CA2217816A1 - Method for producing a highly enriched population of osteoclast cells - Google Patents
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Abstract
Methods of obtaining enriched populations of osteoclast precursor cells which can be released from tissue culture dishes and used for biochemical studies are described. Osteoblastic cells and bone marrow cells are co-cultured. Next a .alpha.v.beta.3 receptor ligand, such as echistatin is used for cell detachment. The result is a 75-95 % pure enriched population of tartrate resistant acid phosphatase (TRAP+) cells, in high yields (2-3 X 106 cells per experiment) can be obtained. These cells are mostly mononucleated and based on their characteristics are considered to be pre-fusion osteoclasts (pOC cells).
The precursor osteoclasts can be reseeded onto osteoblasts to obtain an enriched population of mature, multinucleated osteoclast cells.
The precursor osteoclasts can be reseeded onto osteoblasts to obtain an enriched population of mature, multinucleated osteoclast cells.
Description
W O 96/32469 PCTrUS9~'0q63 , TITLE OF THE INVENTION
METHOD FOR PRODUCING A HIGHLY ENRICHED POPULATION
OF OSTEOCLAST CELLS
DESCRIPTION OF THE INVENTION
This invention relates to a method of producing a population of cells which is highly enriched in it.s content of osteoclast precursor cells, and to the cell populations so produced.
BACKGROUND OF THE INVENTION
O.steocla.sts are termin~lly differentiated cells which play a key role in bone resorption. Due to the low number of m~mm~lian osteoclast~ and the difficulty to isolate them from bone tis~ue, and from other cell.~, their characterization has been limited primarily to immunohi~tochemistry or anatomical and phy~iological mea~;urement.~;
on ~ingle cells.
Several attempts have been made, with limited .~;ucce~, to identify, i~iolate and e~tablish in culture, cell~ capable of difi~'erentiating into o~;teocla~t.s from either bone marrow (Billecoc~l et cll., l9gO P70c .
Nclt. Acacl. Sc i. USA ~7:6470-6474; Prallet et c~l., 1992, J. Bone Mi~?.
Re~. 7:405-414: and Chamber.s etcll., 1993, P70e. Nclt. Acclcl. Sci. USA
90:557~-55~2) or leukemic and promyelocytic cell line.s (Yoneda et cll 1991, Endocrinolo~ 129:683-6~9; Gattei et al., 1992 J. Cell Biol.
116:437-447). Recently, cell~ obtained from hum~n giant cell tumor.~ of bone were reported to forrn osteoclast~ in culture (Grano et cll., 1994 J.
Bone Mi~l. Res. 9:1013-1020; Grano, et cll., 1994 Exp. Cell Re~.
212 :209-21 ~).
To obtain enriched o~iteocl~it preparation~i. Akat~u et cll., 1992, J. Bo~1e Mille7. Res. 7:1297-1306; cultured the o~teobla~tic and bone marrow cell.~ on collagen gel-coated di.~he~. and relea.~ed the cell~
from the collagen matrix u.~ing collagen~ie. However, the yield of o.~teocla~tic cell~ wa~ ~uite low. Recently. Shioi et cll., 1994, Cc~leif:
CA 022l78l6 l997-l0-08 W 096t32469 PCTrUS9f'01~3 Tissue Int. 55:387-394, using a similar co-culture system reported the enrichment of generated osteoclasts by treating the cultured cells with bacterial collagenase to remove the stromal supporting cells. By this procedure they obtained 60% pure tartrate resistant acid phosphate 5 positive (TRAP+) cells, however the osteoclasts were not available except as attached cells to either dishe~ or bone slices.
Oursler et al., 1991, J. Bone Min. Re~. 6:375-3~S5 cultured avian osteoclasts and further purified the osteoclasts by density gradient centrifugation. This method yielded an enriched population of TR~P+
10 cells capable of bone resorption.
To date, however, the only m;~mm~lian osteoclasts obt~ined in high yields (300,000 per rabbit) and purity (9~o) are rabbit osteoclasts (Tezuka etal., 1992,Biochem. B~ophys. Res. Comm. 1~6:
911-917). These cells, however were attached to the plastic culture 15 dishes and cannot be removed.
DESCRIPTION OF THE lNVENTION
This invention relates to a method of obtaining a highly enriched population of osteoclast precur~sor cell~ compri~sing 20 co-cultivation of osteoblastic cells and bone marrow cells and treatment of the culture with an integrin o~v~(33 receptor ligand to rele~e a population which is highly enriched with precursor osteoclasts. Thi.s invention also comprises the process of obtaining a highly enriched population of mature osteoclast cells comprising reseeding the 25 population of highly enriched precursor o~teocla.~t cell.~ so produced on osteoblast cells for a time sufficient for the osteocla~st precursor cell.s to fuse into mature multinucleated olsteoclasts.
This invention also relates to the enriched populations of mammalian precursor o~teoclasts. Yet another a~pect of this invention 30 is ~ population which is highly enriched with mature osteoclasts.
Further aspects of this invention is a suspension culture comprising W 096/32469 PCTrUS~ 1C3 m~mm~ n osteoclast precursor cells, and to suspension cultures comprising mature osteoclast cells.
