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HEMATOPOIESIS
From INSERM Unité 506 and Institut du Cancer et
d'Immunogénétique, Hôpital Paul Brousse, Villejuif,
France.
Development of the full repertoire of
hematopoietic-lymphopoietic cells from a single stem cell
requires specific contacts with stromal cells. The spatio-temporal
organization of these cell associations in the bone marrow in ontogeny
is, however, not well understood. In the adult, 10% of marrow cells
form a cohort of compact aggregates, the hematon. In the hematon
mesenchymal cells (Stro-1+), perivascular lipocytes
(desmin+), endothelial cells (CD34+,
Flk-1+, Sca-1+), and macrophages amalgamate
with the hematopoietic progenitors long-term culture-initiating cells
(LTC-IC), cobblestone area-forming cell (CAFC),
high-proliferative-potential colony-forming
unit (HPP-CFU), granulocyte-macrophage (GM)-CFU,
and burst-forming unit-erythroid (BFU-E). During endochondral
ossification of the femur, GM-CFU and day 7 CAFC numbers increased
progressively from day 17 of gestation, but primitive, day 35 LTC-IC
appeared from postnatal day 2. Unexpectedly, bone marrow (BM) taken
between embryonic day 17 and day 5 was unable to support myeloid cell production in long-term cultures or to support day 35 LTC-IC growth. However, a gain in stromal cell competence occurred between days 7 and
10, which was correlated with the emergence of hematon in the BM. Thus,
acquisition of hematopoietic competence by BM lags behind for
approximately 10 days after the initial hematopoietic cell influx. In
the adult, the hematon fraction was 3.7-fold enriched in day 35 LTC-IC
over the buffy coat. It produced more GM-CFU and HPP-CFU in myeloid
culture and more B cells in lymphopoietic "switch" cultures. It is
reported that stromal hematopoietic units named hematons are specific
morphogenetic structures that emerge at a well-defined postnatal stage
of development in long bones, delineate discrete territories for
hematopoietic stem cell seeding and development, embody the most
productive hematogenous compartment in the BM, and probably
enclose a morphogenetic organizer.
(Blood. 2000;96:3763-3771) Hematopoietic stem cells (HSCs) can ensure 3 vital
functions Recent investigations provide evidence that adult-type HSCs emerge at
E8.5 from a distinct region of embryonic ventrolateral mesoderm, the
para-aortic splanchnopleura (P-Sp), which later develops into the
aorta-gonad-mesonephros region.9,12,14-16 Between E10 and
E13, HSCs migrate to the liver primordium, which becomes the main
hematopoietic organ with overwhelming erythropoiesis. HSCs may expand
significantly in the liver primordium and later in the
spleen,8,15,17 before definitive hematopoietic tissues develop in the bone marrow (BM). The cellular and molecular mechanisms that determine autonomous hematopoietic competence in the BM have not
been characterized yet. BM development is predisposed by bone formation
by the endochondral ossification of cartilaginous
rudiments.18 On approximately E15-16, chondrocytes become
hypertrophic and then undergo apoptotic cell death in the central
diaphysis of long bone rudiments. Hypertrophied chondrocytes secrete
vascular endothelial growth factor (VEGF) that triggers vascular cells to penetrate the perichondrium,19 bringing along
osteoblast precursors and circulating hematopoietic cells, mostly
primitive macrophages. The vasculature provides a fractal-type,
arborized conduit20 for the recruitment of chondroclasts
that resorb hypertrophied chondrocytes and for osteoblasts that depose
a calcified spongioid shaft replacing the cartilage. Increasing numbers
of hematopoietic colony-forming cells (GM-CFU and CFU-S) were detected
in the femora from E17,8,21 suggesting that active
hematopoiesis starts at approximately E17 to E18 in the mouse femur.
