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~~<br />
Loss of Attachment to Fibronectin With Terminal Human Erythroid Differentiation<br />
By M.H. Vuillet-Gaugler, J. Breton-Gorius, W. Vainchenker, J. Guichard, C. Leroy, G. Tchernia, and L. Coulombel<br />
Human erythroblastic progenitors (colony-forming uniterythroid<br />
[CFU-E] and burst-forming unit-erythroid [BFU-<br />
E]) have been shown to attach to fibronectin (Fn), a<br />
property that might be involved in the local regulation of<br />
erythropoiesis. In this study, we have investigated changes<br />
in cell attachment to Fn upon terminal erythroid differentiation.<br />
We first purified CFU-E from human marrow by<br />
avidin-biotin immune rosetting. This negative selection<br />
procedure yielded a cell population containing -80% blasts<br />
that, after characterization by colony-assays and electron<br />
microscopy, appeared to consist of CFU-E (10% to 15%)<br />
and their immediate progeny (85% to 90%). here defined as<br />
“preproerythroblasts.” In the presence of erythropoietin,<br />
purified cells differentiated into reticulocytes in 7 to 10<br />
days. Cell attachment to Fn was inversely correlated to the<br />
N MANY TISSUES acquisition of differentiated func-<br />
I tions is induced by either cell-cell or cell-matrix<br />
interactions.’.* In the bone marrow also, hematopoietic stem<br />
cells depend for their differentiation upon interactions with a<br />
complex environment composed of nonhematopoietic stromal<br />
cells and their extracellular matrix (ECM).3-6 However,<br />
there is little known about the molecular mechanisms involved<br />
in these interactions. Recently attention has been<br />
focused on the role of growth factors that are synthesized by<br />
stromal cells and act either in a soluble or bound to<br />
the cell-surface9 or to ECM components.” However, most of<br />
these studies have been performed with fibroblasts and<br />
endothelial cells from extramedullary sources that may not<br />
help to elucidate lineage- or differentiation-specific interactions<br />
between hematopoietic and stromal cells in the marrow.<br />
These may be accounted for by multiple specialized marrow<br />
stromal cells. Alternatively, hematopoietic cells themselves<br />
may modulate, as differentiation proceeds, the expression on<br />
their surface of receptors involved in stromal cell or matrix<br />
recognition. Likely candidate molecules for such a mechanism<br />
are integrins.” These glycoproteins exist as heterodimers<br />
of noncovalently associated LY and @ transmembrane<br />
subunits and are widely distributed. Three different @<br />
chains exist, which define three integrin families.” In each<br />
family, @ associates with multiple a chains, resulting in<br />
different ligand specificities. Thus, the al-6 81 family, or<br />
VLA family, includes most of the cell surface receptors for<br />
Fn and ECM proteins.”.’*<br />
There are at least two arguments to implicate integrins<br />
and cell-matrix adhesion in the regulation of hematopoietic<br />
differentiation. First, the ECM produced by marrow stromal<br />
cells has been shown to contribute to the function of the<br />
adherent layer in long-term marrow culture^.'^^'^ Second,<br />
we’’ and ~thersl~.’~ have demonstrated direct adhesion of<br />
progenitor cells to ECM proteins. Thus, colony-forming<br />
units-erythroid (CFU-E) and some burst forming unitserythroid<br />
(BFU-E) show significant attachment to Fn,”.I6<br />
whereas CFU granulocyte-macrophage (GM) interact with<br />
a different ECM component.” Furthermore, in the erythroblastic<br />
lineage, adhesion to Fn is a differentiation-regulated<br />
event, as shown by the higher capacity of CFU-E than<br />
BFU-E to bind Fn,” and by the loss of Fn-adhesion and of<br />
stage of differentiation of the erythroid cell: more than 50%<br />
of the CFU-E population reproducibly adhered to Fn,<br />
whereas at most 30% of the preproerythroblasts had the<br />
same capacity. Adhesion was further lost at late maturation<br />
stages, and a constant finding was the inability of<br />
reticulocytes to adhere to Fn. Finally, CFU-E adhesion to Fn<br />
was blocked by polyclonal IgG raised against the Fn<br />
receptor and by a monoclonal antibody against VLA-I.<br />
These results demonstrate that adhesion to Fn is developmentally<br />
regulated during normal human erythropoiesis.<br />
Restriction of its expression to CFU-E and its first divisions<br />
strikingly correlates with the migratory capacity of these<br />
cells.<br />
0 1990 by The American Society of Hematology.<br />
the Fn receptor in murine erythroleukemic cells when induced<br />
by dimethyl sulfoxide<br />
In the present study, we have investigated how adhesion to<br />
Fn is modulated during terminal erythroid differentiation.<br />
For this purpose, we developed a procedure for obtaining a<br />
highly enriched population of very immature erythroid cells,<br />
ie, CFU-E and their immediate progeny. These purified cells<br />
were then induced to differentiate in vitro, and during this<br />
process, we investigated their changing ability to attach to<br />
Fn. We found that CFU-E attach strongly to Fn, and that<br />
this property is lost at specific stages of terminal erythroid<br />
maturation. Moreover, the binding of CFU-E to Fn is<br />
mediated by a receptor bearing some similarities to the<br />
integrin family.<br />
MATERIALS AND METHODS<br />
Bone marrow samples. Normal marrow samples were obtained<br />
after informed consent from hematologically normal patients undergoing<br />
hip surgery. Cells were flushed from the bone fragments with a<br />
medium containing 100 to 200 pg/mL DNAse (type I, Sigma<br />
Chemical Co, St Louis, MO). Cells were separated on Ficoll-<br />
Hypaque (1.077 g/mL) (Eurobio, Paris, France). Light-density<br />
mononuclear cells were washed twice, resuspended at a concentration<br />
of 3 x lo6 cells/mL in a-medium supplemented with 10% fetal<br />
From the Hematology Laboratory and University Paris XI,<br />
H6pitaI BicBtre, Kremlin-BicZtre: and INSERM U.91, H6pital<br />
Henri-Mondor, CrLteil, France.<br />
Submitted April 24,1989; accepted October 24. 1989.<br />
Supported by Grants No. CRE 862007 from INSERM (L.C.);<br />