A further aspect of this invention is an assay for bone resorption activity comprising exposing a population of m~mmalian 5 cells which is highly enriched in osteoclasts, but also comprising osteoblasts, to a bone, in the presence of Vitamin D3 or a biologically active derivative of Vitamin D3 and measuring the bone-resorption which occurs.
In accordance with this invention, it was observed that 10 integrin ocV133 receptor ligands appear to play a role in cell attachment and in the fusion of the precursor osteocla,st,s into multinucleated osteoclastic cells in the bone marrow co-culture system. One aspect of this invention thus u.se,~ (XV,B3 receptor ligands to isolate a highly enriched population of pre-fusion cells. The pre-fusion cells (also 15 referred to as precursor osteoclast,s) can be co-cultured with osteobla.sts ,so that they differentiate into mature o.steoclasts. Thi.s invention also relates to the enriched populations of precursor osteoclasts and mature o,steoclasts produced by the processes described above.
As used throughout the .~ipecification ~nd claim.s, the 20 following definitions will apply:
"~v~3~ receptor ligand" is any molecule which bind.~ to th~
o~V133 receptor. An avl33 receptor ligand can be identified by determining whether it replaces echistatin which ha.s bound to the o~vl33 receptor in a competitive binding a.~ay. Tho~;e compound.~ ~,vhich 25 replace echistatin are considered ocv~133 receptor ligands. Example~ of ocv,133 receptor ligands include RGD-containing pepetides and non-peptides which bind to the .same receptor ~s RGD-peptides.
"highly enriched" mean.~ that ~ population of cell.~ cont~in~;
at least about 75% of a ~;ingle type of cell, more preferably ;~t lea~it 30 about 90%, and more preferably at least about 95'J/~, of a particular cell type.
W 096/32469 PCTrUS~/0 "biologically active derivative of Vitarnin D3" refers to any metabolite of Vitamin D3 which is normally produced by a m~mm~l and contributes to bone metabolism. Examples include 25-hydroxy Vitamin D3, and 1,25-dihydroxy Vitarnin D3.
S "precursor osteoclasts" are pre-fusion osteoclast cells.
Precursor osteoclast cells are characterized by their ability to generate bone-resorbing, giant, multi-nucleated osteoclasts within about 24 hour.s of being plated on osteoblasts, Like mature multi-nucleated osteoclast,s, they may also be defined by the following characteristics: they are TRAP+; have calcitonin (CT) receptors; expres.s ocv~33 integrin; have o,steopontin (OP); carbonic anhydrase II (CAII). The precursor osteoclasts made according to the processes of this invention al,so form pit,s when plated on bone ,slices along with osteobla,st,s in the pre,sence of Vitamin D3 or a biologically active derivative of Vitamin D3.
"suspension culture" means any culture containing precursor o,steoclast,s or mature osteoclast,s which are not attached to the culture vessel.
Any o~v,B3 receptor ligand may be u~ed in accordance with thi,s invention. Known ocv~33 receptor ligand,~ include vitronectin, echi,statin, kir,stin, other ~inake venom,~, other RGD-containing peptide~, and non-peptide,s which bind to ,same receptor as RGD-containing peptide,s. One preferred o~v,133 receptor ligand i.s echi,statin. Echi,~tatin is an ~GD-containing snake venom which inhibit.~ osteoclast attachment and formation (Sato, et al., 1990 J. Cell Bi~ 1713-1723; Tanaka, et al., 1991, J. Bc~ne Min. Re~. 6:S14~ both of which are hereby incorporated by reference.).
One aspect of this invention i,s a convienent procedure to obtain a ,suspen,sion culture of abundant, highly enriched precur,~or osteoclast cells which (except for their mononucleated characteri,stic) have all the characteri~tic,~ of mature multinucleated o,steoclasts. The culture,s of thi,s invention compri~e a population of cell,s, at lea~t about 7:~% of which are precur,~or osteocla~ts. In a preferred embodiment, W 096/32469 PCTrUS~ 3 the population comprises at least about 90% precursor osteoclasts, and in particularly preferred embodiments, the population contains at least about 95% precursor osteoclasts. One important distingllishin~ feature of this invention is that the precursor osteoclasts of the population are in 5 a suspension, i.e. they are not attached to a plastic or glass substrate, and are thus able to be further manipulated by the researcher. This invention thus makes it possible to study the biochemistry of o.steoclast.s, the regulation of genes expressed by these cells and other characteristics .
The culture proce,sses of thi.s invention can be u.sed with any .~pecies of ~nim;~l cells which can form osteoclast.i, and are able to be cultured in vitro. Preferred ~nim~ls include m~mmal~;, particularly those of bovines, rodent~; (such as mou.se and rat), and primate.s (such a~
human and monkey). Particularly preferred are human, and m;lmm~lian .specie~; .such as rat and mouse or other ~nim~ls whose osteocla,sts ~ihare biochemical characteristic,s with human o.steocla~it.s.