Because a reciprocal correlation existed between colony-forming cell
(CFC) contents in the liver primordium and the developing femur at
perinatal stages, it was concluded that BM is colonized progressively
by liver-derived HSCs.8,16 Indeed, fetal liver cells
tagged by the in vivo transfer of a retrovirus-driven marker gene were
later retrieved in the adult BM.22 However, it is not well
established whether, at the initial step of endochondral bone
formation, hematopoietic progenitor cells arise locally from previously
seeded HSCs or are continuously seeded from the liver by the
vasculature irrigating the femora. Clearly, the exact fate
map23 of the BM primordium and spatiotemporal emergence of
specific stromal cells, whereby the BM gains definitive hematopoietic
competence during ontogenesis, have not been determined.
In the adult marrow, HSC development requires the support provided by
bone tissue and by a network of stromal cells. Long-term BM cultures
(LTBMC) display after several weeks rare hematogenous areas
(cobblestone area [CA]) in the adherent layer,24,25 the presence of which is absolutely required for long-term HSC expansion. The enumeration of LTC-IC/CAFC on a competent stromal feeder layer has
became the most reliable functional test to quantify HSCs in
vitro.26-29 Searching for a preferential spatial location
of HSCs in the mouse femur has led to contradictory data, though, leaving open whether HSC-associated stromal cells have a fixed territory allocated in the BM.30-36
We have postulated that critical stromal cells and hematopoietic
progenitors form firm multicellular units under physiologic conditions.
A cohort of preformed cell complexes, termed hematons, has been
isolated in the low-density, floating layer of normal primate BM
aspirates.37,38 Hematon complexes can be also isolated from the gently dispersed BM of mice, using repeated 1g
sedimentations and decanting the buffy coat
suspension.39,40 The hematon is a finely arborized stromal
framework that includes mesenchymal cells, endothelial cells, and
macrophages. These spherical particles are tightly packed with
hematopoietic cells, including primitive cells with marrow-repopulating
ability (MRA), CAFC, CFU-Sd12 in the mouse hematon, and
HPP-CFC, BFU-E, GM-CFU, and differentiated postmitotic cell populations
in both human and mouse.40,41
Stromal cells and hematogenous cells may be generated simultaneously
during BM morphogenesis or may be attracted together by a putative
organizer to form hematon complexes, such as the nephron in the kidney
and the gustatory bud in the tongue or germinal centers in the lymph
nodes. This study was aimed at characterizing the cells that constitute
the hematon core complex, at determining the stage of its emergence in
the femur during ontogeny, and at identifying when in development the
BM stromal microenvironment gains functional autonomy.
Mouse BM preparation
Embryonic tissues.
C57BL/6 mice were purchased from IFFA CREDO (L'Arbresle, France), bred
under a 12-hour light-dark schedule, and fed sterilized food
(UAR, Epinay-sur-Orge, France) and acidified water ad libitum. Vaginal plugs were checked the morning after natural overnight mating.
Mice were killed by cervical dislocation, and uteri were removed
aseptically. Embryos were rinsed in phosphate-buffered saline, and
femora were dissected with no. 55 watchmaker forceps (Drumond,
Poly-Labo, France) and microsurgical knives prepared from 30-gauge
needles. Femora from embryos and young mice were cleaned of muscle and
cartilage, washed, and cut longitudinally into halves. The diaphyseal
shaft was denuded from the ossified shell and dissociated with 2 30-gauge needles under a STEMI SV8 stereomicroscope (Zeiss,
Oberkochen, Germany).
Purification of the buffy coat, hematon, and hematon-core
complex fractions.
Eight- to 16-week-old mice were killed by cervical dislocation. Femora
were cleaned, and the proximal (hip) and distal (knee) parts were
opened using a 23-gauge needle. Then the BM was recovered by forcing
2-mL culture medium through the femoral shaft. BM pooled from 4 to 6 femora was pipetted vigorously 10 times, and particles were allowed to
settle at 1g for 15 minutes. The upper cell suspension was
transferred to a new tube, and the pellet was pipetted again 10 times
in 1-mL culture medium and diluted to 5 mL. After 15-minute sedimentation, the 2 supernatants were combined and centrifuged (400g, 7 minutes) to obtain the "buffy coat." The pellet
was transferred to a Petri dish, and bone and muscle fragments were
removed to obtain the purified hematon fraction. Hematon units were
counted under the stereomicroscope in one-tenth aliquots of the total hematon fraction. Tiny hematon core particles were purified by forcing
either the whole hematon fraction or single hematons through 25-gauge
and 30-gauge needles 10 times each, followed by repeated 1g sedimentations.
