U.S. patent application number 10/691654 was filed with the patent office on 2005-01-06 for methods for isolation of osteoclast precursor cells and inducing their differentiation into osteoclasts.
This patent application is currently assigned to Shionogi & Co., Ltd.. Invention is credited to Maeda, Tomoko, Ochi, Takahiro, Suzuki, Ryuji.
Application Number | 20050003528 10/691654 |
Document ID | / |
Family ID | 26437128 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050003528 |
Kind Code |
A1 |
Maeda, Tomoko ; et
al. |
January 6, 2005 |
Methods for isolation of osteoclast precursor cells and inducing
their differentiation into osteoclasts
Abstract
This invention relates to: a method for differentiating
osteoclast precursor cells (preosteoclasts) into osteoclasts, which
comprises culturing the preosteoclasts in the absence of accessory
cells; a method for isolating preosteoclasts, which comprises
culturing peripheral blood or joint fluid in the absence of
cytokine for 1 to 3 weeks; an preosteoclasts, which is obtainable
by the above method; a method for differentiating the
preosteoclasts obtained by the above method into osteoclasts, which
comprises culturing the preosteoclasts in the absence of accessory
cell; an osteoclast, which is obtainable by the above method; a
method for screening agents for treating metabolic bone diseases,
which comprises using the preosteoclasts or the osteoclasts as
described above; and agents for treating metabolic bone diseases,
which is obtainable by the above screening method.
Inventors: |
Maeda, Tomoko; (Settsu-shi,
JP) ; Suzuki, Ryuji; (Settsu-shi, JP) ; Ochi,
Takahiro; (Kobe-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Shionogi & Co., Ltd.
1-8, Doshomachi 3-chome, Chuo-ku
Osaka-shi
JP
541-0045
|
Family ID: |
26437128 |
Appl. No.: |
10/691654 |
Filed: |
October 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10691654 |
Oct 24, 2003 |
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09646899 |
Oct 10, 2000 |
|
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09646899 |
Oct 10, 2000 |
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PCT/JP99/01803 |
Apr 6, 1999 |
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Current U.S.
Class: |
435/366 |
Current CPC
Class: |
A61P 19/00 20180101;
C12N 5/0643 20130101; C12N 2501/21 20130101; C12N 2502/11 20130101;
C12N 2501/23 20130101; C12N 2501/22 20130101; C12N 2503/02
20130101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 8, 1998 |
JP |
95962/1998 |
Jun 18, 1998 |
JP |
170407/1998 |
Claims
1-20. (Canceled).
21. A method for producing an osteoclast precursor cell, which
comprises: culturing a hematopoietic stem cell-derived cell
obtained from peripheral blood or joint fluid in an essential
medium for mammalian cells, optionally with added serum, in the
absence of any additional cytokine(s) for at least 1 to 3 weeks to
obtain an osteoclast precursor cell.
22. The method of claim 21, wherein said essential medium for
mammalian cells contains serum.
23. The method of claim 21, wherein the hematopoietic stem
cell-derived cell is obtained from the mononuclear fraction of
peripheral blood.
24. The method of claim 21, wherein said hematopoietic stem
cell-derived cell is obtained from the cellular fraction of joint
fluid.
25. The method of claim 21, wherein said hematopoietic stem
cell-derived cell is cultured at a temperature ranging from
35-37.degree. C. in 5-7% CO.sub.2-containing air for 1-3 weeks.
26. An osteoclast precursor cell, which is obtainable by the method
of claim 21.
27. A method for producing an osteoclast, comprising: culturing an
osteoclast precursor cell in a culture medium in the absence of
accessory cells.
28. The method of claim 27, wherein said culture medium comprises
one or more compound(s) selected from the group consisting of IL-3,
IL-7, GM-CSF, eotaxin, eotaxin-2, and eotaxin-3.
29. The method of claim 28, wherein said culture medium comprises
IL-3.
30. The method of claim 28, wherein said culture medium comprises
IL-7.
31. The method of claim 28, wherein said culture medium comprises
GM-CSF.
32. The method of claim 28, wherein said culture medium comprises
eotaxin.
33. The method of claim 28, wherein said culture medium comprises
eotaxin 2.
34. The method of claim 28, wherein said culture medium comprises
eotaxin-3.
35. The method of claim 28, wherein said culture medium comprises a
culture supernatant of nitrogen-stimulated peripheral blood
mononuclear cells.
36. The method of claim 28, wherein said culture supernatant
comprises a supernatant of phytohemagglutinin-stimulated human
peripheral blood mononuclear cells.
37. An osteoclast, which is obtainable by the method of claim
27.
38. A method for screening an agent for treating or preventing a
metabolic bone disease, which comprises: contacting an osteoclast
precursor cell isolated by the method of claim 21, with an agent to
be tested, and measuring the inhibitory activity of said agent on
differentiation of the osteoclast precursor into an osteoclast.
39. A method for screening an agent for treating or preventing a
metabolic bone disease, which comprises using the osteoclast
precursor cell of claim 21.
40. A method for screening an agent for the treatment or prevention
of a metabolic bone disease, which comprises: contacting the
osteoclast of claim 37 with an agent to be tested and measuring
inhibitory activity of said agent on the bone resorption activity
of said osteoclast.