Grant No. ARC 6532 from the Association pour la Recherche<br />
contre le Cancer; and a grant from Ligue contre le Cancer (Comitt<br />
Dkpartemental des Hauls-de-Seine). M.H. K-G. is the recipient of a<br />
fellowship from the French Ministere de I’lndustrie et de la<br />
Recherche.<br />
Address reprint requests to Laure Coulombel, MD, PhD, Institut<br />
de Pathologie Cellulaire, H6pital Bici?tre, 94275 Kremlin-Bici?tre,<br />
France.<br />
The publication costs of this article were defrayed in part by page<br />
charge payment. This article must therefore be hereby marked<br />
“advertisement” in accordance with 18 U.S.C. section 1734 solely to<br />
indicate this fact.<br />
6 1990 by The American Society of Hematology.<br />
0006-4971/90/7504-0028$3.00/0<br />
<strong>Blood</strong>, Vol 75, No 4 (February 15), 1990: pp 865-873 865
866 VUILLET-GAUGLER ET AL<br />
calf serum (FCS) (Flow Laboratories, Paris, France), and incubated<br />
in 75 cm’ flasks for 1 hour at 37OC in a 5% CO, atmosphere in air to<br />
remove plastic-adherent cells. The nonadherent mononuclear cells<br />
(NA-MNC) were subsequently fractionated by immune rosetting<br />
(see below).<br />
Antibodies. Monoclonal antibodies (MoAbs) against human<br />
HLA-DR (OKIal), human T cells (OKT3), and human CALLA<br />
(CD IO; OKB-CALLA) were purchased from Ortho Diagnostic<br />
Systems (Roissy, France). MoAb recognizing HPCA (CD34) was<br />
from Becton Dickinson (Mountain <strong>View</strong>, CA), BI (anti-B cells)<br />
from Coulter Immunology (Hialeah, FL). MoAbs CH3 (IgM<br />
recognizing granulocytes and CFU-GM), CS4 (recognizing glycophorin<br />
A) and CK26 (binding to lymphoid and monocytic cells) have<br />
been previously described,20 and were generously provided by Dr<br />
Berthier (INSERM U.217, Grenoble, France). Anti-human HLA-<br />
DR K5 MoAb was a gift from Dr Fermand (INSERM U108,<br />
Hdpital St Louis, France). FA6-152 MoAb,,’ which recognizes an<br />
88 Kd glycoprotein on platelets, monocytes, and erythroid cells and<br />
belongs to the CD36, was a gift from L. Edelman (Institut Pasteur,<br />
Paris, France). Anti-platelet GPIIIa C17 MoAb and anti-glycophorin<br />
A (GPA) plyclonal antibody used in electron microscopy<br />
were kind gifts from Drs F. Tetteroo22 and L.C. Ander~son.~’<br />
Biotin-conjugated affinity-purified goat anti-mouse IgM (p specific)<br />
and IgG (H -t L) F(ab’)2 fragments were obtained from Cappel-<br />
Cooper Biomedical (Malvern, PA). A rabbit antiserum prepared<br />
against the purified human placental fibronectin receptor (HFnR-<br />
Ab) was purchased from Telios Pharmaceuticals (La Jolla, CA).<br />
This antibody recognizes mainly the 115 Kd 81 subunit of the<br />
receptor, and is less reactive with the 160 Kd a5 subunit.24 A rat<br />
MoAb BlE5, which is directed against the a5 subunit of the human<br />
Fn receptor and which selectively inhibits cell attachment to Fn was<br />
kindly provided by C. Damsky (University of California, San<br />
Francisco, CA) and used as hybridoma s~pernatant.~’<br />
Enrichment of erythroblastic cells by immune rosetting. Cell<br />
purification was performed by negative selection using avidin-biotin<br />
immune rosetting according to the original technique described by<br />
Fiirfang and Thierfelder.26 Briefly, 1.2 mg avidin (Vector Laboratories<br />
Inc, Burlingame, CA) was covalently coupled to 2 x lo9<br />
glutaraldehyde-fixed sheep red blood cells (SRBC-A) as described in<br />
detail elsewhere.26 SRBC-A can be kept at 4OC in sterile conditions<br />
for at least 4 to 6 months without loss of reactivity. The purification<br />
was performed in two steps: NA-MNC (usually 0.5 to 3 x IO8 cells)<br />
were first mixed with a 10-fold excess of SRBC-A. The mixture was<br />
centrifuged 6 minutes at 200g, and the pellet was incubated 45<br />
minutes at 4OC. Cells were then gently resuspended at a concentration<br />
of 1 x lo7 cells/mL in a-medium supplemented with 5% FCS<br />
((~5%) and layered over Ficoll-Hypaque. Mature granulocytes that<br />
had phagocytized SRBC-A were eliminated in the pellet (fraction<br />
P-1). Cells harvested at the interface (fraction 1-1) were washed<br />
twice, resuspended at a concentration of 4 x lo7 cells/mL, and<br />
incubated 45 minutes at 4OC with an excess of the following MoAbs:<br />
OKIal, OKT3, OKB-CALLA, B1, anti-HPCA, K5, CH3, CS4,<br />
CK26. At the end of the incubation period, cells were washed twice,<br />
resuspended at a concentration of 4 x lo7 cells/mL in a5% and<br />
incubated with a 1/40 dilution of a mixture of biotin-conjugated<br />
goat anti-mouse IgM and IgG for 45 minutes at 4°C. Excess<br />
biotinylated immunoglobulin was removed by three washes in 015%.<br />
Finally, labeled cells were incubated with SRBC-A in the proportion<br />
of l:lO, as described above, and rosettes were allowed to form. Cells<br />
were then gently resuspended and adjusted to the initial concentration.<br />
Rosettes were removed by centrifugation over Ficoll-Hypaque<br />
(fraction P-2), and unlabeled cells were recovered at the interface<br />
layer (fraction 1-2). Cells recovered in each fraction (1-1, P-I, 1-2,<br />
P-2) were counted and the number of rosettes was determined.<br />
Purified cells (1-2) were characterized morphologically on May-<br />
Griinwald-Giemsa (MGG) stained cytocentrifuged slides, by electron<br />
microscopy (see below), and by their ability to form colonies in<br />
methylcellulose colony-assays.<br />
Electron microscopy. The simultaneous detection of specific<br />
membrane markers (GPIIIa and GPA) by immunogold and intracellular<br />
peroxidatic activities was determined on the purified erythroid<br />
cell population (1-2) as previously described in detaiL2’ Briefly,<br />
immunolabeling was first performed by incubating cells with specific<br />
antibodies recognizing either GPIIIa or GPA, and then with a<br />
second antibody coupled with 15 nm and 5 nm gold particles,<br />
respectively. Cells were then fixed in 1.25% glutaraldehyde in Gey’s<br />
buffer, washed, incubated in diaminobenzidine medium,28 post-fixed<br />
with osmium tetroxide, dehydrated, and embedded in Epn. Sections<br />
were examined with a Philips electron microscope CM 10 (Eindhoven,<br />