Thi.s invention includes the fir~st detailed characterization of the propertie.i of mononuclear cell.~ which can generate multinucleted osteoclast~; within 24 hours in culture. While not wishing to be bound by theory. ~ince the cells of thi~ invention have all the propertie.~; te.~ited so far which are attributed to highly differentiated o.steocla~t.s. it i~
believed that precur.~or o.steocla~t cell.s represent a itage of differentiation in the o.~teocla.st lineage cell~ just prior to fu~;ion, which is the last .step in o.steocla.st differentiation.
In ~ccordance with thi.s invention, bone marrow ~nd o.steoblast cell~i are co-cultured according to known techni~ue.s, and using known and available cell line~. For example. one mou.se o~teobla~t cell line which may be u~ed i.~ MB l.~s (a mou~e c~lvaria-derived o.steobla.~tic cell line), but any other cell line which ~;upport.i o~teoclaitogene~i~i may al~io be employed, including ST-2 (a publicly available line). other calvaria-derived o.~teobla~tic stromal cell line~, or primary o.~teoblaxt cell line~.
W 096/32469 PCTrUS~
Co-culture of bone marrow and osteoblast cells should continue for approximately 6 days (depending on the particular cell lines used) or until the cell lines have yielded the maximum amount of pre-fusion osteoclast cells. However, if cells are cultured too long, 5 multinucleated osteoclasts will forrn which, due to their attachment to the cell culture plate, cannot be used in this invention. Prior to the formation of giant multi-nucleated osteoclasts, the majority of cells of the osteoblast cell line should be detached from the co-cultured bone marrow cells using an enzyme system which disrupts the co-culture, 10 such as dispase/collagenase. The amount,s of enzymes may vary with the specifc enzyme used and the specific activity, but should be sufficient to break up the attachrnent of osteoblasts to precursor osteoclasts.
Generally, approximately 1 mg/ml of each enzyme is sufficient.
Next, the rem~ining cells are treated with the (xv~33 receptor ligand for a time and an amount sufficent to inhibit the formation of multinucleated osteoclastic cells in the co-culture sy~stem a~
well as to inhibit the attachment and function of multi-nucleated o~steoclasts themselves. In a preferred embodiment of this step, the 20 remaining cells were treated with 10 to 50 nM echistatin for at least about 10 minute~s. In a particularly preferred embodiment, approximately 30 nM of echistatin is used for approximately 20 minutes.
Cells isolated from bone marrow co-culture~s by treatment 25 with the oCV~33 receptor ligand attach to coverslips or tissue culture well~
within one hour. Thi~s ilsolated population of cells i~ well-defined:
based on their TRAP+ st;lining and their ability to form mature osteoclasts when ,seeded back on osteobla.sts, the.se cell.s were identified as precursor osteoclasts (pOC cell.~). Seventy-five to ninety per cent of 30 the cells are mononuclear; the rest are multinucleated cells with two to four nuclei. Almo.st all the cells (~-95%) are TRAP+. A few TRAP+
cells, (approximately 2-3%) may still be attached to alkaline CA 022l78l6 l997-l0-08 W 096132469 PCTnU~I0 phosphatase-positive mononuclear cells, presumably osteoblastic cells.
A small number of cells which (approximately 3-4%) do not stain for " either TRAP or ~lk~line phosphatase may also be present.
Another feature of this invention is that a high yield of cell.s S is possible. For example, yields of about 150,000 cells per 150 cm2 tissue culture dish, or approximately 1000 cells/cm2 are common.
To further characterize the precursor o,steoclast cells so produced, various assay,s were performed on the highly enriched population. Since the presence of abundant calcitonin receptors i,s regarded as a distinctive marker of the osteoclast phenotype, the pOC
cells were examined for thi~s property according to known techniques.
The osteocla.st precursors bound a large number of salmon calcitonin (125I-sCT) molecules, a.s visualized by autoradiography, and binding was displaced by a 100 fold excess of unlabeled sCT. However, ,some TRAP+ cell,s did not bind calcitonin.
Exposure to salmon calcitonin (sCT) for 10 minute,s increa,sed cAMP in pOC cells in a do,se-dependent manner, up to 250 fold at 10 nM sCT, while PGE2 had a small ,stimulatory effect and PTH
wa,~ without effect.
The isolated pOC cells, when plated onto 1,25-dihydroxy D3-treated osteobla,st cells fu~;e rapidly (within 24 hour,s) to form multinucleated TRAP+ osteoclast-like cell,s. The pOC cells which are kept frozen in liquid nitrogen and thawed~ retain the capability to form multinucleated TRAP+cells.
The pOC cell,s pos,ses,s other featurels of multinucleated osteoclasts. On slice,s of steer cortical bone, and in the presence of osteoblast,s and Vitamin D3 or a biologically active derivative of Vitamin D3, the pOC cells formed pits after 24 hour.s of incubation and this activity wa,s inhibited by calcitonin. The resorption pits were heterogenou,s in size, varying from single to compo,~ite pit~,. The re~iorption pit,s coincided with inten.se F-actin st;lining. The actin ring.
W 096/32469 PCTrUS~6/01'~1 could be visualized as early as 2 hours after the pOC cells are plated on r bone slices.