Cell counting, staining, and vitality tests.
BM fractions were dissociated in LTC medium containing 0.1% type II/IV
collagenase for 30 minutes at 37°C. Cells were washed 2 times in 5 mL
medium, counted, and used in quantitative progenitor cell assays. Red
blood cells were lysed in 0.1% acetic acid, and total nucleated cells
(TNCs) were counted in a hemocytometer. BM film and cytospin
preparations were stained with May-Grünwald-Giemsa. Cell vitality
was determined by trypan blue (0.2%) exclusion. Apoptotic cell death
in LTCs was analyzed by fluorescein isothiocyanate (FITC)-labeled
annexin V-propidium iodide (PI) double staining (Coulter, Miami,
Florida). Briefly, 5 × 105 cells were spun down
(400g, 7 minutes), suspended in 490 µL incubation buffer
with 5 µL FITC-annexin V and 5 µL PI, and incubated for 10 minutes
at 4°C in the dark. Cells and apoptotic bodies were analyzed under an
ICM-405 epifluorescence microscope (Zeiss) using a 515- to 565-nm
filter for FITC and a 590-nm filter for PI.
In vitro assays for clonogenic cells
Semisolid cultures for myeloid and erythroid colony-forming
cells.
HPP-CFC, GM-CFU, and BFU-E progenitor cells were determined in a
modified40 double-layer, methylcellulose-agar culture
system.42 Briefly, 0.7 mL 0.5% agar in a 35-mm Petri dish
(ATGC) was overlaid with test cells in 1.5 mL 0.9% complete
methylcellulose medium containing 1% bovine serum albumin (BSA) and
0.1 mmol/L 2-mercaptoethanol (2-ME) (HCC #4230; StemCell Technologies,
Vancouver, British Columbia). Cells were stimulated with a mixture of
murine recombinant hematopoietic growth factors (mrHGFs): 50 IU mrIL-1,
4 ng mrIL-3, 20 ng mrGM-CSF, and 100 ng hrG-CSF (Genzyme, TEBU,
Le-Perray-en-Yuelines, France) for myeloid cultures and with
mrHGFs plus 10 ng mrSCF and 3 U erythropoietin/mL for erythroid
cultures. GM-CFU were counted after 7 days; HPP-CFC was counted on day
28 from the same dishes, and BFU-E was counted on day 14 of incubation.
Assay for long-term culture-initiating cells (LTC-IC/CAFC).
Stromal feeder cells were prepared using the BM hematon fraction 7 to
10 days before limiting dilution analysis (LDA); 2 × 107
dispersed cells were seeded in 10 mL complete LTC medium in a 25-cm2 T-flask (ATGC). The adherent layer was trypsinized
and washed, and aliquots of 3 × 105 cells in 100 µL
LTC medium were seeded in 96-well flat-bottom microplates (ATGC).
Complete LTC medium consisted of Iscove modified Dulbecco
medium (IMDM, Flow Laboratories, Irving, United Kingdom) supplemented with 0.5 mg/L ascorbic acid, 37 mg/L myo-inositol, 10 mg/L
folic acid, 5 × 10 Long-term culture of fractionated bone marrow
Myeloid LTBMC.
The abilities of the buffy coat and hematon fractions to produce cells
were studied in liquid cultures maintained without irradiated stromal
feeder layers. Buffy coat cells and hematons were seeded at
5 × 105 cells per 2 mL LTC medium in 24-well
Nunclon tissue culture dishes (Nunc, Inc, Naperville, IL).
