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HEMATOPOIESIS
From Basel Institute for Immunology, Switzerland;
Biozentrum der Universität Basel, Switzerland;
Unité d'Immunologie de Transplantation, Hôpital cantonal
universitaire de Genève, Geneva, Switzerland.
Self-renewal, pluripotency, and long-term reconstitution are
defining characteristics of single hematopoietic stem cells. Pax5 Mature cells of hematopoietic lineages have limited
lifespans. Hence, to maintain their peripheral pools, they have to be generated throughout life. All cells of the hematopoietic system develop from pluripotent hematopoietic stem cells (HSCs). A single HSC
can repopulate all blood cell lineages.1-3 Two of the 3 basic properties of HSCs, self-renewal and pluripotency, can be tested by in vivo transplantation into hematopoietically deficient, ie, lethally irradiated, hosts4 or by in vitro proliferation of the HSCs, followed by differentiation into distinct hematopoietic lineages under appropriate tissue-culture conditions.5 The third basic property, the capacity for long-term reconstitution, requires that, upon transplantation, HSCs home back to their
appropriate sites in the bone marrow, from where they can be
reisolated, recloned in vitro, and then retransplanted into secondary
and subsequent hosts.
Experimentally, 2 types of HSC have been identified. The one,
short-term HSC (ST-HSC), possesses limited self-renewal
potential, pluripotency, and only short-term reconstitution capacity,
while the other, long-term HSC (LT-HSC) has long-term self-renewal and hematopoietic reconstitution capacity.6 While HSCs have
been found to persist throughout life in almost constant
numbers,7,8 and while 3 to 5 consecutive transplantations
of HSCs into lethally irradiated hosts have suggested that they might
undergo 80 to 200 divisions,9-13 their in vitro capacity
for self-renewal appears limited to about 20 cell
divisions.14 Therefore, the self-renewal capacity of
LT-HSCs appears to be strongly dependent on conditions, which can be
provided by the host in vivo, but are insufficient with current in
vitro culture conditions.
Normal somatic cells have a finite replicative lifespan.15
Human fibroblasts have been found to undergo 50 ± 10 divisions, after which they lose the capacity to replicate and enter a state of
senescence. One of the mechanisms by which this biological clock is
thought to function is the shortening of telomeres with each cell
division. Telomeric DNA capping the ends of chromosomes consists of
TTAGGG repeats, which are thought to protect the chromosomes from
illegitimate recombination, to localize the chromosomes within the
nucleus, and to regulate the replication of chromosomes.16 The normal rate of telomere loss has been measured as 50 to 100 base
pairs (bp) per cell division,17 although accelerated
losses have also been observed.18 Telomere loss can be
counteracted by the enzyme telomerase, which synthesizes telomeric
repeats and, hence, can lengthen telomeres.19
Overexpression of telomere reverse transcriptase, the catalytic subunit
of telomerase, in human somatic cells has been shown to result in
elongation of telomeres and thereby extension of the lifespan of these
cells.20 High telomerase activity has been detected in
germ line cells, in some somatic cells, in cancer cells, and, more
relevant to the present study, in LT-HSCs, while ST-HSCs have
significantly less.21 By compensating for telomere losses,
telomerase is thought to be able to maintain the long-term self-renewal
capacity of LT-HSCs in vivo. However, extensive telomere shortening in
HSCs upon serial transplantation has been reported.22
The chromosomes of different species have telomeres of different
length. While human telomeres are around 6 kb long, mice have telomeres
approximately 10 times longer; telomeres in mice can be 60 kb and
longer in germ cells and primary HSCs.23 Hence, at
normal rates of telomere losses and with insufficient telomerase activity, it would take human cells 60 divisions and mouse cells 600 divisions to lose their telomeres entirely.