In accordance with this invention, the pOC cell.s resorb bone only in the presence of osteoblastic cells and 1,25-dihydroxy vitamin D3. It was also found that the number of pits as well as the number of F-action rings not only correlate with each other, but both increase proportionately when pOC cells are co-cultured with an increasing number of osteoblast cells. There were no F-action rings when pOC cells were cultured in the absence of o,steoblastic cells or a Vitamin D3 derivative.
The pOC cells expre.ss mRNAs for proteins found in multinucleated osteoclasts. For example, high mRNA levels for cxv (7.0 kb) and ,133 (6.5) integrin subunits, calcitonin receptor (4.2 kb), carbonic anhydrase II (1.8 kb) and osteopontin (l.~ kb) can be observed. In addition, they express mRNA for protein tyrosine phosphatase ~ (a major tran,script of about 5 kb and a minor transcript of about 3 kb), OC-2 (l.~ kb), a possible cy,steine proteina.se, and matrix metallo-proteinase 9 or 92 kD type rv collagenase (3.0 kb). All of these mRNAs are known to be highly or preferentially expressed in osteoclast.s. These finding.s illustrate the use of pOC cell.s produced according to thi~ invention for identifying and studying osteoclast-associated proteins.
Isolated osteocla.sts a.s well as multinucleated osteocla~sts derived from the i~1 vitro co-culture.s express high level.s of pp60C-~srC.
pOC cell Iysates immunoprecipitated with the monoclonal antibody mAB 327, which recognize.s C-.SFC 60 kDa protein, and immunoblotted with phosphotyro.sine antibodie.s, show an abundance of phosphorylated pp60C-src The highly enriched population of pOC cell.s can then be re.seeded onto o.steoblalst cell.s in order for the pOC cells to differentiate into multinucleated o.steocla.sts. The osteobla.stic cells may be the same a.s u.sed in the co-culture proces.s, or may be a different o.steoblast line.
CA 022l78l6 l997-l0-08 W O 96/32469 PCTrUS~6/C163 An example of a suitable osteoblast line is the murine MB 1.~ line which has been treated with 1,25-dihydroxy-vitamin D3. Other appropriate lines include: bone marrow stromal lines, primary osteobla.st cells lines from calvaria or any other cell line which supports osteoclast formation. Upon plating, the pOC cells fuse within a short period of time, generally within 24 hours, to form multinucleated, TRAP+
osteoclasts.
Another aspect of this invention is an assay for the bone resorption inhibitory or stimulatory effect of a test substance comprising placing an osteoclast-enriched population of cell,s which al.so comprises osteoblasts on a bone slice, in the presence of Vitamin D3 or a biologically active derivative thereof, and a test substance (which m~y contain a putative inhibitory substance or a putative ,stimulatory substance) and measuring the amount of bone resorption, by, e.g., determining the number of pits formed. The resulting activity can be compared to the activity of a control where no test substance was present. Thus a further aspect of this invention is a method of identifying a drug which inhibits bone resorption comprising placing ;ln osteoclast-enriched population of cells which al.so comprises o~steobla.st~
on a bone slice in the pre.sence of Vitamin D3 or a biologically active derivative thereof, and a putative drug having bone resorption inhibitory activity, and determining whether re~sorption occur~s. Thi~
invention also includes bone resorption-inhibiting drug~; identified by this method.
The following non-limiting Example~s are presented to better illu~strate the invention.
W 096/32469 PCT~US9.r~1C~
Co-Culture And Isolation of Pre-Fusion Cells Mouse bone marrow-osteoblast co-culture system was 5 performed essentially as described by T~k~h~hi et al., 198~
Endocrinology 123:2600-2602, which is hereby incorporated by reference. A cell line (MB 1.8), (Tanaka et al., 1992 J. Bon~ Min. Res.
7:S307) established from neonatal mouse calvaria was found to support bone marrow cell differentiation into osteoclasts. MBl.~ cell~ were plated at 10,000/cm2 in Alpha Modification of ~inim~l Eagle's Medium (a-MEM) con~ining 10% fetal bovine serum andlO nM 1,25(0H)2D3.
(Fetal Bovine Serum was from JRH Biosciences, Lenexa, KS. Other tissue culture reagents, were from Gibco/BRL Life Technologie.s, Inc., Grand I.sland, NY.) Balb/C male mice (six weeks old) were sacrificed under C02; and tibiae and femora were aseptically removed. The bone end~i were cut off with scissors and the marrow cavity was flushed with l ml o~-MEM by using a 27G needle. The bone marrow cell.s were then filtered through 70 mm nylon mesh. Cellls were centrifuged for 7 20 minute~ at 300 x g and washed once with a-MEM and finally re.su~ipended and aliL~uoted at 25,000 cell.s/cm2 onto the MB l .~ culture.~.
Medium wa~ replaced every two day.s.
