for all of the hemopoietic lineages, some of the ES cells within an EB must commit to the hemopoietic system, i.e. they must become hemopoietic stem
cells. Indeed a number of studies has demonstrated the presence of
cells at various stages of hemopoietic development, from hemangioblasts to primitive long-term and short-term repopulating stem cells as well
as committed progenitors of the myeloid and lymphoid lineages, in the
developing EBs (12, 14, 16-18).
We have used a range of in vitro and in vivo
assays to demonstrate a reproducible temporal pattern of emergence of
long-term repopulating and short-term repopulating HSC in EBs post-LIF
removal in vitro. Thus, while at day 3 post-initiation of
differentiation no HSCs are detectable in the developing EBs, by day 4 long-term repopulating stem cells, and by day 5 short-term repopulating stem cells are detectable (19). This stem cell generation requires no
exogenous growth factors and precedes the emergence of mature hemopoietic cells or blood islands. The reproducible time frame of HSC
emergence in this in vitro system and the ease of generation of large numbers of EB-derived cells has prompted us to examine the
usefulness of the day 3/day 5 time frame as a source of known and novel
hemopoietic gene products. We now report the characterization of a
subtracted library generated using cDNA from day 3 and day 5 embryoid bodies. Our results suggest that of the identifiable cDNAs
within the subtracted library, ~46% are predominantly associated with hemopoietic or hemopoietic supportive cells confirming the usefulness of the ES cell system as a source of known hemopoietic gene
products. Characterization of a novel membrane encoding cDNA from
the subtracted library has further emphasized the value of this system
as a source of novel genes of relevance to hemopoiesis. In addition,
analysis of the hemopoietic genes identified in the subtracted library
is indicative of erythroid pre-conditioning of the primitive
hemopoietic stem cell.
ES Cell Culture and Embryoid Body Formation and
Differentiation--
The EFC-1 ES cell line was routinely passaged and
maintained in an undifferentiated state as described previously (19,
20). EBs were generated by hanging drop culture of 10 µl of ES
cells at a concentration of 3 × 104/ml in the
presence of LIF in a humidified, 5% CO2 atmosphere. After
2 days, the EBs were harvested into a Petri dish, washed clear of the
LIF, and differentiation allowed to proceed in LIF-depleted medium at a
concentration of 103 EBs/10-ml culture medium. At days 3 and 5 post-differentiation initiation, EBs were harvested and the
presence of HSCs determined using the CFU-A assay as previously
described (19). The remaining EBs were processed for mRNA and
cDNA generation as described below.
Isolation of mRNA and Generation of cDNA from the Day 3 and Day 5 EBs--
Large scale differentiation of EBs was carried out
on a number of occasions until high level CFU-A generation was achieved between days 3 and 5 post-initiation of differentiation. EBs from this
experiment were harvested and mRNA produced using a Stratagene Messenger RNA isolation kit. Aliquots (5 µg) of the mRNA samples were used to generate day 3 and day 5 full-length cDNA libraries in
ZAP Express (Stratagene) according to manufacturers instructions.
Subtractive Hybridization--
Subtractive hybridization was
performed according to the method of Wang and Brown (21) with the
modification of Balzer and Baumlein (22) which involves the use of
alternative linkers on the driver and tracer cDNA populations thus
minimizing driver cDNA carry-over which may complicate the analysis
of the subtracted library. Briefly this method involved the preparation
of mRNA from day 3 and day 5 EBs which was then converted to
double-stranded cDNA using the Invitrogen Copy Kit. cDNA from
the day 3 and day 5 EBs was then digested separately with
AluI and with a mixture of AluI and
RsaI and these two digests pooled for each time point. Both
enzymes are "blunt end" cutters and have a four-base recognition site resulting in a reduction of the average size of the cDNA population to around 500 base pairs. At this stage,
double-stranded linkers were generated according to the method of
Balzer and Baumlein (22). The oligonucleotides used to generate these
linkers were: (a)
5'-TAGTCCGAATTCAAGCAAGAGCACA-3'; (b)
5'-CTCTTGCTTGAATTCGGACTA-3'; (c)
5'-AGCCATTCTAGACGTGTAACTGATA-3'; (d)
5'-AGTTACACGTCTAGAATGGCT-3'.
Oligonucleotides a, b, c, and d were phosphorylated and ab and cd
pairs allowed to anneal to form double-stranded linkers. These linkers
were ligated onto either the digested day 3 EB cDNA (ab linker
pairing) or the day 5 EB-digested cDNA (cd linker pairing). These
linkers serve both as PCR primer recognition site for amplification of
the cDNA populations but also as restriction enzyme sites for cleaving prior to cloning into sequencing vectors, Thus, and as underlined above, the ab linker pairing is cleavable with
EcoRI and the cd paring with XbaI.
