Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02211380 1997-07-18
WO 96123872 PCT~NL96/00031
Title: Enrichment of hematopoietic stem cells from blood or
bone marrow
F;~ld of the ;nvent;on
The invention is in the area of hematopoietic stem cell
transplantation and is concerned with a novel process for the
preparation of enriched suspensions of hematopoietic stem
cells. The invention relates to a method to deplete non-stem
cells from hematopoietic stem cell suspensions used for
transplantation, without the use of xenogeneic antibodies
directed against cell surface molecules.
The process will be suitable for the processing of auto-
logous transplants, as it will result in a reduction of thevolumes of transplants to be cryopreserved, a reduction of the
amount of (toxic) c yoprotectant to be infused into the
patient, a ~im; n; ~hed risk of transfusion problems related to
lysis of granulocytes and erythrocytes upon freezing and
thawing, and probably a reduction of the number of
cont~m;n~ting malignant cells.
Its use will also be advantageous in both autologous and
allogeneic transplantations when further purification of the
stem cells is desirable. A smaller scale immunoselection
technique, requiring less monoclonal antibody can be used for
final purification after stem cells have been enriched by the
invented procedure.
R~ckgrol]nd of the ;nve~t; on
Hematopoietic stem cell transplantation.
Although results in the treatment of cancer patients have
improved, the prognosis with current treatment protocols in some
patient groups is still poor. One approach to improve cure rate
is intensification of chemo- and/or radiotherapy. However, this
~ 30 is hampered by irreversible damage to hematopoiesis in the bone
marrow, even with the use of hematopoietic growth factors. This
~ problem can be corrected by the administration of bone marrow
cells.
This can be done in an allogeneic setting, with bone marrow
from an HLA-matched related donor. Two problems restrict the
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application of allogeneic bone marrow transplantations: firstly t
the fact that only 30% of the patients have an HLA-identical
sibling, and secondly, the frequent occurrence of Graft-versus-
Host Disease (GvHD). GvHD is caused by T cells from the graft,
which react to the patient's alloantigens, thereby damaging
patient tissues. This is in some cases a severe, sometimes
fatal, disease.
Alternatively, autologous bone marrow transplants are being
used. For this purpose bone marrow is harvested in the remission
phase of the disease, cryopreserved, and reinfused into the
patient after high-dose chemotherapy. However, in some tumors,
bone marrow infiltration with tumor cells is a common feature.
Furthermore, intensive pretreatment (such as pelvic irradiation)
may lead to damage to bone marrow, thereby hampering the harvest
of sufficient bone marrow stem cells.
A few years ago it has been found that hematopoietic
progenitor cells can be mobilized to the blood by treatment with
certain chemotherapeutic drugs. Moreover, it was observed that
the mobilizing effect of chemotherapy can be enhanced by in vivo
a~m; n istration of hematopoietic growth factors, such as G-CSF.
Peripheral blood stem cells (PBSC) are now increasingly used as
an alternative for bone marrow in autologous transplantations.
PBSC are collected by leukocytapheresis. This is a centri-
fugation technique in which low density cells (i.e. mononuclear
cells, including the progenitor cells) are collected. The other
blood components are reinfused (continuously or intermittently)
into the donor.
PBSC transplantation has several advantages over bone
marrow transplantation:
1. The chemotherapy applied before harvesting largely
eradicates circulating tumor cells, thereby reducing the tumor
load in peripheral blood stem cell transplants. However, with
recently developed more sensitive detection techniques, (low
numbers of) malignant cells in the PBSC transplants can still be
detected.
2. Treatment protocols of patients in which mobilized PBSC
were infused, resulted in more rapid hematopoietic recovery as
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compared to bone marrow transplantation, thus reducing the
dangerous period of aplasia.
3. Less morbidity of the harvesting procedure (no general
anesthesia).
Recently, it has been found that considerable numbers of
stem cells are present in umbilical cord blood. Worldwide about
50 (allogeneic) umbilical cord blood transplantations in
children have been performed so far. The majority of these
patients showed hematopoietic recovery. Several centers have
started umbilical cord blood banking. Major advantages of the
use of umbilical cord blood stem cells will be:
1. The ready availability for transplantation of fully
typed material with known progenitor cell content.
2. A harvest procedure without any discomfort to the
mother/child.
Umbilical cord blood stem cell transplantations have mainly
been performed in children, in view of the limited numbers of
stem cells harvested. Expansion in vitro may however facilitate
transplantation of umbilical cord blood stem cells in adults.
Processing of stem cell transplants.
It is generally assumed that purification of stem cells
from the transplants would be desirable. Purification will
provide major advantages:
a. In autologous transplantations, purification will lead
to a reduction in the number of malignant cells.
b. In autologous and umbilical cord blood transplantations,
purification will lead to a reduction in the volume of cells to
be cryopreserved, to a reduction of the amount of (toxic)
cryoprotectant to be infused into the patient, and to diminished
risks of infusion which are related to the lysis of granulocytes
and erythrocytes upon freezing and thawing (such as formation of
aggregates, hemoglobinuria).
c. In allogeneic transplantations, the resulting depletion
of T cells will lead to diminished GvHD.
d. Growth-factor induced expansion and differentiation of
stem cells in vitro is more effective when purified stem cells
are cultured.
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e. For effective transfer of foreign genes into stem cells
(for gene therapy) purified preparations of stem cells may be
necessary.
Several immunological techniques have been developed for
the isolation of stem cells. In all the available methods, CD34
monoclonal antibodies (mAbs), which react specifically with
hematopoietic stem cells, are being used for the isolation of
progenitor cells. One of the first reports was by Berenson et al
tJ. Clin. Invest. 31 (1988): 951) who isolated CD34+ cells from
bone marrow, transplanted these cells into irradiated baboons
and achieved hematological reconstitution. Because leukemic
blasts from patients with acute leukemia also carry the CD34
antigen, this approach of stem cell purification cannot be used
in acute leukemia. However, in solid tumors like breast
carcinoma, neuroblastoma etc. and in other hematological
disorders, the malignant cells are CD34-negative.