After 6 days, many mononuclear and some multinuclear TRAP+ cell.s were pre.sent in the,se cultures. Cultures were wa.shed 25 twice with PBS and treated with collagena~;e/dispase (1 mg/ml each in PBS) at 37~C for 20 minutes. (Collagena.se Cat. # 034-10533 wa.~ from Wako Chemical.s USA, Inc., Dalla~;, TX. Dispase Cat. # 165~59 wa~
from Boehringer Mannheim Corp., Indianapoli,s, IN.) Relea.sed cell.~, mo.~tly o~teoblast~ were removed with a pipet and the plate~s were 30 wa.~hed three time~ with PBS. The remaining cell~ were incubated with ~0 nM echi~tatin in a-MEM cont~ining l % BSA for 20 minute.s at ~7~C.
The~e cell.s were collected, pelleted and wa.shed once with cx-MEM
W O 96/32469 PCTrUS96/04634 cont~inin~; 10% FBS and were plated either on bone, coverslips or in wells, as indicated below.
J
Table 1 s Abundance of TRAP+ cells in enriched populations of pOC cells Experiment number TRAP+ cell,s TRAP- Cell~
~29 (94%) 4g (6%) 2 614 (g8%) ~1 (12%) 3 1006 (91%) 102 (9%) Freshly isolated pOCs were plated at 12,500 cell~s/cm2 on 10 .serum coated wells and allowed to attach for 2 hrs. Cells were fixed and stained for TRAP and in some experiments counterstained for alkaline phosphatase as described. Approximately 1,000 cell.~i were counted per well.
Characterization of pOC Cells Measurement of cAMP: O,~;teoclast precur~;or~ were i~iolated as de.scribed in Example 1 and plated at 240,000 cell~ per well in 24 well di~he~ that were precoated with FBS. Cell~; were allowed to attach for 90 minute~i, wa~shed twice with o~-MEM and treated with I
mM i~obutylmethylxanthine (IBMX) for 10 minute.~; at 37~C. Bovine parathyroid hormone (PTH), ~almon calcitonin (~CT), or PGE2 were added at the indicated concentration~; and the culture.~ were incubated for 10 minute~; at 37~C. (Bovine PTH 1-34 wa~; from Bachem, Inc Torrance, CA. Salmon calcitonin wa~ from Penin~;ula Laboratorie~, W 096132469 PCTrUS96/04634 Belmont, CA. The IBMX and TRAP kit were from Sigma, St. Louis, MO).
The medium was aspirated and the cells were extracted three times with absolute ethanol. The extracts were evaporated to 5 dryness and analyzed for cAMP by radioimrnunoassay according to the manufacturer's instructions (Arnersham Corp.).
Table 2 10 Effects of CT, PTH and PGE2 on cAMP accumulation in pOC cell~s.
Additions cAMP (pmol/l 05 cell.s) Experiment 1 none 0.29 + 0.026 .sCT (10- 1 1 M) 6.~i 1 + 1 . 1 4*
sCT(10-~ M) 70.~ + S.l*
Experiment 2 none 0.15 + 0.025 sCT (10-~ M) 36.9 + 2.5*
PTH (10-7 M) 0.19 + 0.027 PGE2 (10-6 M) 0.27 + 0.00~*
Fre.shly isolated pOCs were plated on serum coated 24-well culture dishes at 1'~0,000 cells/cm2 (Exp. 1) or 65.000 cells/cm2 (Exp.
15 2) and allowed to attach for 2 hours. Cultures were washed and pretreated for 10 mins at 37~C with 1 mM IBMX. Cells were then incubated for 10 min.s with sCT, PTH or PGE2 at 37~C. The medium w~s a.spirated and cAMP wa~ extracted 3x with ethanol. The extracts were analyzed for cAMP by radioimmunoa~ssay as de~icribed. Re,sults 20 given in Table 2~ above are the mean + s.d. for triplicate wells. The asterisk signifies that the amount was significantly greater than control group, p<0.001.
~ = ~
W O 96/32469 PCT~US96104634 Calcitonin Autoradio~raphy Isolated osteoclast precursors from Example 1 were plated on coverslips that were precoated with F13S for two hours at 37~C. The cells were incubated for one hour at room temperature in a-MEM
containing 0.1% BSA and 0.3 nM 125I-sCT with or without 100 fold excess unlabeled sCT. (125I-salmon calcitonin (2000 Ci/mmol) was purchased from Amersharn Corp., Arlington Heights, IL.) Cultures were washed five times with ice cold a-MEM, fixed in 10% folmalin for 10 ~ninutes at room temperature and permeabilized with ethanol/acetone (1:1) for 2 minutes and ~tained for TRAP. Coverslips were mounted on slides, dipped in ILFORD K.2 emulsion (Polysciences, Warrington, PA) diluted 2 parts emulsion:l part 6% glycerol, dried and stored at 4~C for 2 week.s. Slide,s were developed in Kodak D-l9 developer diluted 1:1 with water for 5 minutes followed by fixer for 5 minutes, washed with deionized water for 15 minutes and air dried.