The linker ligated on day 3 and day 5 cDNA populations were
separated from residual linkers and size fractionated on a 1.4% low
melting point agarose gel. From this gel a slice corresponding to a
size range of 200 base pairs to 2 kilobase pairs was cut out for the
day 3 and day 5 cDNA populations. These low melting point-agarose
cDNA preparations were used as templates for PCR amplification of
the day 3 and day 5 cDNA populations using the shorter of the two
primer pairs as PCR primer. Driver cDNAs were biotinylated using
the Photobiotin reagent supplied by Vector Laboratories according to
manufacturers instructions.
The subtractive hybridization involved a combination of short and long
hybridizations and a simultaneous generation of both day 5 and day 3 sequence-enriched libraries (see Ref. 21 for a detailed discussion of
the subtractive strategy). For each, biotinylated driver and
nonbiotinylated tracer were mixed at a 20:1 molar ratio and
co-precipitated. The mixture was then resuspended in 20 µl of 10 nM Tris, 1 mM EDTA, pH 8, and boiled for
3 min. This was then mixed with an equal volume of 2 × hybridization buffer (1.5 M NaCl, 50 mM Hepes,
10 mM EDTA, 0.2% SDS, pH 7.5) overlaid with mineral oil
and then boiled for a further 3 min to ensure denaturation. The
denatured cDNA samples were then allowed to hybridize at 68 °C
for 2 h (short hybridization) or 20 h (long hybridization)
following which 9 volumes of 10 mM Hepes, 1 mM EDTA, pH 7.5, prewarmed to 55 °C was added and the tubes incubated at 55 °C for 5 min. The aqueous phase was then transferred to a
fresh tube to which 20 µl of streptavidin at 2 µg/µl (in 0.15 M NaCl, 10 mM Hepes, 1 mM EDTA, pH
7.6) was added and the mixture incubated at room temperature for 20 min. Protein and protein-DNA complexes were removed by
phenol/chloroform extraction followed by four further streptavidin
incubation and extractions. Finally the subtracted material was
subjected to two more phenol/chloroform extractions and one extraction
with chloroform.
After 6 rounds of short and long subtractions (21), cDNA fragments
were cloned into Bluescript (pSK+) and random clones selected for
sequencing. Sequencing was performed on an Applied Biosystems automated sequencer.
Northern and Southern Blotting--
For Northern blotting, 1.4%
formaldehyde/agarose gels were run with 2 or 20 µg of mRNA or
total RNA, respectively, and blotted according to standard protocols
onto Hybond-N. Similarly for Southern blots, agarose gels with 5 µg
of cDNA per lane were run and blotted onto Hybond-N. All probes
were labeled by random priming using the Amersham Pharmacia Biotech
"Ready to Go" labeling kit.
Isolation of the Full-length cDNA for JB542--
The
full-length JB542 cDNA was isolated from the day 5 EB cDNA
library by PCR using two primers internal to the JB542 sequence and the
flanking T3 and T7 sites in pBK-CMV which can be rescued for the Zap
Express FDCPmix Culture and Differentiation--
The murine FDCPmix cell
line was maintained and induced to differentiate essentially as
described previously (23). Briefly, cells were maintained in Fischers
medium with 10% donor horse serum and interleukin-3 and subcultured
every 3-4 days. Neutrophilic differentiation was induced by addition
of interleukin-3, granulocyte macrophage-colony stimulating factor, and
granulocyte-colony stimulating factor and the cells allowed to develop
for 7 days. Monocytic/macrophage differentiation was induced by
culturing cells in interleukin-3, granulocyte-macrophage-colony
stimulating factor, and macrophage-colony stimulating factor for 10 days. Following completion of the differentiation program cells were
harvested and total RNA prepared using Trizol. The success of the
differentiation was confirmed by morphological analysis of the cells
following cytospinning and staining with May Grunwald and Giemsa (data
not shown).
Generation of cDNA from Lineage Marker-depleted Hemopoietic
Cells--
For generation of lineage-depleted cells, murine bone
marrow cells were depleted of lineage marker-bearing cells using cell surface markers specific for B cells (B220), T cells (CD4 and CD5),
macrophages/monocytes (Mac1), and granulocytes (Gr1). The residual
subpopulation of cells that were negative for the above lineage markers
were therefore enriched for primitive hemopoietic cells. This
population is referred to in the text as the lin Generation of RNA from Lineage Restricted Hemopoietic
Cells--
Separately, individual lineages were enriched using
specific anti-surface marker antibodies. The antibodies used were
TER-119 for erythrocytes (PharMingen), CD41 for megakaryocytes
(PharMingen), Gr-1 for granulocytes (Cambridge Bioscience), CD11b for
monocytes and macrophages (PharMingen), and CD3 for T-lymphocytes
(Cambridge Biosciences). Cells were isolated using Dynabeads according
to the manufacturers instructions, the cells washed, lysed, and RNA prepared using Trizol. JB542 expression was analyzed by PCR using the
following primers which are expected to yield a product of 386 base
pairs: 5'-CACTTCATATCCCCGTGAGG-3' and 5'-CCTCATCAAACTTGGTGCTG-3'. PCR
was carried out for 30 cycles as described previously (24).