The following companies have developed systems for the
enrichment of CD34+ cells:
* Applied Immune Sciences introduced the microCELLector. In
this system, Soybean agglutinin (SBA) or CD34 mAb is covalently
bound to polystyrene flasks. In the first step of the procedure,
mature differentiated cells are (partly) depleted by binding to
SBA. CD34+ cells are then selected from the SBA-negative
population by binding to CD34 mAb. Finally, bound cells are
released by physical agitation.
* CellPro introduced the CEPRATE SC Stem Cell Concentration
System, which is based on the binding of biotinylated CD34 mAb
to progenitor cells, and subsequent separation over a column
with avidin-coated polyacrylamide beads. Progenitor cells are
liberated by mechanical stirring of the gel bed. The procedure
results in a > 50-fold enrichment of CD34+ cells and CFU-C, with
approximately 50~ yield.
* Baxter introduced the Isolex system, comprising immuno-
magnetic beads coated with CD34 mAb for the selection of
progenitor cells. In this system the positively selected stem
cells are treated with chymopapain to strip the beads from the
surface of the cells.
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WO 96l23872 5 PCT/NL96~00031
* Dynal introduced a procedure similar to that of Baxter, but
which differs in the method used to detach the beads from the
cell surface. Cell release is performed using a polyclonal anti-
body directed against murine Fab antibody fragments.
Some of the techniques reported so far have actually been
applied for isolation of progenitor cells in a clinical setting
and transplantations with selected stem cells have been
performed succesfully.
One major disadvantage of the available techniques is the
large amount of CD34 monoclonal antibody needed, which makes
these techniques very expensive. A second disadvantage is the
need of some technique to release the cells, which may result in
damage to the progenitor cells. Moreover, the purity and yield
achieved in isolations of stem cells from PBSC transplants is in
general lower than purity and yield of stem cells isolated from
bone marrow. Therefore, a process leading to substantial
enrichment of stem cells by depletion of non-stem cells without
the use of mAb would be a considerable advantage.
The following non-immunological techniques are used for
processing of stem cell grafts:
* Density separation for depletion of erythrocytes and
granulocytes from bone marrow.
* Counterflow centrifugal elutriation for depletion of
lymphocytes from bone marrow grafts.
However, no non-immunological procedure is available by
which a substantial depletion of all non-stem cell types from
grafts can be obtained. PBSC transplants are in general infused
into the patients without any separation step. PBSC leukocyta-
pheresis products obtained after treatment of the patients with
chemotherapy and G-CSF cannot be enriched by density separation
because all cells collected by an optimal leukocytapheresis
procedure have a low specific density.
Sl~mmAry of the inve~t;on
The invention comprises a procedure for enrichment of
hematopoietic progenitor cells based on selective adherence of
certain non-progenitor cells to a fiber material coated with an
opsonizing substance. In a preferred form, the adherence step is
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preceded by counterflow centrifugal elutriation. The invented
procedure is based on two discoveries:
1. Selective removal of monocytes and myeloid cells from
stem cell preparations can be achieved by passage of the cell
suspensions over scrubbed nylon fibers, provided that first
platelets are rigorously depleted from the graft. Alternatively
a chelating substance (e.g. EDTA or trisodium citrate) may be
included in the filtration medium, thereby preventing the
adherence of platelets to leukocytes. Without platelet depletion
or prevention of the adherence of platelets to leukocytes by
capture of divalent cations, stem cells adhere to nylon fiber.
The optimal way of platelet removal is counterflow centrifugal
elutriation.
2. The selective adherence of monocytes and immature
myeloid cells to nylon fiber is strongly promoted by coating the
fiber with opsonizing human plasma proteins.
When the enrichment step with coated nylon fiber is
preceded by elutriation, substantial depletion of all non-stem
cell types from the transplant can be achieved. By each of these
two techniques different cell types are removed: the elutriation
technique separates platelets, erythrocytes and part of the
lymphocytes from the progenitor cells. The passage over coated
nylon fiber results in depletion of monocytes and myeloid cells
from the transplant. With the combination of these techniques
substantial enrichment of stem cells is achieved without the use
of monoclonal antibodies. In peripheral blood transplants
approximately 10-fold reduction of the total number of non-stem
cells is achieved.
The invention provides a process for the enrichment of
hematopoietic stem cells in a stem cell preparation (usually
umbilical cord blood, a peripheral blood stem cell suspension,
or bone marrow), the process comprising depleting non-stem
cells (more particularly comprising monocytes and myeloid
cells) from the preparation by adherence to an opsonized
material and recovering a hematopoietic stem cell-enriched
preparation.
Preferably, said opsonized material comprises a fiber
material, in particular a polyamide fiber material, treated
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with an opsonizing substance, wherein the opsonizing substance
is preferably selected from the group consisting of IgG, IgA,
C3b, C3bi, C3dg, Clq, iC3, conglutinin, mAnn~n-binding protein
(MBP), CL-43, SP-A and SP-D. Most preferably, the opsonized
material comprises a scrubbed nylon fiber material coated with
human IgG. Furthermore, the opsonized material is preferably
treated with a blocking substance which reduces aspecific cell
binding, wherein the blocking substance is preferably selected
from the group consisting of human serum albumin, animal serum
albumin, gelatin, Ficoll 70 and Ficoll 400.
Preferably, the stem cell preparation is pretreated to
remove platelets (thrombocytes) therefrom. The pretreatment of
the stem cell preparation preferably comprises a counterflow
centrifugal elutriation in which smaller cells including
platelets, erythrocytes and small lymphocytes are removed and
larger cells including hematopoietic stem cells, monocytes and
myeloid cells are recovered. Alternatively, the platelets can
be removed by sequential low-speed centrifugation steps.