The Netherlands) without staining in order to easily identify<br />
ferritin molecules by their own contrast.<br />
Suspension culture of purijed erythroblastic cells. Purified<br />
cells from the 1-2 fraction were cultured at a concentration of 1 x lo6<br />
cells/mL in a-medium containing 20% FCS and 3 U/mL human<br />
urinary erythropoietin (Ep). The batch of FCS used during the study<br />
contained less than 10 pg of Fn per milliliter as measured by an<br />
enzyme-linked immunosorbent assay (ELISA). Cultures were initiated<br />
in 96-flat bottom microtiter plate wells (Falcon, Becton-<br />
Dickinson, France, No. 3072; 200 pL per well) and incubated at<br />
37OC in a water saturated air atmosphere with 5% CO,. Cells were<br />
harvested regularly, and their number, viability (dye exclusion),<br />
morphology, plating efficiency, and ability to adhere to Fn were<br />
determined. Three stages of maturation were defined after MGG<br />
staining: immature (proerythroblasts and early basophilic erythroblasts),<br />
intermediate (late basophilic and polychromatophilic), and<br />
mature (acidophilic and enucleations). Reticulocytes were counted<br />
separately .<br />
Hematopoietic progenitor cell colony-assays. Cells from the<br />
P-1, 1-1, P-2, and 1-2 fractions were plated in a-medium containing<br />
0.8% methylcellulose, 30% FCS, 1% deionized bovine serum albumin<br />
(BSA), mol/L 8-mercaptoethanol, 3 U/mL Ep and 10%<br />
agar leukocyte conditioned medium. Final concentration of cells per<br />
dish was 1.5 x IO’ for NA-MNC and P-1 and 1 to 3 x lo4 for 1-1,<br />
1-2, and P-2. Erythroid colonies were scored as previously described<br />
in detail7<br />
Adhesion assay. The capacity of erythroid cells to adhere onto<br />
Fn was assessed in two different ways: purified 1-2 cells and cells<br />
grown during various periods of time in suspension (see above) were<br />
incubated on Fn-coated 2.5 cm2 tissue culture wells (2 to 4 x 10’<br />
cells per well) in (Y 5% for 2 hours at 37OC in a 5% CO, atmosphere.<br />
Nonadherent cells were then harvested, weakly adherent cells were<br />
removed by two mild washings, and strongly adherent cells were<br />
detached by vigorous pipetting or short trypsinization. Adherent and<br />
nonadherent cells were counted, stained with MGG for morphologic<br />
examination, and plated in methylcellulose colony-assays at 1.5 x<br />
lo4 cells per dish to assess the number of CFU-E in each fraction. In<br />
the second method, cells were grown as described above for suspension<br />
culture, except that they were incubated from the start on<br />
Fn-coated 96-flat bottom microtiter plate wells (1 x lo6 cells/mL,<br />
200 pL per well) and kept on that substrate for the whole culture<br />
period. Every 2 days during the 6 to 10 day-culture period, one well<br />
was sacrificed and nonadherent and adherent cells were harvested<br />
separately, counted, and stained with MGG. Cell viability of both<br />
fractions was checked by dye exclusion (Nigrosin). In both methods,<br />
tissue-culture wells were precoated with 50 to 80 pg/mL of Fn<br />
purified from human plasma by gelatin affinity chromatography, or<br />
human collagens I, 111, and IV in the monomeric form or laminin.<br />
Nonspecific binding sites were blocked by 1-hour incubation in 10<br />
mg/mL BSA.”
I<br />
ERYTHROBLAST ADHESION TO FIBRONECTIN 867<br />
L A<br />
- {<br />
A<br />
Fig 1. Photomicrographs of MGG-stained 1-2 cells before culture (A), and after 7 days (6). and 10 days (C), in suspension culture in the<br />
presence of Ep. There was no difference in the morphology of cells grown in suspension or on an Fn 8ubstrate. (Original<br />
magnification, xs00)<br />
In some experiments, 1-2 cells were first incubated (45 minutes at<br />
4OC) with a 1/10 dilution of the HFnR-Ab or undiluted BlE5<br />
hybridoma supernatant, and then incubated as described above onto<br />
Fn-coated wells for 2 hours in the continuous presence of the<br />
antibody. Negative controls included incubation of cells either with<br />
nonimmune rabbit IgG or with anti-CD36 (FA6-152) or anti-GPA<br />
hybridoma supernatant (CS4).<br />
Indirect immuncfluoresence labeling. Purified erythroblasts were<br />
stained with the HFnR-Ab by indirect immunofluorescence. For<br />
that purpose, 0.5 to 1 x lo6 cells in 100 pL were incubated 30<br />
minutes at 4% with a 1/10 dilution of the HFnR-Ab or control<br />
nonimmune rabbit IgG. Cells were subsequently washed twice and<br />
resuspended in a 1 /50 dilution of fluorescein-labeled goat anti-rabbit<br />
IgG F(ab')2. Incubation was continued for 30 minutes at 4OC. Cells<br />
were washed twice, resuspended at a final concentration of 1 x IO6<br />
cells/mL in phosphate buffered saline (PBS) containing 1% BSA<br />
and examined under a Zeiss microscope equipped for epifluorescence.<br />
RESULTS<br />
Purification to homogeneity of very immature human<br />
marrow erythroblasts. The purification procedure de-<br />
scribed under Materials and Methods yielded in 20 experiments<br />
a population (1-2) containing 79% f 3% of blasts that<br />
were HLA-DR, CD34, and GPA negative by indirect immunofluorescence<br />
(Fig 1). Positive labeling with the FA6-152<br />
MoAb whose expression is restricted to platelets, monocytes,<br />
and erythroid cells suggested the erythroid origin of these<br />
~elIs.2~<br />
The erythroid origin of purified 1-2 cells was clearly<br />
demonstrated by the 1-2 cells' ultrastructural characteristics<br />
and immunophenotype. Eighty percent of 1-2 cells were<br />
classified as erythroid and included only two maturation<br />
stages (Fig 2): the most immature phenotype represented<br />
10% to 15% of this cell fraction. Despite the absence of GPA<br />
and rhopheocytosis, the population was recognized as erythroid<br />
by the presence of Theta granules (8-Gr) and of a few<br />
ferritin molecules located in the 8-Gr and scattered through<br />
the cytoplasm (Fig 2A, B). These cells were presumably<br />
CFU-E, since the 1-2 cell fraction contained the same<br />
proportion of progenitors giving rise to 1-2 hemoglobinized<br />
clusters (see below). Other erythroid cells in the 1-2 fraction<br />
were more mature, as shown by the presence of GPA (Fig<br />