EB in Situ Hybridization--
Procedures for in situ
hybridization of EB sections were derived from hybridization protocols
for mouse embryo sections (25, 26) and from whole mount in
situ hybridization procedures on Ebs.2 Briefly, EBs allowed to
differentiate in vitro for various periods of time were
fixed in 4% paraformaldehyde, embedded into paraffin wax, and cut into
7-µm sections. Hybridization conditions were as described (25).
Single-stranded riboprobes for Generation of a Day 3/Day 5 Subtracted Library--
To investigate
the usefulness of differentiating ES cells as a source of known and
novel hemopoietic gene products we have performed subtractive
hybridization using cDNA populations obtained from day 3 and day 5 embryoid bodies. We have used intact embryoid bodies as our cDNA
source in an attempt to ensure inclusion of both hemopoietic and
stromal/supportive cell gene products in the subtracted library. Prior
to the initiation of the subtractive hybridization, the successful
generation of HSCs within the EBs was confirmed by measurement of
"transiently engrafting" CFU-A stem cell activity in the day 5 EBs
as described previously (19). Subtractive hybridization was carried out
using the "Gene Expression Screen" method of Wang and Brown
(21) as described under "Experimental Procedures." The
success of the subtractive hybridization is shown in Fig.
1a which demonstrates
effective removal of common Differentiating ES Cells Are a Rich Source of Known and Novel
Hemopoietic Gene Products--
We have sequenced 474 cDNAs from
this subtracted library and data base searching has demonstrated 132 of
these sequences to be identical to cDNAs already deposited in the
non-EST data bases. Of these 132 cDNAs, which are representative of
80 discrete gene products, 61 (representative of 31 discrete gene
products) are known to be primarily expressed in, and to
function in, hemopoietic or hemopoietic supportive cells (Table
I). Among the hemopoietic cell-associated
sequences is a number characteristic of primitive hemopoietic cells and
these include the transcription factors Ikaros (31) and
GFI-1b (32), the surface markers CD34 (30), PECAM (33), and the integrins
In addition to the known genes outlined in Tables I and II there
are 78 ESTs for which no expression or functional data is yet
available (Table III) and 264 sequences
that currently have no counterparts in any publicly available data
base. Of these 342 ESTs or unidentifiable sequences, it is likely,
given the percentages of known genes that are of relevance to
hemopoiesis that ~150 of these novel cDNAs/ESTs will represent
genes of importance to the development and maintenance of hemopoiesis.
As many as 125 are likely to be expressed specifically in hemopoietic
cells.
Clearly, it is not feasible to pursue the full-length cDNA cloning
and biological characterization of all of these novel cDNAs and
thus our strategy has been to concentrate on the characterization of
cDNAs that are novel but that have motifs that allow an assessment of likely family affiliation or function. Using this, and a range of
expression analysis strategies, we have identified a number of novel
transcription factors and signaling molecules that are preferentially
expressed in primitive hemopoietic cells. As a demonstration of the
usefulness of this strategy in identifying novel cDNAs expressed in
HSCs and potentially of relevance to HSC generation and function, we
have pursued the cloning and the characterization of the expression
profile of one of the novel cDNAs (JB542). This cDNA was
selected for study as the fragment present in the library had homology
with members of an interferon inducible family of transmembrane
proteins and was thus identified as a potential novel surface marker of
HSCs. A cDNA incorporating the full-length coding sequence for
JB542 was obtained from a day 5 EB cDNA library as described under
"Experimental Procedures." This sequence is shown in Fig.
2a and reveals a 450-base pair cDNA encoding a 134-amino acid peptide with a calculated molecular mass of 14,667 daltons. Interestingly, while this cDNA
incorporates the full-length coding sequence for JB542, the size of the
primary transcript for JB542 in tissues blots (see Fig.