Precursor B cells from Pax5-deficient mice are blocked in
B-cell development at the stage of a
DHJH-rearranged pre-B I cell.24 These Pax5 In this paper, we investigate the replication capacity of clones of
pre-B I cells from Pax5-deficient mice in vitro, and we measure their telomere length and telomerase activity and the relation
of these to the numbers of cell divisions. Moreover, we analyze
the ability of these clones to home back to the bone marrow upon
intravenous tail injection and to repopulate the pre-B cell compartment
as a measure of their long-term reconstitution capacity. Finally, we
determine the in vivo potential of these clones to differentiate to
thymocytes and mature T cells and, in some cases, also to NK cells,
DCs, and other myeloid cells as a measure of their multipotent
hematopoietic capacity during serial transplantations, with in vitro
recloning of the bone marrow-homed donor cells occurring after all
transplantations. We show here the results of 5 such repetitive in vivo
transplantation/in vitro growth steps, which have expanded the original
Pax5 Mice
Establishment of pre-B I cell clones/subclones
Transplantation of Pax5-deficient pre-B I cell clones Balb/c RAG2 / or
RAG2 c/ mice at 8 to 12 weeks
of age were -irradiated with 400 rad, and 107 in
vitro-grown Pax5/ pre-B I cells were
injected intravenously 6 to 8 hours after irradiation. Cell suspensions
of various lymphoid organs were prepared by standard protocols as
described.28
Carboxyfluorescein succinimidyl ester labeling Carboxyfluorescein succinimidyl ester (CFSE) labeling of cells was performed as described.30 Briefly, cells were resuspended in phosphate-buffered saline at 5 × 107/mL. CFSE was added to a final concentration of 2.5 µM, and the cell suspension was incubated for 10 minutes at room temperature. At the end of the incubation period, the cells were washed 3 times.Flow cytometry Flow cytometric analysis were performed by means of a FACSCalibur (BD PharMingen, San Diego, CA). Antibodies used for the analyses are described elsewhere.27,28 Single-cell sorting was done with a FACStar Plus equipped with an automated cell deposition unit (BD PharMingen).Measurement of telomere length The average length of telomere repeats in wild-type and Pax5/ pre-B I cell clones/subclones was
determined by flow fluorescence in situ hybridization as
described.31
Measurement of telomerase activity Telomerase activity in individual wild-type and Pax5/ pre-B I cell clones/subclones was
measured by photometric enzyme immunoassay with the use of the
telomeric repeat amplification protocol as recommended by the
manufacturer (Roche Moleular Biochemicals, Rotkreuz, Switzerland).
Fluorescence intensity was measured as OD405nm-600nm.
In vitro replication capacity of Pax5-deficient pre-B I cell clones To test the capacity of a series of clones of pre-B I cells from Pax5/ mice, CD19
B220+c-Kit+ cells from bone marrow were single
cell-sorted by fluorescence-activated cell sorter (FACS) and grown on
stromal cells in the presence of IL-7 to approximately 109
to 1010 cells within 4 to 5 weeks by subculturing the
expanding cells every 3 days on new stromal cells in fresh medium at a
suitable concentration.29 Under these culture conditions,
approximately 1 of 3 single ex vivo isolated cells establishes good
clonal growth, comparable in plating efficiency to fetal liver-derived
pre-B I cells with the same B220+c-Kit+
phenotype from wild-type mice (Rolink et al27 and data not shown). Growth of Pax5-deficient pre-B I cell clones was
monitored by cell counting, which allowed us to estimate the number of
cell divisions each clone underwent (Figure
1). We conclude that bone marrow-derived
Pax5/ pre-B I cell clones have plating
efficiencies ex vivo and show proliferative capacity in vitro that are
comparable to fetal liver-derived wildtype pre-B I cells.
In vivo repopulation of bone marrow by GFP-expressing
Pax5 /
pre-B I cells from several clones with a GFP-expressing retrovirus.
Previously, we had observed that GFP-expressing
Pax5/ pre-B I cells repopulate the pre-B I
cell compartment of B220+c-Kit+ cells in the
bone marrow of RAG2/ hosts, so that
approximately 5% to 10% of the lymphoid cells appear
GFP+.27 At 3 weeks after
transplantation, this was also observed in the present study with all
analyzed clones of GFP-expressing Pax5/
pre-B I cells (data not shown). This repopulating capacity of the
Pax5/ pre-B I cell clones remained upon
secondary and subsequent transplantations (see below).
The Pax5 We conclude that the Pax5 In vivo dividing, transplanted Pax5 /
pre-B I cell clones is shown in Figure 1 with data obtained by cell
counting in vitro. In Figure 1, it is assumed that these cell clones do
not proliferate in vivo in the bone marrow of the host. However, we
know that, 3 to 4 weeks after transplantation, between 10% and 15% of
the GFP+Pax5/ pre-B I cells in
bone marrow are in S, G2, or M phases of the cell cycle
(Rolink et al27 and data not shown). To estimate the
proliferative capacity of the Pax5/ pre-B I
cell clones in vivo, in vitro-grown cell clones expressing the major
histocompatibility complex allele H-2Kb were labeled with
CFSE and transferred into Balb/c RAG2/ hosts
expressing the major histocompatibility complex allele H-2Kd. To compare the in vivo proliferation capacity of
these cells to the in vitro capacity, an aliquot of the labeled cells
was cultured on stromal cells in the presence of IL-7.