In a second preferred embodiment a chelating substance
(e.g. EDTA or trisodium citrate) is included in the medium in
which the process of depletion of monocytes and myeloid cells
is performed, thereby preventing the adherence of platelets to
the stem cells. In this embodiment of the invention, removal
of platelets is not necessary.
A preferred process comprises providing a stem cell
preparation from bone marrow or, preferably, from peripheral
blood or umbilical cord blood, depleting platelets, erythro-
cytes and small lymphocytes therefrom by a counterflow centri-
fugal elutriation in which smaller cells including platelets,
erythrocytes and small lymphocytes are removed and larger
cells including hematopoietic stem cells, monocytes and
myeloid cells are recovered, depleting monocytes and myeloid
c cells from said recovered larger cells by adherence to a
material coated with an opsonizing substance and blocked
against aspecific cell binding, and recovering a preparation
enriched in hematopoietic stem cells.
In another preferred embodiment a stem cell preparation
from bone marrow, cord blood or peripheral blood is passaged
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directly (without prior removal of platelets) over a material
coated with an opsonizing substance and blocked against
aspecific cell binding, in a medium to which a chelating
substance has been added. Thereby, monocytes and myeloid cells
(and B cells) are depleted and a preparation enriched in hema-
topoietic stem cells will be recovered. In this embodiment,
erythrocytes, lymphocytes and platelets are not depleted, and
the process will result in a more limited enrichment.
Of course, the invention covers the combination of both
methods, i.e. prior depletion, e.g. by counterflow centrifugal
elutriation, of platelets, erythrocytes and small lymphocytes,
and passage of the resulting composition, which is enriched in
the larger cells, over the non-stem cell depletion material in
a medium containing a chelating substance.
The process may also comprise a step of immunoselection
using an antibody which binds hematopoietic stem cells, or an
antibody which binds non-stem cells or malignant cells, to
effect a further enrichment of hematopoietic stem cells.
The invention also provides an assembly of means and
instructions for use in a process for the enrichment of
hematopoietic stem cells in a stem cell preparation, said
means comprising (the constituents of) an opsonized material
capable of binding non-stem cells including monocytes and
myeloid cells present in a stem cell preparation.
The assembly may further comprise at least one member of
the group consisting of elutriation media, cell suspending
media, opsonization media, blocking media, washing media, and
components of said media.
~r;ef descriptio~ of the drawings
Figure 1 shows the principle of counterflow centrifugal
elutriation.
Figure 2 shows the result of separation of PBSC leukocyta-
pheresis product by counterflow centrifugal elutriation. The
figure depicts the recovery of cells (as percentage of input)
in the elutriation fraction containing the large cells (n=14).
CFU-GM = a measure of the outgrowth of monocytic and myeloid
precursors in semi-solid medium during a 12 day culture.
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Figure 3 shows that yield of PBSC CD34+ cells upon passage
over nylon wool in the presence of fetal bovine serum is
enhanced after depletion of thrombocytes, erythrocytes and part
of the lymphocytes by counterflow centrifugal elutriation. The
figure depicts recovery of cells in the filtrate as percentage
of the cells applied to the nylon wool.
Figure 4 shows the effect of an addition of paraformalde-
hyde-fixed resting or thrombin-activated (non-autologous)
platelets on the recovery of various cell types in elutriated
PBSC upon nylon fiber filtration in the presence of fetal bovine
serum. The figure depicts the recovery of cells in the filtrate
as percentage of the cells applied to the nylon wool.
Figure 5 shows the effect of elutriation on platelet
binding to CD34+ cells in PBSC leukocytapheresis products. Adhe-
rence of platelets to CD34+ cells was measured by determinationof CD41 mAb binding to CD34+ cells by FACS-analysis. The figure
depicts the percentage of CD34+ cells binding CD41 mAb (minus the
percentage of CD34+ cells binding non-specific mAb) in
unseparated leukocytapheresis product and in elutriated stem
cell fraction.
Figure 6 shows that the yield of CD34+ cells after
filtration of PBSC samples is enhanced when instead of removing
the platelets by elutriation, a chelating agent is added to the
medium prior to filtration. On the x-axis the composition of the
filtration medium is given. The figure depicts the recovery of
cells in the filtrate as percentage of the cells applied to the
nylon wool. As can be seen, another consequence of the addition
of EDTA to the filtration medium is that myeloid cells are less
well depleted from the cell preparation.
Figure 7 shows recovery of cells upon passage of elutriated
PBSC cells over nylon fiber preincubated with:
a. IMDM supplemented with 10% fetal calf serum and 9 mM MgCl2; b.
IMDM supplemented with human serum albumin (10 mg/ml);
c. IMDM supplemented with human immunoglobulin (1 mg/ml) follo-
wed by incubation with IMDM supplemented with HSA (10 mg/ml).
The figure depicts the percentages of cells applied to the nylon
wool which were recovered in the filtrate.
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Figure 8 shows recovery of cells upon passage of elutriated
PBSC cells over nylon wool preincubated with either IMDM supple-
mented with 10 % fetal calf serum or IMDM supplemented with
immunoglobulin (1 mg/ml), without subsequent saturation with
HSA. The figure depicts the recovery of cells in the filtrate as
percentage of cells applied to the nylon wool.
Figure 9 shows recovery of cells upon filtration of elutri-
ated PBSC over nylon wool preincubated with either IMDM supple-
mented with human serum albumin (10 mg/ml) or IMDM supplemented
with human complement factor iC3 (0.1 mg/ml) followed by satu-
ration of the fiber with HSA. The iC3 was prepared by chemical
cleavage of the intrachain thioester bond which is present in
the ~-chain of the molecule. The figure depicts the recovery of
cells in the filtrate as percentage of cells applied to the
nylon wool.