41. An agent for treating or preventing a metabolic bone disease
which is obtainable by the method of claim 40.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for the
differentiation of osteoclast precursor cells (preosteoclasts) into
osteoclasts, which comprises culturing the preosteoclasts in the
absence of accesory cells; a method for isolating preosleoclasts; a
method for screening agents for metabolic bone diseases, which
comprises using the preosteoclasts or the osteoclasts; and agents
for metabolic bone diseases, which is obtainable by the screening
method.
BACKGROUND ART
[0002] Bone tissues of mammal repeat generation and resorption of
bone. The tissue operates as a central point of calcium metabolism
to keep the balance between bone resorption and generation in the
growth period and even after the period of maturity. Bone
resorption and resorption are well balanced by crosstalk between
osteoclasts and osteoblasts. However, unbalance between bone
resorption and generation lead metabolic bone diseases, including
osteoporosis, rheumatoid arthritis, osteoarthritis, decrease of
bone quantity due to diabetes, many types of hormone abnormalities,
nutritional disorder, osteopetrosis and osteomalacia. Cellular
pathogenesis of the most of above disorders remain to be
elucidated. To resolve the issue and discover therapeutic agents
for the metabolic bone diseases, methods for isolation and
characterization of osteoclasts and osteoblasts have been
required.
[0003] It has been studied well for isolation of osteoclasts of
mice or rats. Recentry, Fujisawa et al. reported that human
osteoclasts were obtained from patients with rheumatoid arthritis
(Annals of Rheumatic Diseases.55;816-822,(1996)). In their study,
human osteoclasts were obtained in the presence of a mouse
osteoblasts-like cell line, since it has been believed that the
presence of accessory cells, such as osteoblasts or bone marrow
stromal cells, were indispensable for differentiation of
preosteoclasts in vitro. The accessory cells have been believed to
play important role for osteoclastogenesisi by close contact with
preosteoclasts.
[0004] On the other hand, JP 7-194373 describes a method for the
differentiation of bone mallow cells into osteoblasts, osteoclasts
or chondrocytes in a medium without bone mallow stromal cells or
osteoblasts. But, they did not succeed in the isolation of
osteoclasts itself. Under the circumstances above, in order to
elucidate the mechanism of differentiation of preosteoclasts into
osteoclasts, it was required to establish a method for isolation of
preosteoclasts, and that for inducing the differentiation into
osteoclasts, which needs few factors for the differentiation, and
particularly, especially in the absence of accessory cells.
DISCLOSURE OF INVENTION
[0005] As a result of intensive studies on the induction of
osteoclastgenesis, the present inventors have accomplished
inventions on a method for differentiating preosteoclasts into
osteoclasts which comprises culturing the osteoclast precursor
cells in the absence of accessory cell, a method for isolating
preosteoclasts, a preosteoclast which is isolated by the isolating
method, a method for differentiating the preosteoclasts isolated by
the isolating method into osteoclasts, a osteoclast which is
obtainable by the differentiating method, a method for screening
agents for metabolic bone diseases an dan agent for metabolic bone
diseases which is obtainable by the screening method.
[0006] The invention relates to:
[0007] {circle over (1)} A method for differentiating
preosteoclasts into osteoclasts, which comprises culturing the
preosteoclasts in the absence of accessory cells;
[0008] {circle over (2)} The method as described in {circle over
(1)}, which uses a culture medium containing IL-3, IL-7, GM-CSF,
eotaxin, eotaxin-2, eotaxin-3 or a mixture of two or more of
them;
[0009] {circle over (3)} The method as described in {circle over
(1)} or {circle over (2)}, which uses a culture medium containing a
culture supernatant of mitogen-stimulated peripheral blood
mononuclear cells;
[0010] {circle over (4)} The method as described in {circle over
(3)}, wherein the culture supernatant of mitogen-stimulated
peripheral blood mononuclear cells is a culture supernatant of
phytohemagglutinin-stimulat- ed human peripheral blood mononuclear
cells;
[0011] {circle over (5)} A method for isolating preosteoclasts,
which comprises culturing peripheral blood or joint fluid in the
absence of cytokine for 1 to 3 weeks;
[0012] {circle over (6)} The method as described in {circle over
(5)}, in which the preosteoslasts are isolated by adding peripheral
blood or joint fluid to essential medium for mammalian cells in the
absence of cytokine and culturing them at 35-37.degree. C. in 5-7%
CO.sub.2-- containing air for 1-3 weeks to perish cells except
preosteoclasts;
[0013] {circle over (7)} An preosteoclast, which is obtainable by
the method described in {circle over (5)} or {circle over (6)};
[0014] {circle over (8)} A method for differentiating
preosteoclasts obtained by the method as described in {circle over
(5)} or {circle over (6)} into osteoclasts, which comprises
culturing the preosteoclasts in the absence of accessory cells;
[0015] {circle over (9)} The method as described in {circle over
(8)}, which uses a culture medium containing IL-3, IL-7, GM-CSF,
eotaxin, eotaxin-2, eotaxin-3 or a mixture of two or more of
them;
[0016] {circle over (10)} The method as described in {circle over
(8)} or {circle over (9)}, which uses a culture medium containing a
culture supernatant of mitogen-stimulated peripheral blood
mononuclear cells;
[0017] {circle over (11)} The method as described in {circle over
(10)}, wherein the culture supernatant of mitogen-stimulated
peripheral blood mononuclear cells is a culture supernatant of
phytohemagglutinin-stimulat- ed human peripheral blood mononuclear
cells;
[0018] {circle over (12)} An osteoclast, which is obtainable by the
method as described in any one of {circle over (1)} to {circle over
(4)} and {circle over (8)} to {circle over (11)};
[0019] {circle over (13)} A method for screening agents for
metabolic bone diseases, which comprises using the preosteoclasts
isolated by the method as described in {circle over (5)} or {circle
over (6)};
[0020] {circle over (14)} A method for screening agents for
metabolic bone diseases, which comprises using the preosteoclasts
as described in {circle over (7)};
[0021] {circle over (15)} A method for screening agents for
metabolic bone diseases, which comprises using the osteoclasts
obtained by the method as described in any one of {circle over (1)}
to {circle over (4)} and {circle over (8)} to {circle over
(11)}.