3) is ~2.1 kilobases suggesting the
presence of extensive 5'- and 3'-untranslated sequences. As mentioned,
data base searching revealed similarities with members of an interferon
inducible gene family typified by the 9-27/leu 13 (40) and 1-8D (41) proteins (Fig. 2b) the roles
for which remain to be elucidated. The highest levels of similarity is
with a membrane protein of undetermined function from the marbled
electric ray, Torpedo marmorata (42). EST data base
searching with this full-length cDNA also reveals a close human
homologue encoded by an embryo-derived EST (accession number AA463818)
which displays ~81% identity with the murine protein (Fig.
2b). This human JB542 sequence is incorporated within a
genomic sequence from chromosome 11q15 (accession number AF015416). The
predicted murine protein is highly charged with acidic and basic
residues accounting for 20% of the total sequence. The overall charge
is +3. To investigate the likely orientation of the JB542 protein within the membrane we have performed Kyte-Doolittle analysis which
reveals (data not shown) a potential transmembrane region, extending
approximately from amino acids 40 to 60. There is a further potential
transmembrane region predicted between amino acids 90 and 110 and thus
determination of the precise orientation of JB542 within the cell
membrane awaits epitope tagging studies which are underway in our
laboratory.
To attempt to implicate JB542 in hemopoietic cell function we have
examined its expression in bone marrow, spleen, and a range of other
tissues. Results from such tissue blot analysis demonstrates that
expression of JB542 is predominantly seen in the brain and bone marrow
with lower level expression in testes and skeletal muscle (Fig.
3a). To further examine the expression of JB542 within the
hemopoietic system and attempt to investigate any primitive cell-restricted expression patterns, we have adopted two cellular models. First, we have examined expression in the primitive murine hemopoietic cell line, FDCPmix, which displays many phenotypic similarities to murine transiently engrafting stem cells (23, 43).
These cells can self-renew under the proliferative stimulus of
interleukin 3 and can be induced to differentiate along a range of
hemopoietic lineages following treatment with appropriate growth factors. For the purposes of the present study we have produced RNA
from parental FDCPmix cells and from these cells following differentiation along the monocytic and granulocytic pathways. Analysis
of expression of JB542 reveals it to be expressed in the parental
FDCPmix cells suggesting expression in primitive hemopoietic cells
(Fig. 3b). Furthermore, this expression appears to be seen
predominantly in undifferentiated cells as upon differentiation along
either the monocytic or granulocytic pathways, expression drops
markedly (Fig. 3b). To further confirm this preferential expression of JB542 in primitive hemopoietic cells, we have examined expression in normal murine bone marrow cells and in populations of
cells enriched for primitive cells by depletion of cells bearing lineage markers (lin
Thus analysis of the expression patterns of JB542, a novel cDNA
identified within the ES cell subtracted library has demonstrated its
preferential brain and hemopoietic expression patterns and furthermore,
has revealed that the hemopoietic expression is preferentially seen in
primitive cell types. This identification of this novel hemopoietic
cDNA confirms the usefulness of the in vitro ES cell system as a valuable source of novel genes of relevance to primitive hemopoietic cell function.
Analysis of the ES Cell Subtracted Library Reveals Preferential
Expression of Erythroid Lineage Gene Products--
As mentioned above,
there is a number of primitive cell-restricted cDNA sequences
identified in the subtracted library confirming its value as a source
of cDNAs of relevance to immature hemopoietic cell generation and
function. In keeping with the previously reported absence of mature
cells or lineage committed progenitors from the EBs at day 5 (19),
there are no cDNA markers of mature myeloid cells and only two
markers of the lymphoid lineages (T cell receptor
In summary these data are consistent with the initiation of erythroid
gene expression coincident with the emergence of the earliest
detectable hemopoietic stem cells in the ES cell in vitro differentiation system. It is our contention that these data supports a
model of erythroid preconditioning of the hemopoietic stem cell in the
developing embryoid bodies.
Adult, Definitive HSCs Are Not Exclusively Preconditioned to
Erythropoiesis--
It is possible that this apparent erythroid
preconditioning is a feature of primitive embryonic type HSCs and may
therefore not be shared by adult definitive HSCs which have previously
been reported to display a multigenic program of gene expression (53). To examine this issue we have arrayed many of the erythroid and primitive cell genes identified in the ES cell subtracted library (columns 1 and 2 and 3 and
4 of Fig. 6a,
respectively) as well as genes that are more typically representative
of differentiating and differentiated myeloid cells (columns
5 and 6 of Fig. 6a). We have used these
arrays to examine gene expression in FDCPmix cells and lin The practical limitations inherent in studies using adult bone
marrow stem cells have precluded directed analysis of the gene products
involved in regulating stem cell production and function. For this
reason we have turned our attention to the ES cell system as an
in vitro model of developmental hemopoiesis. Other studies have also capitalized on the usefulness of this in vitro
differentiation system to identify novel protein kinase cDNAs (56,
57) and to characterize a number of hemopoietic and developmental genes (58). We now report that precise definition of the time frame of
emergence of primitive hemopoietic stem cells in the EBs (19) has
allowed us to use this in vitro differentiation system to generate subtracted cDNA libraries that are abundant sources of both known and novel hemopoietic and hemopoietic related gene products.