Figure 10 shows the effect of addition of trisodium citrate
to the filtration medium, instead of removal of platelets, on
filtration of PBSC samples over IgG-coated nylon wool. The
figure depicts recovery of cells in the filtrate as percentage
of the cells applied to the nylon wool. On the x-axis the
composition of the filtration medium is given.
Figure 11 shows the yields of various cell types present in
PBSC leukocytapheresis products after counterflow centrifugal
elutriation and passage over nylon wool coated with human
immunoglobulin and saturated with HSA (three experiments with
PBSC from different patients). The figure depicts recovery of
cells after the whole procedure as percentage of the cells
started with.
Det~i1ed ~;sclosl~re of the ;nv~nt;on
The invented procedure was developed in the first instance
for enrichment of progenitor cells from peripheral blood stem
cell leukocytapheresis preparations obtained after treatment
with chemotherapy and G-CSF. These preparations contain leuko-
cytes, erythrocytes (10 to 20 fold excess to leukocytes) andplatelets (5 to 50 fold excess to leukocytes). From about 1~ to
about 4% of the leukocyte fraction consists of progenitor cells
~ (CD34+ cells), the remainder mainly consisting of lymphocytes,
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W O 96123872 11 PCT~YL96/00031
monocytes and myeloid cells. We looked for a non-immunological
technique or a combination o~ techniques which would result in
depletion of all these different non-stem cell types, with high
recovery of progenitor cells.
In blood transfusion technology, leukocyte-depleted red
cell concentrates are routinely prepared by passage over fibers
(''filtration''). Such a procedure for removal of leukocytes is
simple, fast, and does not require expensive equipment. A
selective depletion of leukocytes occurs, while erythrocytes are
recovered with high efficiency in these so-called "filtrations".
This is due to differences in size and adherence properties of
the cells (cell sieving and cell adherence). Several types of
leukocyte removal filters are being used, which are composed of
either polyester, cellulose acetate, cotton or nylon fibers.
With nylon fiber not all leukocytes are depleted, but a
selective removal of certain leukocyte types from blood can be
achieved. Passage of blood cells over nylon fiber in the
presence of human plasma (Greenwalt et al, Transfusion 2, 221-
229 (1962); Roos and Loos, Biochim. Biophys. Acta 222, 565-582
(1970)) or fetal calf serum (Litvin and Rosenstreich, Methods in
Enzymology 108, 298-302 (1984)i de Boer et al, J. Immunol.
Methods 43 (1981): 225) is known to result in adherence of
virtually all monocytes, granulocytes and B lymphocytes to the
fibers, while T lymphocytes do not stick to them. Platelets are
retained partially and erythrocytes do not adhere. For optimal
adhesive properties, the finish has to be removed from the fiber
(Greenwalt~ 1962). Nylon fiber from which the finish has been
removed is called: scrubbed nylon fiber.
We have investigated whether nylon fiber can be used for
selective depletion of monocytes and myeloid cells from peri-
pheral blood stem cell leukapheresis suspensions. The most
important question was, whether progenitor cells would adhere to
the nylon fiber or not. We observed that upon passage of PBSC
leukocytapheresis products over nylon fiber in the presence of
~ 35 fetal calf serum, recovery of stem cells (enumerated as CD34+
cells) was strongly variable, ranging from 12 to 112% (Table 1).
The procedure resulted in considerable depletion of monocytes,
but depletion of myeloid cells was only partial and variable.
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The incomplete removal of myeloid cells is in strong
contrast with the nearly complete removal of mature granulocytes
achieved by passage of peripheral blood from normal donors over
nylon fiber. Morphological ~min~tion of the-cells showed that
in PBSC leukocytapheresis products obtained from patients
pretreated with chemotherapy and G-CSF, in contrast to blood
from normal donors, nearly exclusively immature myeloid cells
(band forms, promyelocytes, myelocytes and metamyelocytes) are
present. This is caused by the pretreatment of the patients with
chemotherapy and hematopoietic growth factors. In particular the
more immature myeloid cells did not adhere to the fiber. Because
of their lower density, as compared to mature granulocytes,
these immature myeloid cells are enriched in leukocytapheresis
products compared to whole blood.
To make passage over nylon fiber a suitable technique for
enrichment of stem cells, the recovery of progenitor cells had
to be optimized. We discovered that adherence of CD34+ cells to
the nylon fiber could be prevented by prior depletion of
platelets from the graft or by addition of a chelating substance
to the filtration medium.
Platelets can be separated from leukocytes by sequential
centrifugation washing steps, or by counterflow centrifugal
elutriation. Counterflow centrifugal elutriation is a technique
in which particles are exposed to two opposite forces in a
specially designed flow chamber within a centrifuge: the
centrifugal force and a centripetal hydrodynamic force, caused
by a fluid flow in the opposite direction of the centrifugal
force. By increasing the fluid flow or decreasing the rotor
speed, particles are separated (Figure 1 and Lutz, M.P. et al,
Analytical Biochemistry 200 ( 1992): 376). Separation occurs
mainly according to size and to a lesser extent to density.
In counterflow centrifugal elutriation stem cells co-
separate with the larger blood cells (monocytes and myeloid
cells)~ while the smaller cells (i.e. platelets, erythrocytes
and small lymphocytes) are washed away. Figure 2 shows that a
nearly complete removal of thrombocytes, erythrocytes and a
partial removal of lymphocytes from PBSC leukocytapheresis
products was achieved by elutriation with a >85% yield of CD34+
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cells and of myeloid progenitor cells (colony-forming units
granulocyte-monocyte (CFU-GM)). When the resulting stem cell
preparations were subsequently subjected to nylon fiber
filtration in the presence of fetal calf serum, we observed that
recovery of CD34+ cells in the filtrate was strongly increased in
comparison with filtration of the original unseparated PBSC
leukocytapheresis products (Figure 3).