[0022] {circle over (16)} A method for screening agents for
metabolic bone diseases, which comprises using the osteoclasts as
described in {circle over (12)};
[0023] {circle over (17)} An agent for metabolic bone diseases,
which is obtainable by the method as described in any one of
{circle over (13)} to {circle over (16)}.
[0024] One of the invention relates to a method for differentiating
preosteoclasts into osteoclasts, which comprises culturing the
preosteoclasts in the absence of accessory cell. In this case,
"accessory cell" means mesenchymal cell which can induce the
differentiation of preosteoclasts by producing adhesive factors and
soluble factors. As a "accessory cell", bone mallow stromal cells,
osteoblasts (osteoblast like cells), fibroblasts and tumor cells
are given for examples. "Osteoclast precursor cells" or
"Preosteoclasts" means cells which do not substantially contain any
admixture cell. Concretely, it means human preosteoclasts.
"Osteoclast precursor cells" or "Preosteoclasts" also means
hematopoietic stem cell-derived cells which have an ability of
differentiating into osteoclasts under an appropriate culture
condition. "Osteoclasts" means cells which do not substantially
contain any admixture cells. Concretely, it means human
osteoclasts. "Osteoclasts" also means cells which are multinucleate
(N>3), positive for tartrate-resistant acid phosphatase, and
have an ability of bone resorption.
[0025] Further, this invention relates to a method for
differentiating preosteoclasts into osteoclasts, which uses a
culture medium containing IL-3, IL-7, GM-CSF, eotaxin (Kitaura, M.
et al., J. Biol. Chem., 271, 13, 7725-7730, 1996), eotaxin-2
(Forssmann, U. et al., J. Exp. Med., 185, 12, 2171-2176, 1997),
eotaxin-3 (human CC type chemokine. The nucleic acid sequence is
shown at SEQ ID No.:1, and the amino acid sequence is shown at SEQ
ID No.:2) or a mixture of two or more of them. IL-3, IL-7, GM-CSF,
eotaxin, eotaxin-2 and eotaxin-3, each may be natural type or
recombinant type. As the culture medium which contains IL-3, IL-7,
GM-CSF, eotaxin, eotaxin-2, eotaxin-3 or a mixture of two or more
of them, a culture supernatant of mitogen-stimulated peripheral
blood mononuclear cells can be used. As the culture supernatant of
mitogen-stimulated peripheral blood mononuclear cells, a culture
supernatant of phytohemagglutinin-stim- ulated human peripheral
blood mononuclear cells can be used.
[0026] The invention also relates to a method for isolating
preosteoclasts by culturing peripheral blood or joint fluid in the
absence of cytokine for 1 to 3 weeks. In detail, the invention
relates to a method for isolating preosteoslasts by adding
peripheral blood or joint fluid to essential medium for mammalian
cells in the absence of cytokine, and culturing them at
35-37.degree. C., in 5-7% CO.sub.2-containing air for 1-3 weeks to
perish cells except preosteoclasts. Further, the invention relates
to preosteoclasts which are obtainable by the method.
[0027] "Peripheral blood" means mammalian peripheral blood,
concretely, human peripheral blood. As the peripheral blood,
peripheral blood of healthy donors subjects can be used.
[0028] As "joint fluid", not only joint fluid of healthy donors but
also joint fluid of rheumatoid arthritis (RA) subjects can be
used.
[0029] "Essential medium for Mammalian cells" means an isotonic
buffer solution which contains inorganic salts, essential amino
acids or its derivatives, and vitamin and its derivatives, which
are available for cells to survive. As the "essential medium for
mammalian cells", Dulbecco's Modified Eagle Medium (DMEM), RPMI1640
and AIM-V are given for examples.
[0030] Further, the invention relate to a method for
differentiating preosteoclasts into osteoclasts, which comprises
culturing in the absence of accessory cell the preosteoclasts which
are isolated by culturing the peripheral blood or joint fluid as
described previously in the absence of cytokine for 1 to 3 weeks.