Our analysis of 474 sequences from the subtracted library suggests that
~46% of identifiable sequences are likely to be of relevance to the
processes of hemopoiesis. The hemopoietically derived known genes
include a number of markers of primitive cells such as CD34
(30) and additionally we have shown differential expression of
SCL (28, 29), a transcription factor known to function at a
most fundamental level in hemopoietic development (59, 60).
Intriguingly, despite the absence of detectable mature hemopoietic
cells in the EBs, a large number of the hemopoietic gene products
differentially expressed in concert with the emergence of stem cells in
the EBs are more typically associated with mature elements of the
erythroid lineage. We have interpreted this as indicating that, within
the multigenic program of the differentiating EBs, there is evidence of
an erythroid preconditioning of the primitive cells generated in the
EBs. It remains possible that small numbers of committed erythroid
progenitors are present in the EBs at day 5, however, if this was the
case these would be the only lineage committed cells present in the EBs
and would again argue for some predisposition to erythroid development. We have used in situ hybridization and Northern blotting to
demonstrate that globin gene sequences are detectable at day 4 and even
as early as day 3 although only weakly. These time points precede the
emergence of transiently engrafting stem cells in the in
vitro differentiation system arguing for expression of globin in
relatively uncommitted cells perhaps even hemangioblasts (17). This
erythroid preconditioning appears to be a hallmark of primitive
hemopoietic cells as both analysis of gene expression patterns in
immature adult hemopoietic cells and also assessment of the precise
nature of the globin species identified within the subtracted cDNA
population attests to the primitive nature of the erythropoiesis being
detected. It is intriguing that during development, primitive
erythrocytes appear very rapidly following appearance of the first
primitive HSC within the yolk sac (55) and in differentiating ES cells is the preferred hemopoietic lineage for terminal differentiation. This
preconditioning seen in the EB-derived primitive HSC may help to
account for this phenomenon, i.e. that the stem cells are
generated with a specific predisposition for the erythroid lineage. Why
should this pre-conditioning be lost in the adult HSCs? It is possible
that the reason for the apparent pre-conditioning in the EBs and the
earliest embryonic HSC is that they see no other growth factors at
these early developmental stages and thus are free to spontaneously
commit to their preordained lineage. In the later embryo and in the
adult, the emergence of growth factor expression may subvert this
preconditioning and allow a more multigenic program to be activated
within the HSC. This is clearly what we have seen and mirrors the data
from others indicating a wide gene expression pattern in primitive
adult hemopoietic cells (53, 61, 62).
The data clearly indicate that hemopoiesis is by far the predominant
developmental process occurring over the day 3/5 time frame analyzed
and no other major tissue-specific gene expression patterns are
detected among the known genes outlined in Table II. We have previously
reported that there are few detectable HSC per EB at day 5 (19),
however, the gene expression level detected in this study and
particularly the diffuse nature of the globin staining patterns
observed following in situ analysis suggest that hemopoietic
commitment is considerably more widespread than we had first believed.
It is likely that while limited numbers of HSC are detected by in
vitro assay, hemopoietic gene expression is more widespread within
the EB and a number of cells are expressing hemopoietic genes. It may
be that only a few of the cells express the appropriate cohort of genes
to allow them to be fully functional stem cells although further more
rigorous analysis of EB HSC numbers is required before definitive
conclusions can be reached.
We have also demonstrated the ES cell system to be a valuable source of
novel hemopoietic genes and describe the cloning and characterization
of JB542, a novel hemopoietic cell surface protein with expression
patterns indicative of primitive hemopoietic cell expression.
Embryologically this transcript is detectable by PCR between days 9.5 and 11.5 but the levels are to low to allow precise in situ
localization (data not shown). A JB542 human homologue with 81%
identity to the murine protein is also described and resides on human
chromosome 11. We are currently raising antibodies to JB542 with a view
to examining the specific clonogenic nature of the cells expressing
this cell surface protein.