RNA I.solation and Northern Blot Analysi.s Total cellular RNA was isolated by guanidinium isothio-cyanate and phenol extraction as described in Chomczynski, P ~t ~
19~7 An~ll. Bioc~h~m. 162:156-1~9, which i~i hereby incorporated by reference. Total RNA (25 ,ug) wa~; electrophore~ied through 1 agaro.se-formaldehyde gels and electroblotted onto nylon filter.s (Hybond-N. Amersham Corp., Arlington Height~i, IL). Blot.~ were prehybridized in buffer cont~ining 50% formamide, 5X SSC (I X SSC =
0.15 M NaCI. 0.015 M sodium citrate), 5X Denhardt'.s .solution and 100 rL~/ml ~onicated .s;al.m.oP. spe.rm~ DNA and hybri~d a~42~~ i]~l fre.~h buffer containing the indicated cDNA~; which were labeled with ~
random primer DNA labeling kit (Pharmacia Biotech Inc., Pi~icat~way, NJ) u.sing [o~-32P-dCTP] (Amer.sham Corp.). Blot~i were wa.shed with 0.1 X SSC/O. 1% SDS at 65~C for 30 minute~ ~nd expo.sed to Kod~k XAR
film. The cDNA clone.~ were generated for MMP-9, OC-2, ~nd W 096/32469 PCTnUS~ q osteopontin by using cDNA libraries of rabbit osteoclasts (Tezuka et al., 1992, Biochem. Biophys. Res. Comm. 186: 911-917, 1994 J. Biol.
Chem 269: 15006-15009; and 1994 J. Biol. Chem. 269: 1106-1109 each of which is hereby incorporated by reference). PTP ~ was cloned from S mouse osteoclastic cells (Schmidt ef al., 1993,J. BoneMin. Res. 8:S144, which is hereby incorporated by reference). The following cDNA
probes were cloned by PCR based on the reported sequences: human av (Suzuki et al., 1987 J. Biol. Chem. 262:14080-14085, which is hereby incorporated by reference; mouse calcitonin receptor (Lin et al., 1991 Science 254:1022-1024, which is hereby incorporated by reference);
and human ~3 (Frachet et al., 1990 Molec. Bio. Rep. 14:27-33, which is hereby incorporated by reference). mAB 327 which recognizes pp60C-~rc was a gift of Dr. J. Brugge (Lipsich, et al., 1983 J. Vi~-ology 4~:352-360, which is hereby incorporated by reference).
Measurement of Bone Resorption Bone ,slices (20 mm2) were prepared from bovine cortical bone by a low speed diamond s~w (Buehler, Lake Bluff, IL). Slices were cleaned by ultrasonication (Branson, Shelton, CT) in distilled water (15 mins) three times, rin,sed in distilled water and placed in 96-well culture plates (Costar Co., Cambridge, MA). The bone ,slices were .sterilized under UV light. Bone .slice~; were rehydrated with medium 199 containing 10% FBS and penicillin/streptomycin solution. A
.suspension (20,000 cells) of the TRAP+ bone marrow cell~s were added to each well with or without MB l .~S cells in the presence or absence lOnM 1,25-dihydroxy-Vitamin D3, or in the presence or absence of MBl .~ cells pre-treated for 4g hours or not with 1,25-dihydroxy Vitamin D3 (1,25(0H2)D3). After 24 hr.s, the bone slices are fixed and st~ined with 1 % toluidine blue as described previou~ly (Dem.ster, et ~
19g7 J. Bone Min. Re~. 2:443-44~; 19~7.). Re~;ult.~ are given in TABLE
3, below.
W O 96/32469 PCTrU~ 631 Table 3 Effects of osteoblastic cells and 1,25-dihydroxy - Vitamin D3 on bone resorption by pOC cells s Culture Conditions Number of pits/bone slice Experiment 1Experiment 2 pOCcells O O
+ 1,25 (0H)2D3 0 + MB 1 .g cells ND
+ MB1.8 cells +1,25(0H)2D3 ND 56 + 20 Pretreated with 1,25(0H)2D3:
+ MB1.8 cells 17g + 59 ==57 + 40 + MB1.~ cells + 1,25(0H)2D3 3~7 + 60 196 + 6 ND = not determined Immunofluore,scence Microscopy Bone .~lices containing the pOC cell~i in the pre.sen-ce or absence of MB 1.~ cells or 1 ,25-dihydroxy Vitamin D3 (under the ~ame 10 conditions as described above for measurement of bone re~orption) were fixed in 3% paraformaldehyde, 2% .~iucrose in PBS for 5 min at room temperature. The cells were permeabilized in 0.5% Triton X-100 for 5 min on ice. F-actin was stained with rhod~mine-conjugated phalloidin (Molecular Probes, Inc.), 5U/ml for 15 min at room 1 5 temperature.