Concretely, the invention relates to a method for differentiating
preosteoclasts into osteoclasts, which uses a culture medium
containing IL-3, IL-7, GM-CSF, eotaxin, eotaxin-2, eotaxin-3 or a
mixture of two or more of them. As IL-3, IL-7, GM-CSF, eotaxin,
eotaxin-2 or eotaxin-3, each may be natural type or recombinant
type. As a culture medium which contains IL-3, IL-7, GM-CSF,
eotaxin, eotaxin-2, eotaxin-3 or a mixture consisted by a
combination of two or more of them, a culture supernatant of
mitogen-stimulated peripheral blood mononuclear cells is available.
As a culture supernatant of mitogen stimulated peripheral blood
mononuclear cells, a culture supernatant of phytohemagglutinin
stimulated human peripheral blood mononuclear cells is
available.
[0031] As another embodiment, the invention relates to a method for
screening agents for metabolic bone diseases, which comprises using
the preosteoclasts or the osteoclasts of this invention, and to an
agent for metabolic bone diseases which is obtainable by the
screening method. As the "metabolic bone disease", osteoporosis,
rheumatoid arthritis, decrease of bone quantity due to diabetes,
osteomalacia, and osteopetrosis are given for example.
[0032] As "a method for screening agents for metabolic bone
diseases", (1) a method for measuring inhibitory activities on the
differentiation of preosteoclasts into osteoclasts, and (2) a
method for measuring inhibitory activities on the bone resorption
by osteoclasts are given for example. If a candidate compound for
the therapeutic agent shows a differentiation inhibitory activity
against the preosteoclasts of this invention, the compound is
promising as an antirheumatic drug. Futher, if a candidate compound
for the therapeutic agent shows a bone resorption inhibitory
activity against the osteoclasts of this invention, it is suggested
that the compound is useful for treating osteoporosis caused by
excessive bone resorption, decrease of bone mass due to diabetes,
osteomalacia or the like. Thus, by using the preosteoclasts or the
osteoclasts of this invention, the screening for bone metabolic
diseases agents can easily be performed in vitro.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIG. 1 is photomicrographs which show that the
preosteoclasts and the osteoclasts stained by May-Giemsa
solution.
[0034] FIG. 2 is photomicrographs which show that the TRAP
activities in the preosteoclasts and the osteoclasts.
[0035] FIG. 3 is photomicrographs which show the resorption
activities of the preosteoclasts and the otsteoclasts, which was
tested by using dentine slices.
[0036] FIG. 4 is a photomicrograph which shows resorption pits on
dentine slice formed by the osteoclasts.
[0037] FIG. 5 is photomicrographs which show the resorption
activities of the preosteoclasts and the otsteoclasts, which was
tested by using a hydroxyappatite sintering quartz disc.
BEST MODE FOR CARRYING OUT THE INVENTION
[0038] The human osteoclasts of the invention can be obtained by
the following methods.
[0039] Isolation of Preosteoclasts
[0040] Isolation of osteoclast precursor cells comprises (1)
separation step of cellular fraction and (2) isolation step of
preosteoclasts.
[0041] (1) Separation of Cellular Fraction
[0042] Preotsteolasts can be obtained from peripheral blood of
healthy donors or joint fluid of rheumatoid arthritis subjects. The
volume of bodily fluids necessary for isolating the cells is 50 ml
to 200 ml for peripheral blood of normal subjects, or 1 ml to
several tens ml for joint fluid of rheumatoid arthritis subjects.
In the case of using peripheral blood of healthy donors, the blood
is collected in the presence of heparin or an alternative
anticoagulant. Peripheral blood mononuclear cells (PBMC) are
obtained from the blood by excluding of erythrocyte using a
specific gravity centrifugation or ammonium chloride so on.
[0043] On the other hand, in the case of using joint fluid of
rheumatoid arthritis subjects, the cellular fraction can be
obtained by the centrifugation of the joint fluid, if necessary by
adding essential medium for mammalian cells, such as RPMI1640,
thereto.
[0044] (2) Isolation of Preosteoclasts
[0045] The obtained cellular fraction is cultured in essential
medium for mammalian cells, such as DMEM, at 35-37.degree. C.,
preferably, 37.degree. C., for several weeks, preferably, 1-3
weeks, in 5-7% CO.sub.2-containing air. This culture make cells
except preosteoclasts perish, and the preosteoclasts can be
isolated.
[0046] Isolation of Osteoclasts
[0047] Isolation of osteoclasts comprises (1) preparation step of
culture medium and (2) differentiation-inducing step of
preosteoclasts into osteoclasts.
[0048] (1) Preparation of Culture Medium
[0049] The medium is prepared by an addition of cytokines to
essential medium for mammalian cells, such as AIM-V to induce the
differentiation. Preferably, antibiotics are added to the medium
for preventing the contamination of bacteria. According to the
invention, IL-3, IL-7, GM-CSF, eotaxin, eotaxin-2, eotaxin-3 are
given for example as the cytokine which have an activity to induce
the differentiation of the preosteoclasts. These cytokines may be
added as alone or mixture thereof.