Myeloid LTC medium was identical to that described for LTC-IC/CAFC
assays. Cell production was determined, using semisolid medium colony
assays, in the nonadherent fraction and in adherent cells dispersed
with 0.1% type II/IV collagenase and washed 2 times in Iscove modified
Dulbecco medium-20% FCS.
Myeloid-lymphoid cell production in LTBM switch culture.
HSCs for myeloid and lymphoid cell lineages were assayed using myeloid
cultures (2 × 106 cells in 2 mL medium) in 35-mm Petri
dishes and were maintained for 4 weeks at 33°C, as described above
for standard LTBMC.44 Medium and nonadherent cells were
then removed, and adherent cells were washed 3 times with lymphoid
culture medium containing RPMI-1640 medium (HEPES modification; Sigma),
5% FBS (Hybrimax, Sigma), 50 µmol/L 2-ME, and antibiotics. Cultures
were incubated at 37°C, and TNC and emerging B lymphocytes were
analyzed from the nonadherent suspension on days 7, 14, and 21, from
the adherent layer on day 21 by flow cytometry, and on Giemsa-stained
cytospin preparations.
Organotypic microcultures.
Single hematon units, or hematon core complexes, were isolated under
the STEMI SV8 stereomicroscope (Zeiss). Single aggregates were
transferred in flat-bottom microwells, and the development of native CA
was analyzed over time.
Assay of native and secondary CA-forming cell complexes.
The buffy coat suspension and partially dissociated hematon fractions
were diluted in 24 mL LTC medium and seeded in 384-microwell cloning
plates (Greiner, Nürtingen, Germany). Adherent stroma cell
colonies (F-CFU) and discrete foci containing hematopoietic progenitor
cells under stromal cells (CA) were counted in each microwell over time
under an ICM-405 microscope (Zeiss).
Cell marker studies
Cytochemistry.
Acetylcholinesterase (ACE) reaction was used to identify
megakaryocytes, acid phosphatase was used to identify macrophages, osteoclasts/chondroclasts were detected by tartrate-resistant acidic
phosphatase (#387; Sigma), and osteoblasts were detected by alkaline
phosphatase (#104; Sigma) reaction. Lipocytes were identified by
staining with oil red.
Flow cytometry.
Collagenase-dissociated BM fractions (5 × 105 cells)
were washed in PBS with 1% BSA/0.1% Na azide and incubated for 30 minutes with fluorochrome-labeled antibodies. The mouse B-cell lineage was analyzed using rat FITC-labeled anti-CD45R, B220 antibody (Caltag,
Burlingame, CA) after the adsorption of test cells with 10% whole rat
serum in DPBS for 40 minutes. Cells were washed twice in 1% BSA/DPBS,
and the percentage of labeled cells was determined by flow cytometry on
an Epics II fluorescence-activated cell sorter (Coulter).
Immunostaining.
Hematopoietic progenitor cells were identified in native cell
aggregates on cytospin slide preparations with antibodies to Sca-1
(Ly6A/E, mouse IgG-FITC; Immunotech/Coulter, Marseille, France), CD117
/c-kit (RAM IgG-PE; Immunotech), CD34 (RAM-IgG- FITC; Immunotech),
Thy-1 (RAM IgG2c-FITC; Immunotech), CD41 (RAM IgG-FITC; Immunotech),
CD49d/VLA-4 (RAM IgG-FITC; Caltag), and stroma cells were identified
with antibodies to Stro-1 (RAM pcIgG, kindly provided by Dr Paul
Simmons), desmin 33 (mouse IgG; DAKO, Glostrup, Denmark), Flk-1/KDR
(MAH IgG-FITC; Santa Cruz Biotech, Santa Cruz, CA), VCAM-1 (GAH
IgG-FITC; Santa Cruz Biotech), Histology.