Fig 5. Photomicrographs of<br />
MGG-stained adherent (A) and<br />
nonadherent (6) erythroblasts<br />
observed in a typical experiment<br />
where purified 1-2 cells<br />
were cultured in Fn-coated<br />
wells for 7 days.<br />
t<br />
A
868 VUILLET-GAUGLER ET AL<br />
.<br />
Fig 2. Ultrastructural analysis<br />
of purified 1-2 cells. (A) Pre<br />
sumptive CFU-E. (inset left) At<br />
low magnification (original magnification,<br />
~3,300) a very large<br />
nucleolus is saen in tho nucleus.<br />
Cytoplasmic vacuoles and<br />
granules are clustered in the<br />
golgi zone. At higher magnification<br />
(original magnification,<br />
x 52.600). the structura of 0-Gr<br />
is clearly distinguished. In all of<br />
them, a few ferritin molecules<br />
are identified. There are no gold<br />
particles on the plasma mambrane,<br />
indicating the absence<br />
of GPA. (inset right) High magnification<br />
(original, x 108,300)<br />
of a 0-Gr with its dense rod and<br />
some dense periodic structure.<br />
The typical configuration of ferritin<br />
molecules (f) is recognized<br />
in the cytoplasm and in 0-Gr.<br />
(E) Preproerythroblast. Weak<br />
labaling for GPA is detected at<br />
one place of the cell membrane<br />
(arrow). Two rhopheocytosis invaginations<br />
(r), also called<br />
coated pits, are indicated. In<br />
one of them, ferritin molecules<br />
(f) are presant. Nota also the<br />
presence of intracytoplasmic<br />
free ferritin (f)(original magnification,<br />
~68,800). (C) A more<br />
matura preproarythroblast as<br />
judged by the heavier labeling<br />
(arrow) by GPA-Ab and by the<br />
increased number of free ferritin<br />
molecules (1). and ferritin<br />
molecules associated with theta<br />
granules 0-Gr (original magnification.<br />
~58,800).<br />
2B). However, GPA density was below the threshold of<br />
sensitivity expected for conventional immunofluorescence or<br />
avidin-biotin labeling, which explains why these cells were<br />
not eliminated by immune rosetting. One or 2 sites of<br />
rhopheocytosis were observed (Fig 2B), and the concentration<br />
of ferritin in the 0-Gr and in the cytoplasm was increased<br />
in comparison with the more immature cdls (Fig 2C).<br />
Hemoglobin was absent, as judged by the negative dense<br />
peroxidase reaction. These properties, together with the low<br />
expression of rhopheocytosis and GPA, indicated that this<br />
population was less differentiated than the classical<br />
proerythroblast,*’ and we have therefore designated these<br />
cells “preproerythroblasts” as they appear to represent a<br />
stage intermediate between CFU-E and proerythroblasts.<br />
The 1-2 fraction contained on average 65% preproerythroblasts.<br />
Twenty to twenty-five percent of 1-2 cells were<br />
nonerythroid, as recognized by electron microscopy. Most of<br />
these belong to the megakaryocytic lineage, and a minority<br />
were mast cells, basophil myelocytes, and promyelocytes<br />
(data not shown).<br />
Clonogenic properties of purified 1-2 cells were assessed in<br />
methylcellulose colony-assays (Table 1). If we define a<br />
CFU-E as a progenitor giving rise to a cluster of at least 10 to<br />
15 hemoglobinized cells at day 9 to 10, 11% * 1% of 1-2 cells<br />
were CFU-E (n = 20), whereas preproerythroblasts variably<br />
gave rise to clusters of less than 10 cells that usually had<br />
lysed by day 9. The proportion of CFU-E matched the<br />
proportion of very early GPA negative erythroid cells recognized<br />
by electron microscopy. CFU-E enrichment was 36 2<br />
4-fold relative to NA-MNC and over 50 to 60-fold compared<br />
with native marrow. The enrichment of preproerythroblasts<br />
could not be calculated, as these cells cannot be accurately<br />
quantitated in unpurified marrow cells either by morphologic<br />
or colony-assays criteria. Recovery of CFU-E in the 1-2
ERYTHROBLAST ADHESION TO FIBRONECTIN 869<br />
Table 1. Progenitor Cell Enrichment During the Different Steps of the Purification Procedure<br />
Fraction<br />
NA-MNC<br />
I- 1<br />
P- 1<br />
1-2<br />
P-2<br />
No. Progenitors/l.B x lo5 Cells Plated<br />
CFU-E mBFU-E IBFU-E CFU-GM<br />
490 f 60<br />
1,609 k 314<br />
(3)<br />
62 f 16<br />
17,571 k 1,658<br />
(36)<br />
586 k 123<br />
155 f 24 12 f 2 328 f 54<br />
427 f 57 43 f 7 1,308 f 113<br />
(2.7) (3.5) (4)<br />
27 k 13 924 90 k 26<br />
1,262 f 276 26 f 8 898 f 286<br />
(8) (2) (2.7)<br />
634 f 74 57 f 12 1,316 f 263<br />
Each number represents the mean f SEM of seven different experiments. Numbers in brackets represent the enrichment factor relative to NA-MNC in<br />
the 1-1 and 1-2 fractions.<br />
Abbreviations: mBFU-E, mature BFU-E; iBFU-E, immature BFU-E.<br />
fraction was 41% 3%. In seven experiments where the<br />
different fractions obtained during the purification were<br />
cloned in methylcellulose, 10% CFU-E were found in the P-2<br />
fraction (Table 1). The latter may represent a subset of<br />
HLA-DR+, CD34+ CFU-E. Our results also showed that<br />
contamination by other progenitor cells was negligible since<br />
in 20 experiments, BFU-E and CFU-GM represented 0.7%<br />
and 0.6% of 1-2 cells, respectively (Table 1).<br />
In order to assess the proliferative and differentiative<br />
behavior of the purified 1-2 cells in vitro, we cultured them in<br />
suspension in the presence of 3 U/mL Ep (but no other<br />
hematopoietic growth factors). For the first 2 to 3 days, there<br />
was no net change in the number of cells, whereas during the<br />
next 7 to 9 days, there was a four- to ninefold increase over<br />
the initial number of cells, reflecting the proliferation of the<br />
maturing erythroblasts (Fig 3). Morphologic examination<br />
showed that 1-2 cells differentiated synchronously up to the<br />
enucleation stage, with the production of many reticulocytes<br />
(Fig 1). In the absence of Ep, 1-2 cells did not survive beyond<br />
48 hours. Nonerythroid cells were not maintained in these<br />
cultures.<br />
Adhesion of purified erythroblasts to Fn during their<br />
diferentiation. CFU-E and preproerythroblasts adhesion<br />
to Fn was evaluated in 19 separate experiments using 1-2<br />
cells. The proportion of Fn-adherent CFU-E was precisely<br />