[0050] (2) Differentiation Induction of Preosteoclasts into
Osteoclasts
[0051] The obtained preosteoclsats are cultured at 35-37.degree.
C., for 48-96 hours in the medium described above. The
preosteoclasts are differentiated into osteoclasts by the
stimulation of the cytokines described above. Thus, the osteoclasts
can be isolated.
[0052] Screening of Therapeutic Agents for Metabolic Bone
Diseases
[0053] As a method for screening therapeutic agents for metabolic
bone diseases, (1) a method for measuring inhibitory activities on
the differentiation of preosteoclasts into osteoclasts, and (2) a
method for measuring inhibitory activities on bone resorption by
the osteoclasts are given for example.
[0054] (1) Measuring Method of Differentiation Inhibitory
Activity
[0055] For example, the preosteoclasts obtained by the invention
are prepared as 1.times.10.sup.6 cell/well and cultured on 48 wells
plate. In the cultivation, essential medium for mammalian cells,
contains IL-3, IL-7, GM-CSF, eotaxin, eotaxin-2, eotaxin-3 or a
supernatant of mitogen-stimulated peripheral blood mononuclear
cells, is used. Various concentration of a candidate compound for
therapeutic agents is added into the each well, and the prepared
plate is cultured at 37.degree. C., for 48-96 hours. At the end of
the culture period, the adherent cells are fixed and stained with
tartrate-resistant acid phosphatase (TRAP) which is the specific
maker enzyme for osteoclasts. The multinucleate (N>3) and TRAP
positive cell is counted as an osteoclast in each well. By
calculating the result as IC.sub.50, it is possible to evaluate the
differentiation inhibitory activity of the candidate compound
against the preosteoclasts.
[0056] (2) Measuring Method of Bone Resorption Inhibitory
Activity
[0057] For example, the osteoclasts obtained by the invention are
prepared as 1.times.10.sup.6 cell/well and cultured on a dentine
slice in the essential medium for mammalian cells 48-well culture
plate. Various concentration of a candidate compound for
therapeutic agents is added into the each well, and the cells are
cultured at 37.degree. C., for 48-96 hours. At the end of the
culture period, the dentine slice is stained with hematoxylin.
Osteoclasts resorb hydroxyappatite, and the resorbed parts
(resorption pit) is visualized by the hematoxylin staining when the
bone resorption is occurred. The formation of resorption pit on
dentine slice is observed microscopically or an
electron-microscopically. Otherwise, the change of calcium
concentration in the culture supernatant can be assayed. By
calculating the result as IC.sub.50, it is possible to evaluate a
bone resorption inhibitory activity of the candidate compound
against the osteoclasts.
[0058] The inhibitory activity on the osteoclasts formation is
becoming important as an indicator for assessing an antirheumatic
activity. Consequently, a compound which has a differentiation
inhibitory activity against preosteoclasts, is hopeful as an
antirheumatic drug. Further, osteoporosis is caused by an excessive
bone resorption. Thus, a compound with a bone resorption inhibitory
activity against osteoclasts is hopeful as a therapeutic agent for
osteoporosis.
EXAMPLE
Example 1
Isolation of Human Osteoclasts from Joint Fluid of Patients with
Rheumatoid Arthritis
[0059] (1) Separation of a Cellular Fraction
[0060] Joint fluids were obtained from patients with rheumatoid
arthritis. The joint fluids were kept in tubes at 4.degree. C. The
following procedures were generally under sterile conditions. The
joint fluid, 1 ml to several tens ml, was added to equal volume of
RPMI 1640 medium (Gibco BRL, #22400 or equivalent). The mixture was
centrifugated at 1,000-2,000 rpm for 5 minutes at 4.degree. C. to
obtain a cellular fraction containing granulocytes and
lymphocytes.
[0061] (2) Isolation of Preosteoclasts
[0062] The obtained cellular fraction was cultured in DMEM medium
(Gibco BRL, #12430-21 or equivalent) supplemented with 10% (v/v) of
fetal calf serum (FCS) in 5-7% CO.sub.2-containing air at
37.degree. C. for several weeks. During the culture period, all
cells except preosteoclasts died out, and only preosteoclasts
survived (FIGS. 1, 2).
[0063] (3) Preparation of Medium
[0064] The medium was prepared for the differentiation of
preosteoclasts into osteiclasts . Four hundred ml of 400 ml of
AIM-V medium (Gibco BRL, #87-0112) was supplemented with 60 ml of
RPMI1640 medium (Gibco BRL, #22400), 40 ml of human T-STIM (10
BRMP/ml; BRMP, Biological Response Modifier Program Jurkat IL-2
reference reagent), 50 ml of 10% FCS (inactivated in advance by
heating at 56.degree. C. for 30 minutes), and antibiotics (100 U of
penicillin and 100 .mu.g/ml of streptomycin; Gibco BRL, #15140-015
or equivalent), and used as Medium A. Human T-STIM is a culture
supernatant of human peripheral blood mononuclear cells stimulated
with phytohemagglutinin (Human T-STIM with PHA, Becton Dickinson,
#40045).