Embryos or dissected tissues were fixed in 2% paraformaldehyde-0.1%
glutaraldehyde in PBS for 60 minutes at 20°C, then washed 3 times for
60 minutes in PBS at 4°C. Selected specimens were further cleaned;
they were then dehydrated in 30% and 50% (vol/vol) ethanol in water
for 10 minutes, in 50% ethanol in Unicryl (British BioCell, Cardiff,
United Kingdom) for 30 minutes, and in 100% Unicryl overnight at
Statistical analysis
Histologic structure of the developing femur
Emergence and expansion of hematopoietic progenitor cells and
hematon units
The emergence of multicellular hematon units was assessed in the
sediment fraction during microscopic examination and in liquid cultures. Close associations of osteoblasts and hematopoietic cells
(mainly macrophages and osteoclasts) could be discerned from E17. These
cells were tightly compacted in the alveolar ossicle, from which they
could be released but could not form adherent CAs or maintain long-term
hematopoiesis in culture (Figure 3A-D). Hematon complexes emerged and could be purified and counted in the
femora of mice from day 7 to day 8, after 1g sedimentation of mechanically dissociated femoral shafts. The number of hematons increased rapidly with age and reached steady values from day 35 to day
48 (Figure 2). In native isolates many hematons are joined together,
resembling a bunch of grapes, by fine capillaries (Figure 3E-F) but
are, however, disrupted during mild mechanical shearing. Further
disruption through a 30-gauge needle was used to obtain the most
compact core of hematons (Figure 3G) that form CAs in liquid culture
(Figure 3H).
In adult mice 5% to 10% of BM cells in a femur form the hematon
aggregate fraction, whereas most cells constitute the buffy coat
suspension.39,40,45 In young adult mice the frequency of
day 7 LTC-IC/CAFC was 1/2200 in the hematon and 1/9000 in the buffy
coat, and the frequency of day 35 LTC-IC/CAFC was 1/6800 in the hematon
and 1/22 000 in the buffy coat fraction (n = 5; 3.7- ± to 1.2-fold enrichment;
P < .01). However, there were approximately twice as many LTC-IC/CAFC per femur in the buffy coat (Table
1).
Gain of stromal cell competence correlates with hematon emergence in the bone marrow Because blood carries progenitor cells, the presence of hematopoietic cells in the femur between E17 and postnatal day 2 does not prove that these arise locally from previously seeded HSCs. First, LTBMC that imitates many functional aspects of in vivo hematopoiesis was used to assess the competence of femoral BM during ontogenesis. The expansion of TNCs and GM-CFU populations in LTBMC was observed over time. Figure 4 shows that the femoral shaft taken from fetuses or mice until day 5 was not able to sustain the expansion of hematopoietic progenitor cells in culture. Femoral BM cells gained autonomous hematopoietic competence from day 5 to day 8. This function was correlated with the emergence in the femur of compact hematon units containing native CA-forming cell complexes.
Next, we addressed whether competence acquisition resulted from a
gain of stromal cell function or from HSC homing. Stromal feeder layers
were prepared from 5-, 7-, 8-, and 10-day-old and 2-month-old femora,
and their competence was determined by LDA using BM progenitor cells.
Stromal cells established from the femur before day 8 supported poorly
the proliferation of late day 7 CAFC (Figure
5A) and could not support the long-term
expansion of primitive day 35 LTC-IC/CAFC (Figure 5B). These results
indicate that until day 5, the lack of competent stromal cells or their putative progenitors may represent a limiting factor for HSC seeding, expansion, or both. This stage corresponds to the onset of HSC (day 35 LTC-IC) seeding and expansion in the femur (Figure 2).