In conclusion, therefore, we have demonstrated the murine ES cell
system to be a very valuable source of both known and novel hemopoietic
and hemopoietic related genes. Furthermore analysis of known genes is
suggestive of erythroid preconditioning of primitive hemopoietic stem cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
vector. The internal JB542 primers used were: 1)
5'-TTCCCAGCCATCTTCTGGTCTCGGGCC-3'; 2)
5'-GGCCCGAGACCAGAAGATGGCTGGGAA-3'. Following sequencing of the
full-length cDNA, it was re-derived from murine FDCPmix RNA by
reverse transcriptase-PCR using PFU Taq polymerase and three
independent clones were analyzed to verify sequence.
population. mRNA
was generated from bone marrow and the lin
population using the
Invitrogen microfast track mRNA kit and cDNA produced using the
Invitrogen copy kit. Each of the cDNA preparations were sheared
with AluI and linkers were ligated on to allow PCR
amplification of the cDNA populations. It is important to note that
different linkers were used for each cDNA preparation to minimize
the chances of carry-over or cross-contamination between the different
cDNAs during PCR. The cDNAs were amplified by PCR and used in
Southern blotting experiments to assess gene expression. Success of the sorting and cDNA generation exercise has been confirmed previously (24).
-globin were synthesized as run-off
transcripts from linearized plasmid templates under standard
conditions, essentially as described (27). Signal was visualized with
alkaline phosphatase-conjugated anti-DIG antibodies and NBT/BCIP color substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin sequences after 4 rounds of
subtraction and substantial up-regulation of hemopoietic specific
-globin gene sequences over the six rounds of
subtraction. Slot-blot analysis (Fig. 1b) of a number of
cDNAs from the subtracted library has demonstrated only low numbers (less than 10%) of non-day 3/day 5 differentially expressed cDNAs, thus further confirming the success and completeness of the
subtraction. Also shown in Fig. 1b is differential
expression of the primitive hemopoietic markers Scl (28, 29)
and CD34 (30) confirming the usefulness of the day 3/day 5 time frame as a source of known genes of importance to hemopoietic stem
cell generation and function.
View larger version (41K):
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Fig. 1.
Confirmation of the success of the
subtractive hybridization. a, Southern blots of
cDNA from alternate stages of the subtractive hybridization showing
actin suppression and globin enrichment over the six rounds of
subtraction. Agarose gels were run of 5 µg of cDNA from every
second round of the subtractive hybridization. The gels were blotted
and probed with actin or
-globin, both of which were labeled by
random priming. b, slot-blot analysis of day 3/5
differential expression of unknown and known cDNAs in the
subtracted library. Slot-blots were prepared with 200 ng of day 3 or
day 5 EB-derived cDNA. These blots were probed with clone specific
probes labeled by random priming.
4 and
3 (34) and the primitive cell associated GDP
dissociation inhibitor (35). In addition, the transcription factor
Tel identified in the library has a known role in HSC
migration during development and may functionally relate to the CXCR4
chemokine receptor in this context (36, 37). The presence of these
cDNAs in the subtracted library, along with the demonstration of
differential expression of CD34 and Scl (Fig.
1b) further confirms the usefulness of the ES cell differentiation system as a source of known genes of importance to
primitive hemopoietic cell function. The hemopoietic supportive cell-associated cDNAs include a number of sequences associated with
bone marrow stromal cells such as the integrin ligands collagen, fibronectin, and laminin (34), endothelial cell-associated sequences such as VE-cadherin (38) and angiopoietin (39), and the adipocytic cDNAs Paf-1 and apolipoprotein-B. The remaining 71 (46 discrete gene products) identifiable sequences show no clear
commonality in their tissue affiliations and appear to have
little in common in either their known functions or expression patterns
(Table II). Thus this preliminary
sequencing data suggests that ~46% of identifiable cDNAs in the
subtractive library are of relevance to the process of hemopoiesis with
38% being of hemopoietic cell origin and 8% being derived from
presumed hemopoietic supportive cells. The fact that there is no other
major tissue-specific gene expression represented in the library
indicates that hemopoiesis is the predominant developmental process
occurring in the EBs between days 3 and 5 of differentiation.
List of known haemopoietic and haemopoietic related gene products in
the subtracted library (for this and subsequent tables, numbers in
brackets represent numbers of repeats of the individual sequences)
List of known non-haemopoietic gene products in the subtracted library
List of EST sequences in the subtracted library (sequences marked by
asterisks represent murine homologues of the indicated human ESTs)
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Fig. 2.
cDNA sequence and homology comparisons
for JB542. A, the full-length cDNA for JB542 is
shown. This has been derived from a day 5 EB cDNA library using
primers internal to the cloned fragment and primers from vector
sequences flanking the cDNA insert (accession number AJ009781).
Note that the putative transmembrane regions are underlined.
B, homology between murine and human JB542 and other members
of the interferon inducible family of membrane proteins. The human and
murine JB542 consensus sequence is shown (JB542 con) as well
as the overall family consensus (Family con).