The adherence of CD34+ cells to nylon fiber appeared to be
mediated specifically by platelets. Particularly, activated, but
not resting platelets were found to be effective in promoting
retention of stem cells on nylon wool. When thrombin-activated
platelets were added to the elutriated stem cells, recovery of
CD34+ cells after passage over nylon wool was drastically
lowered. Conversely, resting platelets had no effect (Figure 4).
The effect of platelets on adhesion of stem cells to nylon
wool may be caused by two mechanisms:
1. Adherence of platelets to the nylon fiber may
secondarily lead to trapping of stem cells. We found that during
passage of PBSC leukocytapheresis products over nylon fiber 44 +
20 22% (n=5) of platelets adhered to the nylon wool.
2. Platelets or platelet fragments may adhere to the stem
cells after which the stem cells adhere to the nylon wool. In
our institute, Dercksen et al (Blood, in press) have observed
that in PBSC leukocytapheresis products platelets and platelet
25 fragments are bound to part of the CD34+ cells. This was
reflected in binding of mAb directed against CD41 to the CD34+
cells. CD41 is an antigen which is expressed nearly exclusively
on platelets. Upon extensive washing of the cells in the
presence of EDTA, CD41 mAb binding to CD34+ cells was strongly
30 ~im;n;~hed. It was proposed that the residual CD41 mAb binding
after washing reflected cellular CD41 expression on
megakaryocyte progenitors. Figure 5 shows that elutriation not
A only results in removal of free platelets but also in removal of
bound platelets or platelet fragments from the CD34+ cells, as
35 indicated by the lowered percentage CD34+ cells which bind CD41
mAb.
In a preferred embodiment of the invention, platelets are
removed from the stem cell graft by a counterflow centrifugal
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W O 96/23872 14 PCT~L96/00031
elutriation. Alternatively, the same objective may be achieved
by sequential differential (low-speed) centrifugation steps.
However, elutriation has in the invented procedure the following
advantages:
1. Non-bound platelets are more effectively removed by
elutriation than by differential centrifugation.
2. Platelets which are bound to cells are more efficiently
removed by elutriation than by low-speed centrifugation.
3. Additional cell types (erythrocytes and a larger part of
lymphocytes) are removed from the graft by counterflow centri-
fugal elutriation.
Since the adherence of platelets to CD34+ cells (Dercksen et
al, Blood, in press) is dependent on the presence of divalent
cations, it was investigated whether the mere addition of
chelators like EDTA or trisodium citrate to the filtration
medium could prevent the adherence of CD34+ cells to nylon fiber.
As is shown in Figure 6, inclusion of EDTA in the filtration
medium resulted in enhanced yield of CD34+ cells in the filtrate
without prior removal of platelets from the PBSC preparations.
Unfortunately, EDTA also resulted in strongly ~;m;n;.~hed
adherence of the myeloid cells to the nylon fiber. Similar
results were obtained with addition of trisodium citrate to the
filtration medium. However, as is shown in Figure 10, lowered
adherence of myeloid cells in the presence of a chelating
substance does not occur when the fiber is coated with an
opsonizing substance. Thus, in a second preferred embodiment,
when only removal of monocytes and myeloid cells is desired, a
chelating substance is included in the filtration medium to
prevent the adherence of CD34+ cells to the nylon fiber, thereby
circumventing the need of removal of platelets from the stem
cell transplants.
To obtain reasonable enrichment of progenitor cells upon
passage of PBSC transplant over nylon fiber, it was necessary to
enhance the adhesive properties of the fiber for the (immature)
myeloid cells. We discovered that this can be achieved by
coating the fiber with an opsonin, in its most preferred form
the opsonin being human IgG.
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Myeloid cells are phagocytotic cells. Binding of phagocytes
to foreign particles, such as bacteria, is strongly enhanced by
opsonization, i.e. coating of the particle with specific
proteins. Proteins with the ability to enhance the binding of
phagocytotic cells to foreign particles are called opsonins. In
vivo, the main opsonins are immunoglobulins (especially IgG and
to a lesser extent IgA) and complement factors C3b, C3bi and
C3dg. These complement factors are enzymatic cleavage products
of complement factor C3, which are generated upon activation of
the complement cascade. Moreover, complement factor Clq is
thought to have opsonizing properties. We hypothesized that
coating of the fiber with opsonins would result in enhanced
adherence of the (immature) myeloid cells.
To investigate this possibility we used a human immuno-
globulin product prepared from plasma, containing at least 90%
IgG (CLB Immunoglobulin I.M.). C3b, C3bi and C3dg cannot be
purified from plasma. However, the opsonizing capacity of C3
cleavage products can be mimicked by chemical cleavage of the
thioester bond between a cysteine and glutamine residue in the
20 a- chain of C3. The resulting molecule is called iC3. Due to a
conformational change of the molecule phagocytotic cells can
bind to it via their complement receptors with high affinity.
When we replaced the fetal calf serum (FCS) by a non-
opsonizing protein (1% (w/v) human serum albumin (HSA)), both in
the medium used for preincubation of the fiber and in the
filtration medium, no influence on the adherence pattern of
cells was found (Figure 7). However, when the nylon fiber was
preincubated with immunoglobulin I.M. and subsequently saturated
with human serum albumin (HSA), strongly enhanced depletion of
myeloid cells (and also further enhancement of the adherence of
monocytes) from elutriated PBSC was observed (Figure 7). We
found that after coating with immunoglobulin, saturation of the
~fiber with a non-opsonizing protein was necessary to prevent
aspecific adherence of all cell types. Omission of such a
~35 protein addition (from the preincubation and from the filtration
medium) led to binding of almost all cells, including the CD34+
cells, to the immunoglobulin-coated nylon fiber (Figure 8).
CA 02211380 1997-07-18
W 096/23872 16 PCT~L96/00031
Coating of fiber with iC3, followed by saturation with HSA,
resulted in moderately enhanced binding of monocytes and myeloid
cells to the nylon fiber (Figure 9).