The hematon has higher cell-production ability than the buffy coat in LTC Relative richness of the hematon in stromal cell-associated progenitor cells suggested that it produced more progenitor cells in standard LTBMC over time. Aliquots (5 × 105 cells) of the buffy coat and hematon fractions were seeded in 2 mL LTC medium. Although there were initially fewer primary F-CFU in the buffy coat cultures, both cell fractions developed a semiconfluent layer after 8 to 10 days. Cell vitality studies using annexin V/PI double-labeling showed that double-labeled apoptotic bodies accounted for 5% to 15% during the first week in both culture systems. The TNC, GM-CFU, and HPP-CFC populations in the nonadherent and adherent cell compartments were monitored. TNC and GM-CFU declined in the nonadherent fraction, but those in the adherent layer of the buffy coat cultures gradually expanded (Figure 6A); those in both compartments expanded in hematon cultures (Figure 6B). Hematon cultures generated 6 times more nucleated cells, 16 times more HPP-CFC, 55 times more GM-CFU, and 18 times more BFU-E (not shown) than did the buffy coat on day 28 in culture.
Development of B cells from the hematon in myelopoietic-lymphopoietic switch cultures The development of B lymphocytes in LTC was studied to quantify putative HSCs in the buffy coat and hematon fractions. B lymphocytes rapidly die in myeloid LTBMC containing hydrocortisone, but HSCs survive for several weeks and can be commuted to B-cell development. For 4 weeks 2 × 106 cells were maintained in myeloid LTC at 33°C, then adherent layers were washed and cultures were maintained in B-lymphocyte culture medium at 37°C. B lymphocytes emerged in both buffy coat (Figure 6A) and hematon (Figure 6B) cultures, but there were 8.6 ± 2.4 times more B lymphocytes (B220+) in the hematon cultures than in the buffy coat after 21 days (n = 4; P < .001). B lymphocytes appeared in CAs shortly after commutation but later spread over and under a larger number of stromal cells. Immunostaining in situ was used to further characterize developing B cells. Oct-2 and Hox-11 were detected in B cells that expanded over stromal cells 7 days after commutation, but there were only few positive cells among those that grew under stromal cells (not shown).Isolation and analysis of native hematon core complexes The hematon core complex was isolated on dissociation through a 30-gauge needle. This technique produced approximately 1500 to 2000 tiny cell aggregates (20-50 cells per aggregate), which corresponded to approximately 30 to 50 × 103 cells in a femur. This fraction contained almost all native, early, developing day 7 CA and 30 times more day 14 CA than did the buffy coat suspension.The functional heterogeneity of hematon core complexes in day 8 mice and in young adults was analyzed using individual organ microcultures. In day 8 femora 52% ± 8% (n = 3) of hematons contained the core complex that yielded 3 ± 1.5 (range, 1-5) native CA by day 7. In the adult, 87% ± 5% of microaggregates (n = 6) developed native CA by day 7; however, the number of core complexes per hematon was similar to what it was in the day 8 femur (3.2 ± 1.5; range, 1-6). The percentage of core complexes that remained productive for more than 35 days was 35% ± 10% in day 8 femora and 56% ± 12% in adult femora. Immunostaining showed distinct stromal cells building up
hematons
During the past decade major progress has been achieved in the elucidation of the emergence, development, and function of primitive and adult-type HSCs in avian11,53 and mammalian ontogeny.9,12-15,17,46-48 The cellular mechanisms and molecular stimuli that determine gain of autonomous hematopoietic activity in bone cavities remain, however, unidentified. Decipherment of the BM fate map during ontogeny and identification of specific territories in which stromal cells interact with HSCs is hampered by the lack of a technique permitting the isolation, quantification, and functional characterization of native stromal-hematopoietic cell complexes. Yet it was shown previously that adult BM contains the hematon, a cohort of preformed aggregates of stromal cells and hematopoietic progenitors.38-40 We investigated the development of fetal and early postnatal mouse