Immunoprecipitation and Immunoblot~i The pOC cells from Example 1 were allowed to attach to dishes for 3 hour.~; and Iysed in RIPA buffer (150 mM NaCl, 10 mM
Tri.<" 1% Triton X-100, 1% deoxycholate, I mM EDTA, I mM ~odium W 096/32469 PCTnUS~J'~
orthov~n~ te, 10 mg/ml leupeptin, 1 TIU/ml aprotinin, and 1 mM
PMSF, pH 7.3) 100 ,uV100,000 cells. The lysate was preincubated with sepharose 4B-200 for 1 hr at 4~C, then divided into 2 aliquots. One was incubated with 2 ,ul pp60C-srC antibody (mAb327) overnight at 4~C, the 5 other was left untreated. The lysates were reacted with goat-anti-mouse sepharose for 1 hr at 4~C, the beads were washed 5 times with RIPA
buffer, then treated with Laemmli sample buffer for 3 minutes at 95~C
and run on a 10% SDS polyacrylamide gel (Laemmli, 1970 Nature 227:680-685, which is hereby incorporated by reference). The proteins 10 were electro-transferred to I~lmobilon P overnight, the membrane was blocked in 100 mM NaCl, 10 mM Tris, 0.1% Tween, 1% BSA, incubated with anti-phophotyrosine antibody hor.seradish peroxidase conjugate RC20H (Transduction Laboratories, Lexington, KY) diluted 1:10,000, washed exten.sively, reacted with ECL reagent.s (Amer.sham) 15 and exposed to XAR5 film (Kodak). Each lane contained protein from 100,000 cells.
METHOD FOR PRODUCING A HIGHLY ENRICHED POPULATION
OF OSTEOCLAST CELLS
DESCRIPTION OF THE INVENTION
This invention relates to a method of producing a population of cells which is highly enriched in it.s content of osteoclast precursor cells, and to the cell populations so produced.
BACKGROUND OF THE INVENTION
O.steocla.sts are termin~lly differentiated cells which play a key role in bone resorption. Due to the low number of m~mm~lian osteoclast~ and the difficulty to isolate them from bone tis~ue, and from other cell.~, their characterization has been limited primarily to immunohi~tochemistry or anatomical and phy~iological mea~;urement.~;
on ~ingle cells.
Several attempts have been made, with limited .~;ucce~, to identify, i~iolate and e~tablish in culture, cell~ capable of difi~'erentiating into o~;teocla~t.s from either bone marrow (Billecoc~l et cll., l9gO P70c .
Nclt. Acacl. Sc i. USA ~7:6470-6474; Prallet et c~l., 1992, J. Bone Mi~?.
Re~. 7:405-414: and Chamber.s etcll., 1993, P70e. Nclt. Acclcl. Sci. USA
90:557~-55~2) or leukemic and promyelocytic cell line.s (Yoneda et cll 1991, Endocrinolo~ 129:683-6~9; Gattei et al., 1992 J. Cell Biol.
116:437-447). Recently, cell~ obtained from hum~n giant cell tumor.~ of bone were reported to forrn osteoclast~ in culture (Grano et cll., 1994 J.
Bone Mi~l. Res. 9:1013-1020; Grano, et cll., 1994 Exp. Cell Re~.
212 :209-21 ~).
To obtain enriched o~iteocl~it preparation~i. Akat~u et cll., 1992, J. Bo~1e Mille7. Res. 7:1297-1306; cultured the o~teobla~tic and bone marrow cell.~ on collagen gel-coated di.~he~. and relea.~ed the cell~
from the collagen matrix u.~ing collagen~ie. However, the yield of o.~teocla~tic cell~ wa~ ~uite low. Recently. Shioi et cll., 1994, Cc~leif:
CA 022l78l6 l997-l0-08 W 096t32469 PCTrUS9f'01~3 Tissue Int. 55:387-394, using a similar co-culture system reported the enrichment of generated osteoclasts by treating the cultured cells with bacterial collagenase to remove the stromal supporting cells. By this procedure they obtained 60% pure tartrate resistant acid phosphate 5 positive (TRAP+) cells, however the osteoclasts were not available except as attached cells to either dishe~ or bone slices.
Oursler et al., 1991, J. Bone Min. Re~. 6:375-3~S5 cultured avian osteoclasts and further purified the osteoclasts by density gradient centrifugation. This method yielded an enriched population of TR~P+
10 cells capable of bone resorption.
To date, however, the only m;~mm~lian osteoclasts obt~ined in high yields (300,000 per rabbit) and purity (9~o) are rabbit osteoclasts (Tezuka etal., 1992,Biochem. B~ophys. Res. Comm. 1~6:
911-917). These cells, however were attached to the plastic culture 15 dishes and cannot be removed.
DESCRIPTION OF THE lNVENTION
This invention relates to a method of obtaining a highly enriched population of osteoclast precur~sor cell~ compri~sing 20 co-cultivation of osteoblastic cells and bone marrow cells and treatment of the culture with an integrin o~v~(33 receptor ligand to rele~e a population which is highly enriched with precursor osteoclasts. Thi.s invention also comprises the process of obtaining a highly enriched population of mature osteoclast cells comprising reseeding the 25 population of highly enriched precursor o~teocla.~t cell.~ so produced on osteoblast cells for a time sufficient for the osteocla~st precursor cell.s to fuse into mature multinucleated olsteoclasts.
This invention also relates to the enriched populations of mammalian precursor o~teoclasts. Yet another a~pect of this invention 30 is ~ population which is highly enriched with mature osteoclasts.