View larger version (49K):
[in a new window]
Fig. 3.
JB542 is expressed in hemopoietic tissues
with preferential expression in immature cells. a,
expression of JB542 in murine tissues: RNA from the tissues shown was
prepared and 20 µg of each run on a denaturing agarose gel. This gel
was then blotted and the blot probed with JB542-specific probes. As
indicated normalization was by comparison with the ethidium bromide
stained 18 S ribosomal RNA band. b, expression of JB542 in
primitive hemopoietic cells: FDCPmix cells were grown and allowed to
differentiate along either the granulocytic or monocytic pathways.
Total RNA was prepared and Northern blotted in an attempt to detect
JB542 in parental or differentiated FDCPmix cells. c, also
JB542 expression was examined in sorted primitive (lineage negative)
murine hemopoietic cells. cDNA was prepared from lineage negative
cells and from bone marrow and this was run out on an agarose gel and
Southern blotted. Both the Northern and Southern blots in this figure
were probed with random primed JB542 sequences.
). As can be seen in Fig. 3c, JB542 is
expressed at much higher levels in the lin
cells than in the total
bone marrow cells and while the lin
population does represent a
purified population of stem cells, it is enriched for primitive stem
and progenitor cells and thus expression in this population is further suggestive of preferential JB542 expression in primitive hemopoietic cells. To examine the hemopoietic expression further, we have separated
hemopoietic cells into their component lineages using immunomagnetic
techniques and have assessed JB542 expression in these sorted cell
populations. As shown in Fig. 4, PCR
again reveals preferential expression in lineage negative cells
compared with lineage positive cells. The expression in lineage
positive cells appears to be accountable for by maintained expression
in erythrocytes, megakaryocytes, and granulocytes. In contrast
expression is lost following commitment to and differentiation down the
macrophage or T cell lineages. These data therefore confirms the
primitive cell expression of JB542 but suggests that its
down-regulation is lineage dependent.
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Fig. 4.
Examination of JB542 hemopoietic
expression. Murine bone marrow cells were sorted into individual
lineages and into lin and lin+ cell populations using immunomagnetic
separation techniques. RNA was prepared from thes cell populations and
expression of JB542 assessed by PCR. PCR was allowed to run for 30 cycles following which the products were visualized on a 1.2% agarose
gel. Actin has been included as a loading control.
and plasma
cell membrane glycoprotein). Curiously, however, a large cohort of the
identifiable cDNAs is representative of genes that are not
characteristic of primitive hemopoietic cells but are more typically
associated with maturing and mature erythrocytes. These erythroid genes
include the transcription factors NRF1 (44) and
NFE2 (45), the enzymes 5-ALAS (46) and
procathepsin E (47), the surface markers (see Ref. 48 for a review of
erythrocyte surface markers) erythrocyte membrane protein 4.2 (49),
50-kDa plasma membrane glycoprotein (50), and Band-3 anion exchange protein (51), the tropomodulin structural gene (52) as well as multiple
globin sequences. We have previously demonstrated the absence of
erythroid progenitor cells and markers of mature erythropoiesis in the
day 5 EBs and have used benzidine staining to demonstrate that mature,
benzidine-positive erythrocytes do not emerge in the EB differentiation
system until day 8. The absence of evidence of maturing and mature
erythroid cells in the EBs at day 5 has led us to tentatively conclude
that while a multigenic program is evident in the differentiating ES
cells, the relative wealth of erythroid genes is suggestive of
erythroid lineage preconditioning of the ES cell-derived hemopoietic
stem cell. It remains possible that this erythroid lineage gene
expression is indicative of emergence of small numbers of committed
erythroid cells in the EBs at day 5 that may be hard to detect using
conventional bioassays. However, Northern blot (data not shown) and
in situ (Fig. 5) analyses of
-globin expression in the developed EBs has revealed expression at
time points even earlier than day 5. Indeed Fig. 5 shows that
-globin species are readily detectable in day 4 EBs and weakly in
day 3 EBs. In our hands these time points are prior to the emergence of
transiently engrafting stem cells or any identifiable committed
progenitors. Indeed day 3, at which time low levels of globin
expression is detectable, precedes the emergence of long-term
repopulating stem cells in the ES cell system (19) and is more in
keeping with the time of emergence of hemangioblasts (17).
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Fig. 5.