quantitated by colony-assays of adherent and nonadherent<br />
cells and that of Fn-adherent preproerythroblasts was estimated<br />
from the proportion of total Fn-adherent 1-2 cells. The<br />
mean (+SEM) proportion of Fn-adherent CFU-E was<br />
58% f 5%. It was over 65% in 10 of 19 experiments, and<br />
below 30% in only three experiments. In contrast to the high<br />
proportion of adherent CFU-E, only 31% 3% of total<br />
nucleated 1-2 cells were Fn-adherent. As CFU-E represents<br />
10% to 15% of the 1-2 population, and preproerythroblasts<br />
65%, this gives a proportion of Fn-adherent preproerythroblasts<br />
below 25%. This difference in the ability of CFU-E<br />
and preproerythroblasts to attach to Fn was seen in each<br />
experiment, and suggests loss of adhesion to Fn as CFU-E<br />
matures in preproerythroblasts. No significant attachment of<br />
CFU-E and preproerythroblasts occurred onto either human<br />
collagen I, 111, IV or laminin.<br />
The effect of differentiation on the adherence to Fn was<br />
next investigated: seven experiments were performed using<br />
cells removed after varying periods of time from 7 to 10<br />
day-cultures of highly CFU-E-enriched population, and six<br />
experiments were performed by culturing cells 7 to 10 days<br />
on an Fn substrate. Results from these 11 experiments have<br />
been pooled in Fig 4. In each experiment, adhesion to Fn<br />
decreased with maturation and was completely lost at late<br />
maturation stages. Even though the rate of decline varied<br />
between experiments, the pattern observed was similar in<br />
each experiment, ie, in the purified 1-2 population before<br />
culture (day 0), the proportion of adherent CFU-E was<br />
higher (58%) than that of total purified cells (30%) and thus<br />
preproerythroblasts, and slowly declined over the next 8 days<br />
in culture to reach 20% at day 8 to 9 (polychromatophilic<br />
stage). At that stage, the capacity to adhere to Fn appeared<br />
to rapidly decline and was completely lost at the enucleation<br />
- 10-<br />
E<br />
\ 9-<br />
(D<br />
-; 8-<br />
z 7-<br />
6-<br />
LL<br />
s 5-<br />
W<br />
z 4-<br />
0<br />
3-<br />
W 2 2-<br />
1 1 1 1 1 1 1 1 1 1 ,<br />
1 2 3 4 5 6 7 8 9 10 11<br />
DAYS IN CULTURE<br />
2 Imm 81 87 30 3 0<br />
0 0 60 59 22<br />
Mat 0 0 10 38 78<br />
2 1 inter<br />
c<br />
Fig 3. Changes in the number and cytology of cells present<br />
after varying periods of time when purified 1-2 cells were cultured<br />
in suspension in the presence of Ep. Each point represents the<br />
mean f SEM of the cell concentration observed in three to seven<br />
experiments. The SEM is not indicated for the day 10 point since<br />
only two experiments were available. Morphologic examination of<br />
the cells was performed after MGG staining at the indicated day<br />
(see Materials and Methods).<br />
1
870 VUILLET-GAUGLER ET AL<br />
adhere to Fn was paralleled by the disappearance of a<br />
cell-surface receptor (data not shown).<br />
2<br />
W<br />
V<br />
5 40-<br />
W<br />
K W<br />
8 30-<br />
a<br />
I-<br />
Z<br />
W<br />
p 20- Purified 1-2<br />
W 0<br />
10 - cells<br />
DAYS IN CULTURE<br />
Fig 4. Loss of adhesion to Fn with terminal erythroid differentiation.<br />
The percentage of adherent cells and CFU-E was first<br />
determined in the 1-2 population at day 0. Cells were then cultured<br />
either in suspension (n = 7) or on Fn-coated wells (n = 6). and<br />
the proportion of adherent cells was measured sequentially as<br />
described in the text. Each point represents the mean c SEM of<br />
the percent adherent cells. 1-2 cells differentiated synchronously<br />
up to day 7. However, beyond that point, the population was<br />
heterogeneous, and reticulocytes never represented 100% of the<br />
cell population. Therefore, the day 11 point and the dashed line<br />
represent the percent adhesion expected if 100% of day 7 cells<br />
had matured into reticulocytes.<br />
stage. Reticulocytes were always found exclusively in the<br />
nonadherent fraction. This was best appreciated in experiments<br />
in which purified 1-2 cells were cultured on Fn during<br />
several days in the presence of Ep. In a representative<br />
experiment, shown in Fig 5, 20% to 30% of the day 7 cells<br />
were still strongly adherent to Fn. Most of these adherent<br />
cells had a phenotype of polychromatophilic erythroblasts,<br />
and no reticulocytes were seen (Fig 5). However, some<br />
reticulocytes were already present in this culture, but they<br />
had been released from the Fn substrate and represented<br />
25% of the nonadherent population (Fig 5). The same<br />
partitioning was seen for enucleating cells. Failure of enucleating<br />
erythroblasts and reticulocytes produced in culture<br />
(kFn) to attach to Fn was not attributable to any change in<br />
their viability as assessed by dye exclusion. Furthermore,<br />
assessment of enriched reticulocytes, freshly isolated from<br />
the peripheral blood from three patients with hemolytic<br />
anemia showed that these also did not attach to Fn in our<br />
assay (data not shown).<br />
Effect of anti-HFnR antibodies on the adhesion of purified<br />
erythroblasts to Fn. In seven separate experiments,<br />
CFU-E and preproerythroblasts (1-2 cells) were first preincubated<br />
with either a 1/10 dilution of polyclonal IgG against<br />
HFnR or with undiluted B1E5 hybridoma supernatant and<br />
then incubated in Fn-coated wells. As shown in Fig 6, both<br />
antibodies specifically inhibited CFU-E and 1-2 cells (ie,<br />
preproerythroblast) adhesion to Fn, as nonimmune rabbit<br />
IgG, anti-CD 36 (FA6-152), and anti-GPA had no effect.<br />
HFnR-Ab labeled 100% of erythroid 1-2 cells by indirect<br />
immunofluorescence, whereas enucleating cells and reticulocytes<br />
stained negatively, suggesting that the disappearance<br />
of the functional capacity of differentiating erythroblasts to<br />
DISCUSSION<br />
One of the major questions regarding hematopoiesis is the<br />
understanding of the mechanisms whereby progenitor cells<br />
interact with their marrow environment. Our recent<br />
observationls that human marrow CFU-E and mature BFU-<br />