[0065] (4) Induction of Differentiation of Preosteoclasts into
Osteoclasts
[0066] The preosteoclasts obtained in Example 1(2) were stimulated
with Medium A described in Example 1(3) at 37.degree. C. for 48-96
hours. In the culture condition, the differentiation of
preosteoclasts into osteoclasts was observed (FIGS. 1, 2).
[0067] (5) Identification of the Cytokine Possesing a
Differentiation Inducing Ability
[0068] Human T-STIM contains a variety of cytokines. In order to
identify the cytokine which stimulates differentiation of
preosteoclasts into osteoclasts, the differentiation was examined
by modifycation the contents of Medium A described as previously in
Example 1(3).
[0069] {circle over (1)} Preparation of the Medium
[0070] The following 16 kinds of medium (B-R) were prepared by
using the cytokines shown below, alone or a mixture thereof,
instead of human T-STIM contained in Medium A (Example 1(3)). The
other materials than human T-STIM, namely, AIM-V medium, RPMI 1640
medium, 10% FCS and antibiotics (100 U of penicillin and 100
.mu.g/ml of streptomycin; Gibco BRL, #15140-015 or it's equivalent)
were the same as those contained in Medium A.
[0071] Medium B: 0.5-5 ng/ml of recombinant human IL-1 (R & D
Systems, #201-LB, #200-LA or equivalent)+400 ml of AIM-V medium+60
ml of RPMI 1640 medium+50 ml of FCS+antibiotics
[0072] Medium C: 50-200 U/ml of recombinant human IL-2 (R & D
Systems, #202-IL, or equivalent)+400 ml of AIM-V medium+60 ml of
RPMI 1640 medium+50 ml of FCS+antibiotics
[0073] Medium D: 2-10 ng/ml of recombinant human IL-3 (R & D
Systems, #403-ML-010 or equivalent)+400 ml of AIM-V medium+60 ml of
RPMI 1640 medium+50 ml of FCS+antibiotics
[0074] Medium E: 50-200 U/ml of recombinant human IL-4 (Genzyme,
#2181-01 or equivalent)+400 ml of AIM-V medium+60 ml of RPMI 1640
medium+50 ml of FCS+antibiotics
[0075] Medium F: 10-20 ng/ml of recombinant human IL-6 (R & D
Systems, #206-IL or equivalent)+400 ml of AIM-V medium+60 ml of
RPMI 1640 medium+50 ml of FCS+antibiotics
[0076] Medium G: 10-20 ng/ml of recombinant human IL-6 (R & D
Systems, #206-IL or equivalent)+100-300 ng/ml of recombinant human
soluble IL-6 receptor (R & D Systems, #227-SR or it's
equivalent)+400 ml of AIM-V medium+60 ml of RPMI 1640 medium+50 ml
of FCS+antibiotics
[0077] Medium H: 5-20 ng/ml of recombinant human IL-7 (Genzyme,
#1587-00 or equivalent)+400 ml of AIM-V medium+60 ml of RPMI 1640
medium+50 ml of FCS+antibiotics
[0078] Medium I: 1-4 ng/ml of recombinant human IL-11 (R & D
Systems, #218-IL or equivalent)+400 ml of AIM-V medium+60 ml of
RPMI 1640 medium+50 ml of FCS+antibiotics
[0079] Medium J: 2.5-100 ng/ml of recombinant human M-CSF (R &
D Systems, #216-MC-010 or equivalent)+400 ml of AIM-V medium+60 ml
of RPMI 1640 medium+50 ml of FCS+antibiotics
[0080] Medium K: 1-5 ng/ml of recombinant human GM-CSG (R & D
Systems, #215-GM-010 or equivalent)+400 ml of AIM-V medium+60 ml of
RPMI 1640 medium+50 ml of FCS+antibiotics
[0081] Medium L: 2-10 ng/ml of recombinant human IL-3 (R & D
Systems, #403-010 or equivalent)+5-20 ng/ml of recombinant human
IL-7 (Genzyme, #1587-00 or it's equivalent)+400 ml of AIM-V
medium+60 ml of RPMI 1640 medium+50 ml of FCS+antibiotics
[0082] Medium M: 5-20 ng/ml of recombinant human IL-7 (Genzyme,
#1587-00 or equivalent)+1-5 ng/ml of recombinant human GM-CSF (R
& D Systems, #215-GM-010 or equivalent)+400 ml of AIM-V
medium+60 ml of RPMI 1640 medium+50 ml of FCS+antibiotics
[0083] Medium N: 2-10 ng/ml of recombinant human IL-3 (R & D
Systems, #403-ML-010 or equivalent)+5-20 ng/ml of recombinant human
IL-7 (Genzyme, #1587-00 or equivalent)+1-5 ng/ml of recombinant
human GM-CSF (R & D Systems, #215-GM-010 or equivalent)+400 ml
of AIM-V medium+60 ml of RPMI 1640 medium+50 ml of
FCS+antibiotics
[0084] Medium P: 10-1000 ng/ml of recombinant human eotaxin (R
& D Systems, #220-EO or equivalent)+400 ml of AIM-V medium+60
ml of RPMI 1640 medium+50 ml of FCS+antibiotics
[0085] Medium Q: 10-1000 ng/ml of recombinant human eotaxin-2 (R
& D Systems, #343-E2 or equivalent)+400 ml of AIM-V medium+60
ml of RPMI 1640 medium+50 ml of FCS+antibiotics
[0086] Medium R: 10-1000 ng/ml of recombinant human Eotaxin 3+40 ml
of AIM-V medium+60 ml of RPMI 1640 medium+50 ml of
FCS+antibiotics
[0087] (Eotaxin-3 was found in the human genome sequencing data (H
RG356E01) published by Washington University Genome Sequence
Center, an exson sequence, which is considered to exhibit a
significant homology but to encode a different chemokine protein,
compared to known CC type chemokines. According to the sequence,
cDNA of eotaxin-3 was obtained as shown in SEQ ID No.1. Recombinant
baculovirus containing the sequence was prepared to infect insect
cells, then the recombinant eotaxin-3 protein was purified from the
culture supernatant.)