Dividing Pax5
We conclude that when we include the in vivo analyses, the
proliferation capacity of Pax5 Reduction of telomere length in Pax5 / pre-B I cells, like HSCs, possess
long-term self-renewal capacity, we determined their telomere length
with increasing time of proliferation in vitro and in vivo. Data
obtained by flow fluorescence in situ hybridization clearly demonstrate
that in Pax5/ pre-B I cells, telomere
lengths decrease with time of proliferation (Figure
4). On the basis of the in vitro cell
proliferation only (Figure 1), the Pax5/
pre-B I cell clones reduced their telomere length by 70 to 90 bp per
cell division. If telomere length were also reduced by cell divisions
in vivo and if the cells continued to divide throughout the entire time
that they reside in bone marrow and at the same rate as in vitro, then
the rate of telomere reduction could be as little as half, ie, 35 to 45 bp per division. Normal somatic cells shorten their telomeres by 50 to
100 bp per division.17 Therefore, mechanisms counteracting
telomere shortening seem not to be strong in
Pax5/ pre-B I cells.
Telomerase activity in Pax5 / pre-B I cell clones using HEK293
cancer cells as high telomerase-expressing control cells. The results
of our analyses indicate that telomerase activity in
Pax5/ pre-B I cells is about 30-fold lower than in
HEK293 cells (Figure 5). Furthermore,
telomerase activity in wild-type and Pax5/ pre-B I cell
clones is not significantly different and does not change with in vitro
or in vivo proliferation (Figure 5, and data not shown). We
conclude that this low telomerase activity is apparently unable to
avert telomere shortening at normal or even slightly elevated levels in
Pax5/ pre-B I cells.
Stability and changes in multipotency of hematopoietic
differentiation capacity of Pax5 / pre-B I cell clones, which were
recloned ex vivo after each in vivo transplantation step, hematopoietic
differentiation capacity was measured by in vivo differentiation to
T-cell receptor (TCR)  - and TCR -expressing
thymocytes, to NK T cells, and, in some cases, to NK cells, DCs, and
even myeloid cells or, rarely, erythrocytes.
In all serial transplantations, the B220+c-Kit+
pre-B I cell compartment in bone marrow is repopulated by the
transplanted GFP-expressing Pax5
Previously, we have shown that 4 to 6 months after transplantation of
GFP-expressing Pax5 Of the Pax5 We conclude that the self-renewing long-term reconstituting
Pax5 In summary, our experiments show that Pax5
This study has investigated the self-renewal capacity, long-term
bone-marrow reconstitution capacity, and in vivo multipotency of 5 randomly chosen Pax5 The efficiency of cloning of the
CD19 While Pax5 Wild-type and Pax5 Are these properties of HSCs retained or lost upon repeated in vitro
proliferation/in vivo homing steps? The first property, self-renewal,
appears to be retained at constant rates of proliferation with a stable
phenotype and with essentially unchanged high cloning efficiencies.
This also appears to be the case for the long-term reconstitution
capacity in the bone marrow pre-B I cell compartment, with even a
suggestion that the strength of the Pax5 Overall, Pax5 In a normal steady-state condition, about 8% of LT-HSCs enter cell
cycle each day and approximately 5% of LT-HSCs are in S, G2, or M phases of the cell cycle.35 However,
the number of cycling HSCs is increased to approximately 10% upon
transplantation and remains elevated for several months.22
Our observation that 10% to 15% of Pax5 We have recently shown that the Pax5
Submitted October 9, 2001; accepted November 30, 2001.
The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche, Basel, Switzerland.
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: Christoph Schaniel, Department of Molecular Biology, Lewis Thomas Laboratory, Rm 216, Princeton University, Princeton, NJ 08544; e-mail: schaniel{at}molbio.princeton.edu; or Antonius G. Rolink, Institut für Immunologie, Universität Basel, Zentrale Dienste, Klingelbergstrasse 70, CH-4056 Basel, Switzerland; e-mail: antonius.rolink{at}unibas.ch.
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Top
Abstract
Introduction
) the primary
Pax5
) hexagonary Pax5
) HEK293 cells (positive control)
or (
) heat-inactivated HEK293 cells (negative control).