E selectively attach to ECM prepared from marrow fibroblasts<br />
and to purified Fn suggests that progenitor cellsmatrix<br />
interactions might represent one such mechanism. In<br />
the present study, we have demonstrated that attachment to<br />
Fn is a property of most CFU-E and early erythroblasts in<br />
normal human marrow, and that this property is subsequently<br />
lost upon terminal erythroid differentiation.<br />
We have approached this question by designing an in vitro<br />
model that reproduces the successive steps followed by a<br />
CFU-E when it differentiates into reticulocytes. Our strategy<br />
was first to purify human CFU-E directly from fresh marrow<br />
samples. Negative selection by avidin-biotin immune rosetting<br />
reproducibly yielded a homogenous population of very<br />
immature cells identified as erythroid by their positive<br />
labeling with FA6-1 52,29 and by ultrastructural criteria. The<br />
procedure described was nontoxic, and more powerful and<br />
reliable in our hands than panning.30s31 Moreover, large<br />
numbers of marrow cells could be easily processed within a<br />
few hours with minimal cell loss, in contrast to cell sorting.'*<br />
The availability of a highly enriched population of very<br />
immature normal erythroblasts gave us the opportunity to<br />
delineate the ultrastructural changes that are associated with<br />
human erythroid cell differentiation. Previous information<br />
has relied on analyses of cells from patients with erythroblastic<br />
leukemia or of partially purified populations of normal<br />
CFU-E.31 The earliest markers detected in 1-2 cells were<br />
0-Gr and ferritin molecules (Fig 2). Most likely, cells that<br />
exhibited these two features only represent CFU-E. This<br />
4 60<br />
W<br />
I<br />
5 30<br />
g 20<br />
: 10<br />
a.<br />
CFU-E<br />
T<br />
T<br />
PURIFIED CELLS<br />
Fig 6. Inhibition of CFU-E and 1-2 cell attachment to Fn by<br />
anti-HFnR antibodies. 1-2 cells were incubated 2 hours on Fncoated<br />
wells after preincubation with either polyclonal IgG against<br />
the human Fn receptor (a5Dl; ) or B1E5 MoAb against the a5<br />
chain (W). Controls included preincubation of the cells with no<br />
antibody (0). nonimmune rabbit IgG (&I, or with hybridoma<br />
supernatant from an unrelated erythroid-specific antibody (B).<br />
Histograms show the mean * SEM of the percentage of adherent<br />
cells (n = 6).
ERYTHROBLAST ADHESION TO FIBRONECTIN 87 1<br />
conclusion is based on our finding that this morphology<br />
identified the same proportion of cells in the purified population<br />
as were detected by colony-assay as CFU-E. Surprisingly,<br />
these cells did not express platelet-peroxidase-like<br />
activity previously described in presumptive CFU-E.27,31<br />
However, after a few hours in culture, this activity was<br />
recovered (data not shown), suggesting its possible inactivation<br />
by the purification procedure. Cell-surface GPA and<br />
images of rhopheocytosis were seen on the other 1-2 erythroid<br />
cells, but at a much lower density than what has been<br />
described for proerythr~blasts.~~.~~ These cells, or “preproerythroblasts,”<br />
therefore appear to represent an intermediate<br />
stage between CFU-E and proerythroblasts.<br />
Human CFU-E have been purified only indire~tly~’.~~~~~<br />
through the proliferation of partially purified blood or<br />
marrow BFU-E cultured 7 to 9 days either in liquid<br />
su~pension,~~ or in semisolid assay” in the presence of Ep.<br />
This approach also yielded 80% pure populations of very<br />
immature erythroblasts containing up to 50% CFU-E. However,<br />
in contrast to cells directly purified from marrow by<br />
avidin-biotin immune rosetting, which appear homogeneous<br />
with respect to their stage of maturation, BFU-E-derived<br />
cells are more heterogeneous and include many hemoglobin-<br />
ized proerythrobla~ts.~~ CFU-E and early erythroblasts have<br />
also been purified from either thiamphenicol-treated,36 or<br />
Friend virus-infected mice spleen cells,37 but it is difficult to<br />
compare them with human cells.<br />
We have previously shown that the primitive BFU-E,<br />
which give rise to multiclustered colonies, do not attach to<br />
Fn, whereas 30% of CFU-E, which represents the most<br />
mature type of progenitor, express this property.” In this<br />
study, we assessed the attachment to Fn of purified CFU-E,<br />
preproerythroblasts, and their progeny, generated in cultures<br />
that support their terminal erythroid differentiation. Interestingly,<br />
the majority of CFU-E recovered in the purified<br />
population selected for these studies showed a higher proportion<br />
of Fn-adhering members. Although a direct comparison<br />
of Fn-adherence for purified and unpurified CFU-E was not<br />
undertaken, it is possible that cells defined as CFU-E by<br />
colony-assays are heterogeneous with respect to their Fnadherence<br />
properties, and that our purification procedure<br />
isolated a subset of CFU-E with higher Fn-adherence potential.<br />
Alternatively, it is possible that the adhesion assay may<br />
give different absolute results according to the composition of<br />
the population being tested. What the present studies have<br />
revealed is that adherence to Fn also clearly changes during<br />
the later stages of erythroid cell differentiation: (1) CFU-E<br />
attached to Fn in the highest proportion, close to 60% (Fig 4),<br />
(2) the proportion of adherent preproerythroblasts was<br />
consistently half that observed with the copurified CFU-E,<br />
and (3) enucleating erythroblasts and reticulocytes derived<br />
from these immature erythroblasts were unable to attach to<br />
Fn. These data demonstrate that the Fn-adhesion property is<br />
lost in differentiating erythroblasts in two sequential stages:<br />
an initial decline during the transition from CFU-E to<br />
preproerythroblasts, with a subsequent loss of all remaining<br />
adhesive properties at the time of enucleation.<br />
Expression of Fn-adhesion during human erythroid differentiation<br />
appears to be strikingly correlated to the migratory<br />
capacity of these cells. In semisolid culture media, all BFU-E<br />