-Globin is detected at the earliest time
points of hemopoietic commitment in developing EBs. EBs at
days 2, 3, 4, 5, 6, and 8 were sectioned and probed for
-globin expression using an in vitro transcribed
digoxigenin-labeled probe.
primary
hemopoietic cells with a view to assessing the similarities in the
multigenic gene expression pattern between these adult cells sources
and the ES cell-derived HSC. RNA was prepared from FDCPmix cells,
converted to cDNA, labeled, and used to probe the dot blots
outlined on Fig. 6a. The results shown in Fig. 6b
reveal that in the parental FDCPmix cell line, in contrast to the ES
cell-derived hemopoietic cells, a multigenic program of gene expression
is evident with as expected, a good representation of stem
cell-associated gene expression alongside expression of a number of
erythroid and myeloid genes but with no clear predisposition to
erythroid gene expression. A similar multigenic expression pattern
without evidence of erythroid pre-conditioning is seen in primary
murine lineage-negative cell populations (Fig. 6c) further
indicating that, in contrast to the ES cell-derived HSCs, adult HSCs
either from primary sources or as a self-renewing cell line, do not
display significant erythroid preconditioning but instead exhibit a
multigenic expression program. These data is therefore in agreement
with that of Hu et al. (53) who have used PCR to demonstrate
a similar multigenic program in FDCPmix and sorted primary stem cells
(53). The FDCPmix gene expression pattern changes on differentiation
along the neutrophilic lineage with a loss of erythroid and stem cell
gene expression and a concomitant increase in expression of some
myeloid genes, most notably lysozyme (data not shown). These data
suggests that the apparent erythroid specific preconditioning of the ES
cell-derived stem cells may be an indication of their primitive rather
than definitive nature and that definitive stem cells, either as a
homogenous cell line or as an enriched population of lineage-negative
cells display a more multigenic gene expression pattern. Further
evidence pointing to the primitive nature of the EB-derived hemopoiesis
is the predominance of fetal, or primitive,
- and
-globin species
(Ref. 54 and Table I). These results therefore suggest that erythroid
gene expression detected at the earliest time of emergence of primitive hemopoietic cells in the in vitro ES cell system is
indicative of erythroid preconditioning of the primitive hemopoietic
stem cell and may go some way to explaining the rapidity of mature primitive erythrocyte generation from primitive HSC and the apparent preference for erythroid differentiation in nongrowth factor-treated ES
cells (9, 55).
View larger version (51K):
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Fig. 6.
Dot-blot analysis of erythroid, stem cell,
and myeloid cell cDNA expression reveals a multigenic pattern of
gene expression in FDCPmix and lineage negative primary cells.
a, template showing the layout of the dot blots. Each dot
has 100 ng of cDNA per spot. cDNAs are abbreviated as outlined
in the text. Actin and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) are included as loading controls with actin being
loaded at 50, 5, and 0.5 ng/spot and glyceraldehyde-3-phosphate
dehydrogenase at 100, 10, and 1 ng/spot. Thus the differential
intensity of the actin and glyceraldehyde-3-phosphate dehydrogenase
spots correlates with these different loadings. B, dot-blot
probed with radiolabeled total cDNA from FDCPmix cells.
c, dot-blot probed with radiolabeled total cDNA from
sorted lineage negative primary murine hemopoietic cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Prof. John Wyke for helpful comments on the manuscript. Thanks are also due to Prof. Jeremy Brockes and Phill Gates (Ludwig Institute for Cancer Research, London) for advice and assistance with the subtractive hybridization procedure.
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FOOTNOTES |
---|
* This work was supported by grants from the Medical Research Council and the Leukemia Research Fund. Work at the Beatson Institute was supported by grants from the Cancer Research Campaign.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF009781.
Present address: Regulation of Cell Growth Laboratory, Bldg. 560, Rm. 22-45, NCI-Frederick Cancer Research and Development Center,
Frederick, MD 21702.
§ Present address: EiRx Therapeutics Ltd., Bldg. 2800, Kinsale Road, Cork, Eire, United Kingdom.
¶ Present address: Academic Unit of Obstetrics, Gynaecology and Reproductive Healthcare, St. Mary's Hospital, Manchester, M13 0JH United Kingdom.
Present address: Patterson Institute for Cancer Research,
Wilmslow Road, Manchester, M20 4BX United Kingdom.
** Present address: John Hughes Bennett Laboratory, Dept. of Oncology, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
Present address: Biological Sciences, University of Durham,
South Rd., Durham DH1 3LE, United Kingdom.
§§ To whom correspondence should be addressed: Beatson Institute for Cancer Research, Cancer Research Campaign Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United Kingdom. Tel.: 44-141-330-3982; Fax: 44-141-942-6521; E-mail: g.graham@beatson.gla.ac.uk.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008354200
2 U. Menzel, personal observations.
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ABBREVIATIONS |
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The abbreviations used are: HSC, hemopoietic stem cell; ES, embryonal stem; EB, embryoid body; PCR, polymerase chain reaction; LIF, leukemia inhibitory factor.
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