After coating of the nylon fiber with human IgG, myeloid
cells also adhered to the fiber in the presence of a chelating
substance. Figure 10 shows the effect of addition of trisodium
citrate to the filtration medium on the yield of CD34+ cells from
PBSC leukocytapheresis preparations after filtration over IgG-
coated nylon wool, without prior removal of platelets. Yield of
CD34+ cells was enhanced in the presence of trisodium citrate,
while depletion of monocytes or myeloid cells was similar to
that in filtrations in which no chelating substance was included
in the filtration medium. Similar results were obtained upon
addition of EDTA to the filtration medium.
Thus, the invention shows that phagocytic cells can be
removed from stem cell products by passage over-a fiber coated
with an opsonin. It can be used for the enrichment of stem cells
from bone marrow, PBSC grafts, and umbilical cord blood grafts.
In a preferred embodiment, the passage over the opsonized fiber
material is preceded by counterflow centrifugal elutriation, by
which not only platelets, but also erythrocytes and a larger
part of the lymphocytes can be removed from the grafts. By
counterflow centrifugal elutriation similar depletions of non-
stem cells can be achieved from bone marrow, from umbilical cord
blood and~from PBSC leukocytapheresis product. Therefore, the
combination of these techniques may result in a significant
enrichment of progenitor cells from all these types of grafts.
This is illustrated for PBSC leukocytapheresis products in
Figure 11. PBSC grafts were subjected to counterflow centrifugal
elutriation and subsequently subjected to passage over human
immunoglobulin-coated nylon fiber. This resulted in nearly
complete removal of erythrocytes and thrombocytes, in more than
95% removal of myeloid cells, monocytes, and B lymphocytes from
PBSC, and in at least 80~ removal of T and NK cells. The mean
enrichment of stem cells obtained by this procedure was 9-fold.
No procedure is known which results in similar enrichment of
stem cells from peripheral blood stem cell leukocytapheresis
products, without the use of specific monoclonal antibodies.
CA 02211380 1997-07-18
W 096/23872 17 PC~L96/0~31
In another preferred embodiment, stem cell containing
preparations are directly passaged, without prior removal of
platelets, over a fiber material coated with an opsonin in a
medium containing a chelating substance. This will result in
removal of monocytes, myeloid cells and B lymphocytes, and
therefore in a lower enrichment of stem cells than after
processing stem cell grafts by consecutive elutriation and
filtration. However, the removal of these cell types may be of
great advantage for further (immuno)selection steps, since
especially monocytes are sticky cells, which have a tendency to
adhere to many types of material, thereby interfering with
purification. Furthermore, myeloid cells are very susceptible to
freezing/thawing damage, and removal of them will probably lead
to better quality of stem cell grafts after thawing. These
reasons, together with the fact that a filtration technique is
in principle a simple and cost-effective technique, may make it
very attractive to introduce it into current stem cell
processing protocols as such.
It has been reported that in separations of human bone
marrow by counterflow centrifugal elutriation, a primitive,
pluripotent stem cell type, which does not grow out in colony
assays but may be responsible for long-term engraftment, co-
elutriates with the smaller lymphocytes. A fraction enriched for
such primitive stem cells, and depleted of colony-forming cells,
has been isolated from human bone marrow by separating by
counterflow centrifugal elutriation and collecting the smallest
cells, followed by isolation of CD34+ cells from this fraction
~International patent application WO 94/11493i Wagner, J.E. et
al (1995) Blood 86, 512-523). This raises the question whether
pluripotent stem cells from PBSC products might be lost together
with the platelets, erythrocytes and lymphocytes in elutriation
separations. To answer this question, the frequency of primitive
~ hematopoietic stem cells in elutriation fractions was assayed in
a limiting dilution type long-term stem cell culture on FBMD-l
stromal feeder cells by determination of cobble-stone-area-
forming cells (CAFC) at week 4 and week 6 (according to Breems
et al (1994) Leukemia 8, 1095-1104). In mouse studies, CAFC week
4 to 6 frequencies have been found to correlate with the ability
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W O 96/23872 18 PCTANL96/00031
of bone marrow cells to induce long-term hematopoietic
repopulation. PBSC leukocytapheresis samples were separated into
a fraction containing the small cells and a fraction containing
the large cells, which include the majority of CD34+ cells and
CFU-GM. It was shown that the majority of week 4 and week 6 CAFC
were recovered in the elutriation fraction containing the large
cells. Moreover, as a control on the quality of the primitive
stem cells in these fractions, the production of CFU-GM was
assessed in parallel long-term cultures in flasks. For this
purpose, cells from these long-term cultures were replated in
appropriate semisolid medium at week 4 and week 6 and colony
formation was determined after 14 days. These measurements
demonstrated that all CFU-GM producing long-term-culture-
initiating cells resided in the fraction containing the larger
cells. No CFU-GM formation was observed in the cultures inocu-
lated with cells from the elutriation fraction containing the
small cells. Together, these results indicate that in peripheral
blood stem cell grafts at least the majority of the stem cells
capable of initiating long-term hematopoietic reconstitution is
recovered in the elutriation fraction containing the large cells
(together with the majority of CD34+ cells and CFU-GM).