[0088] {circle over (2)} Induction of the Differentiation of
Preosteoclasts into Osteiclasts
[0089] The preosteoclasts obtained by Example 1(2) were cultured in
the Medium B-N described above at 37.degree. C. for 48-96 hours.
The differentiation of preosteoclasts into osteoclats was observed
in the Medium D, H, K, M, N, P, Q and R. However, the
differentiation was not occurred in the Medium B, C, E, F, G, I and
J. From these results, it has become clear that IL-3, IL-7, GM-CSF,
eotaxin, eotaxin-2, eotaxin-3 and the mixture thereof have an
ability for inducing the differentiation of preosteoclasts. And, it
was confirmed that IL-1, IL-2, IL-4, Il-6, IL-11 and M-CSF do not
have an ability for inducing the differentiation of
preosteoclasts.
[0090] On the differentiation requiring the presence of accessory
cells, it has been known that IL-1, IL-6, IL-11, and M-CSF induce
the differentiation of preosteoclasts into osteoclasts. But it has
not been clear that the differentiation was induced by the result
of the stimulation of preosteoclasts or accessory cells.
[0091] On the other hand, on the differentiation of preosteoclasts
in this invention which does not require the presence of accessory
cell, it becomes clear that IL-3, IL-7, GM-CSF, eotaxin, eotaxin-2,
and eotaxin-3 have the differentiation-inducing ability.
Furthermore, IL-1, IL-6, IL-11 or M-CSF does not effect to the
differentiation.
Example 2
Isolation of Human Osteoclasts from Peripheral Blood of Healty
Donors
[0092] (1) Separation of Cellular Fraction
[0093] Fifty ml to 200 ml of peripheral blood of healthy donors
were collected in the presence of heparin or an alternative
anticoagulant. Peripheral blood mononuclear cells (PBMC) were
obtained by a specific gravity centrifugation using a Ficoll-paque
(Pharmacia Biotech). 10.sup.7 cell/ml of PBMC were suspended in
RPMI1640 medium containing 10% (v/v) FCS, then 1-1.5 ml/dish of the
cell suspension was cultured on 60 mm culture dish at 37.degree.
C., in 5-7% CO.sub.2-containing air, for 1-2 hours. After the
culture, the non-adherent cells were rinsed out from the dish with
RPIM1640 medium containing 10% (v/v) of FCS at 37.degree. C.
Adherent cells to the dish were washed with cold (4.degree. C.),
serum-free RPIM1640 medium, then the cells were collected as a
peripheral blood monocytes (about 3-8% of total PBMC).
[0094] (2) Isolation of Preosteoclasts
[0095] The obtained monocytes (0.5-1.times.10.sup.6/ml) was
cultured in DMEM medium (Gibco BRL, #12430-21 or equivalent)
supplemented with 10% of FCS in 5-7% CO.sub.2-containing air at
37.degree. C. for several weeks likewise. Example 1 (2). During the
culture period, all cells except preosteoclasts died out, and only
preostetoclasts survived. In cocultivation of the peripheral blood
monoctyes and synovial nurse cells-derived rheumatoid arthritis
subjects, the proliferation efficiency of the preosteoclasts
increased. At the time, the same medium as Example 1 (2) was
used.
[0096] (3) Induction of Differentiation
[0097] The preosteoclcasts were stimulated with Medium A described
in Example 1(3) at 37.degree. C. for 48-96 hours. The
differentiation of preosteoclasts was occurred and osteoclasts were
obtained.
[0098] It was confirmed that the cells obtained at Example 1(4) and
Example 2(3) were osteoclasts by the following testing
examples.
Testing Example
[0099] (1) Morphologic Observation
[0100] The cells were stained by May-Giemsa solution, and were
examined microscopically. The stained preosteoclasts and the
stained osteiclasts are shown in FIG. 1 instead of photomicrograph.
Form the result, it was confirmed that the preosteoclasts before
the differentiation had a monocyte like morphilogy and the
osteoclasts were multinucleated (over 3-100) giant cells.
[0101] (2) TRAP Stain
[0102] The preosteoclasts before the differentiation and the
osteoclasts after the differentiation were stained by using a TRAP
(tartrate-resistant acid phosphatase) staining kit (Sigma Co.) and
examined by a microscope. The cells before the differentiation
(preosteoclasts) and the cells after the differentiation
(osteoclasts) are shown in FIG. 2 instead of photomicrograph. The
figures shows that the cells before the differentiation
(preosteoclasts) were positive for TRAP, and the cells after the
differentiation (osteoclasts) were positive for TRAP especially
around the nuclei.