exhibit an extensive migratory capacity, which is abruptly<br />
lost at the CFU-E level.38 In vivo, marrow cell egress occurs<br />
at the reticulocyte stage, and young reticulocytes are motile,<br />
whereas very few CFU-E circulate. It is thus tempting to<br />
speculate that these changes in erythroid cell motility reflect<br />
changes in cell attachment to Fn and possibly other adhesive<br />
glycoproteins of the marrow ECM. This is strengthened by<br />
the recent observation that glycoprotein IV, an 88 Kd<br />
glycoprotein identified as a thrombospondin re~eptor,’~ is<br />
present on mature BFU-E, CFU-E and erythroblasts, and is<br />
similarly lost during terminal erythroid differentiati~n.’~<br />
Loss of attachment to Fn with terminal differentiation has<br />
been previously observed in DMSO-induced murine erythroleukemic<br />
cells,” and hemin-induced human K 562 cells,”<br />
and ascribed to the loss of the 140 Kd Fn receptor molecule<br />
identified as VLA-5 on uninduced K562.12,40*41 Results obtained<br />
with human reticulocytes isolated from patients with<br />
hemolytic anemia or recently splenectomized have shown<br />
that these cells can attach to Fn?’ which appears to contradict<br />
the results presented in this study. However, reticulocytes<br />
produced in response to hemolysis are not identical to<br />
those produced during steady-state erythropoie~is,”~ and<br />
their adhesion properties might be altered by the underlying<br />
disorder.” Furthermore, the marked susceptibility of cell<br />
adhesion to the conditions in which the functional attachment<br />
assay is performed might explain quantitatively different<br />
results.<br />
Both the anti-a5/31 polyclonal IgG and the anti-d MoAb<br />
inhibited CFU-E and preproerythroblast adhesion to Fn.<br />
This strongly suggests that VLA-5 (a5pl) functions as an Fn<br />
receptor on normal human erythroblastic cells. The Fn<br />
receptor was undetectable from reticulocytes by indirect<br />
immunofluorescence. However, changes in the expression of<br />
the Fn receptor during in vitro erythroblastic differentiation<br />
could not be analyzed in more detail by immunofluorescence:<br />
the polyclonal antibody recognizes not only VLA-5, but all<br />
integrins sharing the /3l subunit, and some of these molecules<br />
may be expressed by erythroblasts, and the B1E5 MoAb is<br />
not suitable for fluorescence labeling (C. Damsky, personal<br />
communication, January 1989).<br />
The above results demonstrate that adhesion to Fn is<br />
precisely regulated during normal human erythroid differentiation<br />
and may influence cell migration in the bone marrow.<br />
A challenging question will be to determine if stage-specific<br />
expression of integrins will influence cell behavior by triggering<br />
early events associated with erythroid cell proliferation<br />
and differentiati~n.~’.~~<br />
ACKNOWLEDGMENT<br />
We gratefully acknowledge the help of the surgeons who provided<br />
marrow samples: Drs Brunet, Missenard, and Lapresle of Clinique<br />
Arago and Drs Bonnet, Judet, and Koechlin of HBpital de la<br />
Croix-Rouge and their nursing staff. We thank Dr A. Eaves (Terry<br />
Fox Laboratory, Vancouver, Canada) for the gift of erythropoietin;<br />
Drs L.C. Anderson, R. Berthier, C. Damsky, L. Edelmann, J.P.<br />
Fermand, and F. Tetteroo for their generous gifts of antibodies; C.<br />
Eaves, D. Baruch, and M. Rosemblatt for helpful discussions and<br />
critical reading of the manuscript, and M. Laroche for excellent<br />
secretarial assistance.
VUILLET-GAUGLER ET AL<br />
1. Edelman GM: Morphoregulatory molecules. Biochem 27:<br />
3533,1988<br />
2. Li ML, Aggeler J, Farson A, Hatier C, Hassel J, Bissell M:<br />
Influence of a reconstituted basement membrane and its components<br />
on casein gene expression and secretion in mouse mammary epithelial<br />
cells. Proc Natl Acad Sci USA 84:136, 1987<br />
3. Dexter TM, Allen TD, Lajtha LG: Conditions controlling the<br />
proliferation of haemopoietic stem cells in vitro. J Cell Physiol<br />
91:335, 1977<br />
4. Lichtman MA: The ultrastructure of the hematopoietic environment<br />
of the marrow. Exp Hematol9:391, 1981<br />
5. Dexter TM, Spncer E, Toksoz D, Lajtha LG: The role of<br />
cells and their products in the regulation of in vitro stem cell<br />
proliferation and granulocyte development. J Supramol Struct<br />
13513, 1980<br />
6. Coulombel L, Eaves C, Eaves A: Enzymatic treatment of long<br />
term human marrow cultures reveals the preferential location of<br />
primitive hemopoietic progenitors in the adherent layer. <strong>Blood</strong><br />
62:291, 1983<br />
7. Tsai S, Emerson SG, Sieff C: Isolation of a human stromal cell<br />
strain secreting hemopoietic growth factors. J Cell Physiol 127:137,<br />
1986<br />
8. Zsebo KM, Yuschenkoff VN, Schiffer S, Chang D, McWall E,<br />
Dinarello C, Brolun MA, Altrock B, Bagby GC: Vascular endothelial<br />
cells and granulopoiesis: Interleukin-1 stimulates release of<br />
G-CSF and GM-CSF. <strong>Blood</strong> 71:99,1988<br />
9. Rettenmeier CN, Roussel MF Differential processing of<br />
colony-stimulating factor 1 precursors encoded by two human<br />
cDNAs. Mol Cell Biol8:5026, 1988<br />
10. Gordon MY, Graham PR, Watt SM, Greaves MF: Compartmentalization<br />
of a haematopoietic growth factor (GM-CSF) by<br />
glycosaminoglycans in the bone marrow microenvironment. Nature<br />
326:403, 1987<br />
11. Ruoslahti E, Pierschbacher M: New perspectives in cell<br />
adhesion: RGD and integrins. Science 238:491, 1987<br />
12. Hemler ME, Huang C, Schwarz L: The VLA protein family.<br />
Characterization of five distinct cell surface heterodimers each with<br />
a common 130,000 molecular weight @ subunit. J Biol Chem<br />
262:3300,1987<br />
13. Campbell A, Wicha MS, Long M: Extracellular matrix<br />
promotes the growth and differentiation of murine hematopoietic<br />
cells in vitro. J Clin Invest 75:2085, 1985<br />
14. Spooncer E, Gallagher JT, Krizsa F, Dexter TM: Regulation<br />
of haemopoiesis in long-term bone marrow cultures. IV: Glycosaminoglycan<br />
synthesis and the stimulation of haematopoiesis by @-<br />
xylosides. J Cell Biol96:510, 1983<br />