Instead of nylon, also other fiber materials may be used to
remove phagocytotic cells from stem cell preparations. Appropri-
ate fibers should not bind progenitor cells, or, alternatively,
should not bind progenitor cells after being coated with a non-
opsonizing protein (i.e. blocked with a blocking substance)~
Enhancement of the adherence of phagocytotic cells is
achieved by coating with an opsonin. In a preferred embodiment
of the invention the opsonizing substance is human IgG. We
observed that upon coating with human immunoglobulin the removal
of (immature) myeloid cells and monocytes from PBSC transplants
was more efficient than upon coating with human complement
factor iC3. However, the enhanced adherence of phagocytotic
cells to fiber may also be achieved by other opsonizing
proteins. In principle, also IgA may be used, or the complement
proteins C3b, C3bi, C3dg or Clq. Recently, a new class of
opsonizing proteins has been discovered, called collectins
(Holmskov, U. et al., Immunology Today 76 (1994): 67). These are
CA 02211380 1997-07-18
W O 96123872 19 PC~L96~0003~
the plasma lectins conglutinin, mAnn~n-binding protein (MBP),
CL-43 and the pulmonary surfactant proteins SP-A and SP-D. These
proteins are ligands for the collectin receptor on phagocytes
and can bind via their lectin domains to carbohydrates on
microorganisms. These proteins may also be able to enhance
binding of phagocytes to fibers.
For most fiber materials, it may be necessary to cover the
fiber with some non-opsonizing substance to prevent aspecific
cell loss (i.e. as a blocking substance). This can be done with
biocompatible substances, which do not have adherent properties
for cells and bind easily to the fiber. Several kinds of human
or animal proteins (e.g. human serum albumin, bovine serum
albumin) or colloids (e.g. gelatin) may be used. However, it is
preferred to cover the fiber with human serum albumin. This is a
plasma protein of human origin which is non-immunogenic and has
very good coating properties for many types of material.
The invented procedure as such, or in combination with
elutriation, may result in removal of malignant cells from the
graft in several types of malignancies. Malignant cells are
different in adherence properties and/or size from normal blood
cells. For example, malignant B cells may be depleted by passage
over opsonized fiber, but also by counterflow centrifugal
elutriation. Carcinoma cells are generally more adhesive and
larger than blood cells, and may also be separated from the
progenitor cells during elutriation and/or passage over
opsonized fiber.
Several procedures have been described for depletion of Fc~-
receptor bearing cells from stem cell containing cell prepa-
rations, e.g. by using magnetic beads coated with immunoglobulin
(international patent application WO 94/02016) or sheep red
blood cells coated with rabbit anti-sheep red blood cell IgG
(Sawada, K. et al (1990) J. Cell. Physiol. 142, 219-230). It may
be possible to deplete monocytes and myeloid cells from stem
cell grafts by such techniques, since a large proportion of
~ 35 these cells expresses Fc~-receptors. However, there are many
obvious advantages of a filtration technique, as described here.
No foreign particles are introduced into the stem cell prepara-
tion, the technique is simple and can be performed rapidly, is
CA 02211380 1997-07-18
W O 96/23872 20 PCT~NL96/00031
relatively inexpensive, and can be easily brought to a clinical
scale on the basis of existing blood-filtration technology.
Moreover, depletion of phagocytotic cells by the filtration
technology described here, may be more efficient than by using
immunoglobulin-coated beads or cells. The adherence in the
filtration technique is not only based on affinity of the cells
for immunoglobulin, but also on the natural affinity of phago-
cytotic cells for polyamide fiber.
When further purification of the progenitor cells is
necessary, or further removal of malignant cells is necessary,
the invented procedure may be followed by an immunoselection
step. The immunoselection may be performed by a positive
selection of progenitor cells (e.g. by using CD34 mAb) using any
of the available immunoselection techniques described above, or
by new immunoselection techniques. Alternatively, residual non-
progenitor cells may be removed by negative selection using mAb
directed against the various types of non-progenitor cells
and/or the malignant cells.
The immunoselection can be performed after applying the
invented procedure. Alternatively, an immunoselection may be
integrated in the "filtration" procedure. For example, beads
suited for capture of cells which are labeled with a specific
antibody, may be integrated in a column of opsonized fiber
material. Alternatively, beads to which cell-specific antibodies
have been attached, can be applied.
The invention will be illustrated by the following examples
of experimental work. It is noted, however, that these examples
merely serve to a better understanding of the invention and do
not intend, nor should be construed so as to limit the scope of
the invention.
~x~ples
1. Counterflow centrifugal elutriation of stem cell grafts.
In case of PBSC leukocytapheresis products no preceding
processing of cells before elutriation is necessary. Cells (a
preparation containing 3X108 - 7X108 leukocytes) were suspended
in 10-20 ml phosphate-buffered saline (PBS) supplemented with
CA 02211380 1997-07-18
W0 96123872 2 1 PC'r~96~001>31
20 mg/ml human serum albumin (HSA) and 5 mM EDTA. The human
serum albumin preparation used was from the CLB and prepared by
ethanol fractionation of human plasma and contained more than
95% albumin. Counterflow centrifugal elutriation was performed
in a Curamé 3000 elutriation system (Dijkstra Vereenigde BV,
Lelystad, The Netherlands) equipped with disposable polycarbo-
nate separation chambers (International Medical BV, Zutphen, The
Netherlands). A fluid counterflow within the elutriation chamber
was achieved by a roller pump. In the inlet tubing, a pulse
flattening air chamber was placed. The cell suspension was
introduced into the system at a fluid flow of 12.5 ml/min and a
rotor speed of 3000 rpm. The elutriation medium consisted of PBS
supplemented with 4 mg/ml HSA and 5 mM EDTA. Alternatively,
instead of EDTA, 0.38% (w/v) trisodium citrate was added. After
introduction of the cells, the liquid flow was increased to
15 ml/min. The separation was carried out at 6~C. Rotation speed
was decreased by steps of 50 to 100 rpm until a rotor speed of
1800 rpm was reached. At each rotation speed, fractions of 50 to
100 ml were collected from the outlet. After that the speed was
slowed down to 1000 rpm and an additional fraction of 50-100 ml
was collected. Subsequently, the size of the cells in each
fraction was determined with a Coulter Counter equipped with a
Channelyzer. Fractions containing the cells with larger size
(= monocytes, myeloid cells and progenitor cells) were pooled,
sedimented by centrifugation and suspended in a medium
appropriate for filtration over nylon fiber.