[0103] (3) Bone Resorbing Activity
[0104] The preosteoclasts were cultured on dentine slices under the
condition which cause the differentiation (Example 1(4)), then the
dentine slices were stained with hematoxylin (Sigma Co.) and
examined by a microscope. FIG. 3 shows the surfaces of the slice
instead of the photomicrograph. The preosteoclasts before the
differentiation had no effect on the dentine slices. But the
osteoclasts after the differentiation resorbed calcium phosphate
and the resorption pit on the slices were strongly stained with the
dye. The dentine slices resorbed by the osteoclasts were examined
by a scanning electron microscopy. FIG. 4l shows the figure instead
of the scanning electron photomicrograph. The formation of the
resorption pit is noted at the center of the figure. The resorption
of calcium phosphate formed the pit and collagen fibers were
exposed outside.
[0105] Futher, the preosteoclasts were differentiated on calcium
phosphate sintering quartz discs (Osteologic.TM., Sumisho Pharma)
and the discs were examined by a phase-contrast microscope. FIG. 5
shows the figure instead of the phase-contrast photomicrograph. The
preosteoclasts before the differentiation had no effect on the
calcium phosphate sintering quartz discs. But the resorption (the
transparent area of the disc) by the osteoclasts after the
differentiation was observed.
[0106] From the results shown in the testing examples, it was
confirmed that the cells before the differentiation were
preosteoclasts and the cells after the differentiation were
osteoclasts.
[0107] Industrial Applicability
[0108] According to the invention, osteoclasts can be obtained from
the same individual repeatedly, thus the pathophysiological or
immunological investigation of the osteoclasts can be performed by
using them. The screening of candidate compounds useful for
treating metabolic bone diseases can be examined easily by using
the osteoclast precurosor cells or osteoclasts of the invention.
For example, it is effective to use a compound, which is obtained
by the screening for bone resorption-inhibitory activity against
the osteoclasts and shows a bone resorption-inhibiting ability
against the osteoclasts, to treat the metabolic bone disease,
including osteoporosis caused by an excessive bone resorption,
decrease of bone quantity due to diabetes, and osteomalacia.
Sequence CWU 1
1
2 1 467 DNA Homo sapiens CDS (28)..(309) 1 cctgagaagg gcctgatttg
cagcatc atg atg ggc ctc tcc ttg gcc tct gct 54 Met Met Gly Leu Ser
Leu Ala Ser Ala 1 5 gtg ctc ctg gcc tcc ctc ctg agt ctc cac ctt gga
act gcc aca cgt 102 Val Leu Leu Ala Ser Leu Leu Ser Leu His Leu Gly
Thr Ala Thr Arg 10 15 20 25 ggg agt gac ata tcc aag acc tgc tgc ttc
caa tac agc cac aag ccc 150 Gly Ser Asp Ile Ser Lys Thr Cys Cys Phe
Gln Tyr Ser His Lys Pro 30 35 40 ctt ccc tgg acc tgg gtg cga agc
tat gaa ttc acc agt aac agc tgc 198 Leu Pro Trp Thr Trp Val Arg Ser
Tyr Glu Phe Thr Ser Asn Ser Cys 45 50 55 tcc cag cgg gct gtg ata
ttc act acc aaa aga ggc aag aaa gtc tgt 246 Ser Gln Arg Ala Val Ile
Phe Thr Thr Lys Arg Gly Lys Lys Val Cys 60 65 70 acc cat cca agg
aaa aaa tgg gtg caa aaa tac att tct tta ctg aaa 294 Thr His Pro Arg
Lys Lys Trp Val Gln Lys Tyr Ile Ser Leu Leu Lys 75 80 85 act ccg
aaa caa ttg tgactcagct gaattgtcat ccgaggacgc ttggaccccg 349 Thr Pro
Lys Gln Leu 90 ctcttggctc tgcagccctc tggggagcct gcggaatctt
ttctgaaggc tacatggacc 409 cgctggggag gagagggtgt ttcctcccag
agttacttta ataaaggttg ttcatagt 467 2 94 PRT Homo sapiens 2 Met Met
Gly Leu Ser Leu Ala Ser Ala Val Leu Leu Ala Ser Leu Leu 1 5 10 15
Ser Leu His Leu Gly Thr Ala Thr Arg Gly Ser Asp Ile Ser Lys Thr 20
25 30 Cys Cys Phe Gln Tyr Ser His Lys Pro Leu Pro Trp Thr Trp Val
Arg 35 40 45 Ser Tyr Glu Phe Thr Ser Asn Ser Cys Ser Gln Arg Ala
Val Ile Phe 50 55 60 Thr Thr Lys Arg Gly Lys Lys Val Cys Thr His
Pro Arg Lys Lys Trp 65 70 75 80 Val Gln Lys Tyr Ile Ser Leu Leu Lys
Thr Pro Lys Gln Leu 85 90 ?2?
* * * * *