15. Coulombel L, Vuillet MH, Leroy C, Tchernia G: Lineageand<br />
stage-specific adhesion of human hematopoietic cells to extracellular<br />
matrices from marrow fibroblasts. <strong>Blood</strong> 71:329, 1988<br />
16. Tsai S, Patel V, Beaumont E, Lodish HF, Nathan DG, Sieff<br />
CA: Differential binding of erythroid and myeloid progenitors to<br />
fibroblasts and fibronectin. <strong>Blood</strong> 69:1587, 1987<br />
17. Campbell A, Long MW, Wicha MS: Haemonectin, a bone<br />
marrow adhesion protein specific for cells of the granulocytic<br />
lineage. Nature 329:744, 1987<br />
18. Patel VP, Lodish HF: Loss of adhesion of murine erythroleukemia<br />
cells to fibronectin during erythroid differentiation. Science<br />
224:996, 1984<br />
19. Virtanen I, Ylanne J, Vartio T: Human erythroleukemia cells<br />
adhere to fibronectin: Evidence for a Mr 190000 receptor protein.<br />
<strong>Blood</strong> 69:578,1987<br />
20. Mouchiroud G, Berthier R, Newton IA, Chapel A, Hollard<br />
D: Monoclonal antibodies against human hematopoietic cells and<br />
REFERENCES<br />
the separation of progenitor cells from bone marrow. Exp Hematol<br />
13:566, 1985<br />
21. Edelman P, Vinci G, Villeval JL, Vainchenker W, Henri A,<br />
Miglierina R, Rouger R, Reviron J, Breton-Gorius J, Sureau C,<br />
Edelman L: A monoclonal antibody against an erythrocyte ontogenic<br />
antigen identifies fetal and adult erythroid progenitors. <strong>Blood</strong><br />
67:56, 1986<br />
22. Tetteroo PAT, Lansdorp PM, Leeksma OC, Von Dem Borne<br />
AEG: Monoclonal antibodies against human platelet glycoprotein<br />
IIIa. Br J Haematol55:509, 1983<br />
23. Anderson LC, Gahmberg CG, Teerenhovi L, Vuopio P<br />
Glycophorin A as a cell surface marker of early erythroid differentiation<br />
in acute leukemia. Int J Cancer 23:717, 1979<br />
24. Argraves WS, Suzuki S, Arai H, Thompson K, Pierschbacher<br />
M, Ruoslahti E: Amino acid sequence of the human fibronectin<br />
receptor. J Cell Biol 105:1183, 1987<br />
25. Werb Z, Tremble P, Behrendtsen 0, Crowley E, Damsky C:<br />
Signal transduction through the fibronectin receptor induces collagenase<br />
and stromelysin gene expression. J Cell Biol 109377, 1989<br />
26. Furfang J, Thierfelder S: Effective avidin coating of erythrocytes<br />
by disulfide bound formation. J Immunol Methods 91:123,<br />
1986<br />
27. Breton-Gorius J, Van Haeke D, Pryzwansky KB, Guichard J,<br />
Tabilio A, Vainchenker W, Carmel R: Simultaneous detection of<br />
membrane markers with monoclonal antibodies and peroxidatic<br />
activities in leukaemia: Ultrastructural analysis using a new method<br />
of fixation preserving the platelet peroxidase. Br J Haematol58:447,<br />
1984<br />
28. Graham RC, Karnovsky MJ: The early stages of absorption<br />
of injected horseradish peroxidase in proximal tubules of mouse<br />
kidney. Ultrastructural cytochemistry by a new technique. J Histochem<br />
Cytochem 14:291, 1966<br />
29. Kieffer N, Bettaieb A, Legrand C, Vainchenker W, Breton-<br />
Gorius J, Edelman L: Immunochemical identification of the 88 Kd<br />
platelet glycoprotein IV on human erythroid progenitor cells. <strong>Blood</strong><br />
70:156a, 1987 (abstr)<br />
30. Greenberg PL, Baker S, Link M, Minowada J: Immunologic<br />
selection of hematopoietic precursor cells utilizing antibodymediated<br />
plate binding (“panning”). <strong>Blood</strong> 65:190, 1985<br />
31. Breton-Gorius J, Villeval JL, Mitjavila MT, Vinci G, Guichard<br />
J, Rochant H, Flandrin G, Vainchenker W: Ultrastructural<br />
and cytochemical characterization of blasts from early erythroblastic<br />
leukemias. Leukemia 1:173, 1987<br />
32. Loken MR, Shah VO, Dattilio KL, Civin CI: Flow cytometry<br />
analysis of human bone marrow: I. Normal erythroid development.<br />
<strong>Blood</strong> 69:255,1987<br />
33. Breton-Gorius J, Reyes F Ultrastructure of human bone<br />
marrow cell maturation. Int Rev Cytol46:251, 1976<br />
34. Fibach E, Manor D, Oppenheim A, Rachilewitch EA: Proliferation<br />
and maturation of human erythroid progenitors in liquid<br />
culture. <strong>Blood</strong> 73:100, 1989<br />
35. Sawada K, Krantz SB, Kans JS, Dessypris EN, Sawyer S,<br />
Click AD, Civin CI: Purification of human erythroid colony-forming<br />
units and demonstration of specific binding of erythropoietin. J Clin<br />
Invest 80357,1987<br />
36. Nijhof W, Wierenga PK Isolation and characterization of<br />
the erythroid progenitor cells CFU-E. J Cell Biol96:386, 1983<br />
37. Koury MJ, Bondurant MC, Rana SS: Changes in erythroid<br />
membrane proteins during erythropoietin-mediated terminal differentiation.<br />
J Cell Physiol 133:438, 1987<br />
38. Eaves AC, Eaves CJ: Erythropoiesis in culture, in McCulloch<br />
EA (ed): Cell Culture Techniques. Clinics in Haematology, vol 13.<br />
Philadelphia, PA, WB Saunders, 1981, p 371
ERYTHROBLAST ADHESION TO FIBRONECTIN<br />
a73<br />
39. Asch AS, Barnwell J, Silverstein RL, Nachman RL: Isolation<br />
of the thrombospondin membrane receptor. J Clin Invest 79:1054,<br />
1987<br />
40. Takada Y, Huang C, Hemler M: Fibronectin receptor structures<br />
in the VLA family of heterodimers. Nature 326:607, 1987<br />
41. Patel VP, Lodish H F The fibronectin receptor on mammalian<br />
erythroid precursor cells: Characterization and developmental regulation.<br />
J Cell Biol 102:449, 1986<br />
42. Patel VP, Ciechanover A, Platt 0, Lodish HF: Mammalian<br />
reticulocytes lose adhesion to fibronectin during maturation to<br />
erythrocytes. Proc Natl Acad Sci USA 82:440, 1985<br />
43. Coulombel L, Tchernia G, Mohandas N: Human reticulocyte<br />
maturation and its relevance to erythropoietic stress. J Lab Clin Med<br />
94:467, 1979<br />
44. Mohandas N, Evans E: Sickle erythrocyte adherence to<br />
vascular endothelium: Morphologic correlates and the requirement<br />
for divalent cations and collagen binding plasma proteins. J Clin<br />
Invest 76:1605, 1985<br />
45. Dike LE, Farmer SR: Cell adhesion induces expression of<br />
growth-associated genes in suspension-arrested fibroblasts. Proc<br />
Natl Acad Sci USA 85:6792,1988<br />
46. Patel VP, Lodish HF A fibronectin matrix is required for<br />
differentiation of murine erythroleukemia cells into reticulocytes. J<br />
Cell Biol 105:3105, 1987