marrow, with special reference to hematon emergence, and established, first, that chondrogenesis, endochondral bone formation, and the formation of a primary, vascularized spongy bone shaft occur in sequence and clearly before the onset of autonomous hematopoiesis. Our study results confirm a gradual increase in GM-CFU and day 7 CAFC numbers in the femur, starting from E17.8 Surprisingly, however, day 35 LTC-IC/CAFC were not detectable by LDA (using the MS-5 stroma cell line as a support) until 2 days after birth, and long-term culture femur marrow cells could not sustain hematopoiesis in Dexter culture until approximately 5 days after birth. Such cultures contained chondrocytes, vascular endothelial cells, osteoblasts, osteoclasts, and adherent colony-forming macrophages that expanded slowly over time but failed to switch to a productive stage. Earlier works postulated that marrow hematopoiesis starts by HSC seeding to the femora at E17.8,9,21,49 BM stroma from newborn mice did maintain the expansion of adult GM-CFU in LTBMC, but this activity was approximately 4 times weaker than in adult stroma.50,51 Our own results clearly show that day 5 to day 7 stroma could not support day 35 LTC-IC/CAFC growth, which is in contrast to day 8, day 10, and adult stroma that had gained this functional activity. These observations argue against the development of stromal cell-associated hematopoietic stem cells in the bone marrow until approximately day 5. Increasing CFU numbers encountered in the femur between E17 and day 5 may therefore reflect blood volume enlargement caused by the development of marrow vasculature rather than true marrow seeding by HSCs. The fetal bloodstream indeed carries stem cells, clonogenic progenitors,49,52 and monocytes/macrophages that play a role in tissue remodeling. Committed progenitors may seed the fetal and early postnatal marrow and establish a first line of terminal hematopoiesis therein before true stem cells colonize bone cavities after the first week of life. The spongy bone that develops transitorily during limb formation at the metaphysis provides a base plate for the development of BM tissue. MMP-9-gelatinase B is a key regulator of growth plate angiogenesis and chondrocyte apoptosis,53 and VEGF, secreted by hypertrophic chondrocytes, couples cartilage remodeling, ossification, and angiogenesis during endochondral bone formation.19 Interestingly, Flt-1 activation in monocytes is associated with chemotaxis.54 This mechanism may explain invasion of the femur by primitive macrophages and chondroclast-osteoclast progenitors at a very early stage of endochondral ossification.18 These data and our results suggest at this time that enzymes involved in osteolysis and lysis of matrix proteins prevent the formation of marrow microenvironment. Second, we documented in this study that the acquisition of autonomous
hematopoietic competence in BM is not continuous but that it coincides
with the cessation of hematopoiesis in the liver.8 Importantly, this gain of BM function is correlated with the emergence of hematon units in the femur, the enumeration for which we have developed a quantitative assay. Once compact hematon particles emerge
at days 5 to 7, their number rapidly reaches a near plateau value,
indicating that BM gain of function follows a stepwise "one-hit"
kinetics. This suggests that one or several cell types migrate to the
femora at a well-defined developmental stage and that resultant
cell-cell interactions play an inductive role in the organization of
specialized HSC regulatory territories. Stereomicroscopic observations
of the BM shaft, prepared by careful microdissection and mild erosion,
revealed the intimate association of hematons with the vasculature
(Figure 2D-F). Immunostaining showed overlapping expression patterns of