2. Passage over nylon fiber.
Filtration over IgG-coated nylon wool: Elutriated stem cell
preparations were suspended in Iscove's modified Dulbecco's
medium (IMDM) supplemented with 10 mg/ml HSA. 1.7 Gram nylon
wool (type 200 combed, scrubbed nylon wool; Robbins Scientific
Corporation, CA) was tightly packed in a 20 ml syringe. A
plastic tubing was connected to the outlet of the syringe via a
luer connection. A roller pump was used to obtain a fluid flow.
The syringe was placed in a 37~C water bath and approximately
20 ml of IMDM containing 1 mg/ml human immunoglobulin (Immuno-
globuline I.M., CLB, Amsterdam; obtained from normal human
CA 022ll380 l997-07-l8
W 096/23872 22 PCT~L96/00031
plasma, and containing at least 90% IgG) was pumped into the
syringe from below until the nylon wool was completely wetted.
The nylon fiber was incubated at 37~C during 30 min with the
IgG, and subsequently 30 ml IMDM supplemented with 10 mg/ml HSA
was pumped through it in the opposite direction (from above)~
The nylon wool was incubated at 37~C during 20 min. Then the
cells (45x106 - 150X106 leukocytes), suspended in IMDM with
10 mg/ml HSA, were applied to the nylon wool and pumped through
with a flow rate of 1.0 to 2.5 ml/min. The non-adherent cells
were washed from the fiber at the same flow rate with at least
30 ml IMDM supplemented with 10 mg/ml HSA. Cells present in the
filtrate were sedimented by centrifugation and suspended in PBS
supplemented with 0.38% trisodium citrate and 2 mg/ml HSA.
It was found that IMDM could be replaced by phosphate
buffered saline (PBS), without any effect on the outcome of the
filtration process. For enrichment of non-elutriated stem cell
preparations by filtration, cells were suspended in PBS supple-
mented with HSA plus EDTA (5 mM) or trisodium citrate (13 mM).
Likewise, as filtration medium, PBS supplemented with HSA plus
EDTA or trisodium citrate was used.
Protocol using fetal bovine serum: In the first series of
experiments, the nylon fiber was not coated with IgG, but IMDM
containing 10% fetal bovine serum and 9 mM MgCl2 was used as
filtration medium. The nylon wool was also preincubated with
this medium for 30 min at 37~C. Cells were suspended in IMDM
supplemented with 10% heat-inactivated fetal bovine serum and
9 mM MgCl2 (de Boer et al, J. Immunol. Methods 43 (1981): 225).
The nylon wool was preincubated during 30 min at 37~C with this
medium. No subsequent saturation of the fiber with IMDM
supplemented with HSA was done. After the cells were applied,
the non-adherent cells were removed by washing with at least
30 ml IMDM supplemented with 10% heat-inactivated fetal bovine
serum and 9 mM MgCl2.
To investigate the effect of chelating substances on the
yield of stem cells in filtrations of non-elutriated stem cell
preparations, the filtration medium was either or not supple-
mented with 13 mM trisodium citrate or 5 mM EDTA.
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W 096/23872 23 PCT~NL96100031
Protocol using iC3: Similar to the protocol with human
immunoglobulin, except that the nylon wool was preincubated with
0.1 mg/ml iC3 in IMDM instead of immunoglobulin; the iC3 was
prepared from complement factor C3 purified from human plasma.
C3 was incubated with 0.2 M methylamine in PBS (37~C, 60 min).
Non-reacted methylamine was removed by dialysis.
Protocol using HSA: Similar to the protocol using human
immunoglobulin, except that the nylon wool was preincubated with
IMDM supplemented with 10 mg/ml HSA instead of immunoglobulin.
3. Analysis of samples.
The PBSC leukocytapheresis samples, the fractions obtained
after elutriation and the filtrate obtained after passage of
cells over coated nylon wool were analyzed as follows:
* Leukocytes and erythrocytes were counted using a Coulter
Counter (model ZF).
* Thrombocytes were counted using a Cell-Dyn 100 thrombocyto-
meter (Sequoia-Turner).
* Leukocytes were differentiated by direct immunofluorescence
and FACS analysis with a FACSscan flow cytometer (Becton and
Dickinson, Mountain View, CA). The following mAb were used,
conjugated either with fluorescein isothiocyanate (FITC) or
phycoerythrin (PE): Leu-4 (CD3) for T cells, Leu-M3 (CD14) for
monocytes, Leu-16 (CD20) for B cells, HPCA-2 (CD34) for
progenitor cells, Leu-l9 (CD56) for NK cells (all obtained from
Becton and Dickinson), and CLB-gran/2 (CD15) for myeloid cells.
Platelet adhesion to progenitor cells was studied using non-
conjugated CLB-thromb/7 (CD41) followed by incubation with FITC-
conjugated goat anti-mouse immunoglobulin (CLB).
CA 022ll380 l997-07-l8
W 096/2387224 PCT~L96100031
experiment lymphocytes monocytes myeloid CD34~
cells cells
1 89 27 50 103
2 78 2 51 69
3 112 3 83 64
4 36 5 8 29
63 0 10 12
6 71 22 48 112
7 40 3 18 42
8 69 10 42 36
9 80 3 11 29
83 1 5 21
11 71 8 41 37
12 83 1 73 114
13 65 1 15 34
Table l: Recovery of cells in the filtrate after nylon wool
filtration of PBSC leukocytapheresis products. Nylon wool (1. 7 g
in 20 ml syringe) was pre-incubated with IMDM supplemented with
10 % FCS and 9 mM MgCl2. Cells were suspended in this medium and
allowed to pass through the column at a controlled flow rate
(1-2. 5 ml/min.). The non-adherent cells were removed from the
nylon wool by washing with at least two column volumes of
medium. The recovery of cells in the filtrate is expressed as
the percentage of cells which were applied to the nylon wool.