several cell markers Third, quantitative analysis of buffy coat suspensions and hematon fractions in young adult mice indicates that though the latter contains fewer cells (5%-10% of TNC in a femur), hematons enclose a wide spectrum of stromal cell-associated HSC/multipotent hematopoietic progenitor cells and almost all native CA-forming cells. The hematon core complex may be a primordial niche in BM because it can generate hematopoietic progenitor cells autonomously in long-term microculture in the absence of irradiated feeder cells and any "community effect" from neighboring cells. The cell production capacity of buffy coat and hematon fractions was assessed in LTBMC. The hematon generated 55 times more GM-CFU and 15 times more HPP-CFC than did the buffy coat on day 28 of LTC. Mouse LTBMC provides a tool to study the commutation of myelopoiesis to B lymphopoiesis at the level of primitive HSCs.44,56 In the current experiments, hematon LTC generated 8 times more B lymphocytes than did the buffy coat cultures 3 weeks after commutation. Funk et al45 detected no significant difference in early B-lymphocyte colony-forming potential among BM aggregate and suspension fractions immediately after isolation. Because committed B-cell progenitors rapidly die in myeloid LTC conditions, our results indicate that the hematon contains more stromal cell-associated myelopoietic-lymphopoietic HSCs than does the buffy coat fraction. Restricted location of hemopoietic niches (native CA), greater frequency of LTC-IC/CAFC, and presence of common myelopoietic-lymphopoietic HSCs in the hematon explain the higher cell production observed in hematon LTC than in buffy coat LTC and suggest that stromal cells in the hematon provide specific factors that control the expansion and differentiation of HSC/multipotential hematopoietic cells. These specialized niches are limited in number, so they may constitute a rate-limiting regulatory system in maintaining HSC self-renewal and commitment. Our data support earlier notions suggesting that specific regulatory territories for HSC/hematopoietic progenitor cells exist30 and are saturable in the femur.33 This study also implies that the technique used for BM recovery
and dissociation before quantitative analysis may significantly influence the outcome of experiments
We thank Dr Paul Kincade (Oklahoma Cancer Center, Oklahoma City, OK), Dr Nydia G. Testa (Paterson Institute for Cancer Research, Manchester, United Kingdom), Dr Georges Uzan and Dr Michèle Souyri (INSERM U-506), and Dr Jean-Paul Thièry (CHB, Hôpital Paul Brousse, Villejuif) for helpful discussions. We also thank Dr Laure Coulombel (Hôpital Cochin, Paris) for kindly providing the MS-5 stroma cell line and Dr Pierre Charbord (INSERM U 506) for help with antibodies to Stro-1.
Submitted January 10, 2000; accepted August 7, 2000.
Supported in part by Institut du Cancer et d'Immunogénétique grants 07/1998 and 03/1999 and by subventions from Hôpital Paul Brousse and from Université Paris-Sud XI, UFR Kremlin-Bicêtre. Supported also by a grant from the Groupe d'Etude Hémostase et Thrombose (J.C.).
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: Istvàn Blazsek, INSERM Unité 506, Hôpital Paul Brousse, 12-14, Avenue Paul Vaillant-Couturier, 94807 Villejuif Cedex, France; e-mail: U506{at}infobiogen.fr.
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Top
Abstract
Introduction
self-renewal, expansion, and development toward the full
spectrum of hematolymphoid cell lineages.
5 mol/L 2-ME, 10
SM actin (MAH IgG-Cy3; Caltag),
ICAM-1 (RAH IgG; Caltag), Notch-1 (GAH IgG; Santa Cruz Biotech), and
MMP-9-gelatinase-B (GAH IgG; Santa Cruz Biotech). Cells were washed in
DPBS, fixed in methanol at 
20°C for 5 minutes, and, before
staining, rehydrated in PBS/0.5% albumin/0.05% NaN3.
Nuclear factors were detected after permeabilization by adding 0.2%
NP-40 to the staining solution. Anti-Hox-11 (RAM IgG), anti-Oct-2 (RAH
IgG), and CBF-A (GAH IgG) were from Santa Cruz Biotech. They were
detected at 1:100 to 1:500 dilutions with FITC-labeled secondary
antibodies. Preparations were washed, mounted with the antifading
compound ProLong (Molecular Probes, Eugene, OR), and analyzed under a
Leica DMR epifluorescence microscope.
),
total GM-CFC and HPP-CFC (
), and day 7 CAFC (
) increased
logarithmically from E17. Day 35 LTC-IC/CAFCs (
) appear at day 2, and hematon units containing native CA (
) emerge from days 5 to 7.
), NB (
).
). The MS-5 cell line (*) was used as a control
reference.
), HPP-CFC-NA (
), HPP-CFC-AD (
),
B220+ B lymphocytes (*). Representative experiments
repeated 3 times with similar results are shown. Data points represent
the mean (±1 SEM) of 4 determinations.