Further aspects of this invention is a suspension culture comprising W 096/32469 PCTrUS~ 1C3 m~mm~ n osteoclast precursor cells, and to suspension cultures comprising mature osteoclast cells.
A further aspect of this invention is an assay for bone resorption activity comprising exposing a population of m~mmalian 5 cells which is highly enriched in osteoclasts, but also comprising osteoblasts, to a bone, in the presence of Vitamin D3 or a biologically active derivative of Vitamin D3 and measuring the bone-resorption which occurs.
In accordance with this invention, it was observed that 10 integrin ocV133 receptor ligands appear to play a role in cell attachment and in the fusion of the precursor osteocla,st,s into multinucleated osteoclastic cells in the bone marrow co-culture system. One aspect of this invention thus u.se,~ (XV,B3 receptor ligands to isolate a highly enriched population of pre-fusion cells. The pre-fusion cells (also 15 referred to as precursor osteoclast,s) can be co-cultured with osteobla.sts ,so that they differentiate into mature o.steoclasts. Thi.s invention also relates to the enriched populations of precursor osteoclasts and mature o,steoclasts produced by the processes described above.
As used throughout the .~ipecification ~nd claim.s, the 20 following definitions will apply:
"~v~3~ receptor ligand" is any molecule which bind.~ to th~
o~V133 receptor. An avl33 receptor ligand can be identified by determining whether it replaces echistatin which ha.s bound to the o~vl33 receptor in a competitive binding a.~ay. Tho~;e compound.~ ~,vhich 25 replace echistatin are considered ocv~133 receptor ligands. Example~ of ocv,133 receptor ligands include RGD-containing pepetides and non-peptides which bind to the .same receptor ~s RGD-peptides.
"highly enriched" mean.~ that ~ population of cell.~ cont~in~;
at least about 75% of a ~;ingle type of cell, more preferably ;~t lea~it 30 about 90%, and more preferably at least about 95'J/~, of a particular cell type.
W 096/32469 PCTrUS~/0 "biologically active derivative of Vitarnin D3" refers to any metabolite of Vitamin D3 which is normally produced by a m~mm~l and contributes to bone metabolism. Examples include 25-hydroxy Vitamin D3, and 1,25-dihydroxy Vitarnin D3.
S "precursor osteoclasts" are pre-fusion osteoclast cells.
Precursor osteoclast cells are characterized by their ability to generate bone-resorbing, giant, multi-nucleated osteoclasts within about 24 hour.s of being plated on osteoblasts, Like mature multi-nucleated osteoclast,s, they may also be defined by the following characteristics: they are TRAP+; have calcitonin (CT) receptors; expres.s ocv~33 integrin; have o,steopontin (OP); carbonic anhydrase II (CAII). The precursor osteoclasts made according to the processes of this invention al,so form pit,s when plated on bone ,slices along with osteobla,st,s in the pre,sence of Vitamin D3 or a biologically active derivative of Vitamin D3.
"suspension culture" means any culture containing precursor o,steoclast,s or mature osteoclast,s which are not attached to the culture vessel.
Any o~v,B3 receptor ligand may be u~ed in accordance with thi,s invention. Known ocv~33 receptor ligand,~ include vitronectin, echi,statin, kir,stin, other ~inake venom,~, other RGD-containing peptide~, and non-peptide,s which bind to ,same receptor as RGD-containing peptide,s. One preferred o~v,133 receptor ligand i.s echi,statin. Echi,~tatin is an ~GD-containing snake venom which inhibit.~ osteoclast attachment and formation (Sato, et al., 1990 J. Cell Bi~ 1713-1723; Tanaka, et al., 1991, J. Bc~ne Min. Re~. 6:S14~ both of which are hereby incorporated by reference.).
One aspect of this invention i,s a convienent procedure to obtain a ,suspen,sion culture of abundant, highly enriched precur,~or osteoclast cells which (except for their mononucleated characteri,stic) have all the characteri~tic,~ of mature multinucleated o,steoclasts. The culture,s of thi,s invention compri~e a population of cell,s, at lea~t about 7:~% of which are precur,~or osteocla~ts. In a preferred embodiment, W 096/32469 PCTrUS~ 3 the population comprises at least about 90% precursor osteoclasts, and in particularly preferred embodiments, the population contains at least about 95% precursor osteoclasts. One important distingllishin~ feature of this invention is that the precursor osteoclasts of the population are in 5 a suspension, i.e. they are not attached to a plastic or glass substrate, and are thus able to be further manipulated by the researcher. This invention thus makes it possible to study the biochemistry of o.steoclast.s, the regulation of genes expressed by these cells and other characteristics .
The culture proce,sses of thi.s invention can be u.sed with any .~pecies of ~nim;~l cells which can form osteoclast.i, and are able to be cultured in vitro. Preferred ~nim~ls include m~mmal~;, particularly those of bovines, rodent~; (such as mou.se and rat), and primate.s (such a~
human and monkey). Particularly preferred are human, and m;lmm~lian .specie~; .such as rat and mouse or other ~nim~ls whose osteocla,sts ~ihare biochemical characteristic,s with human o.steocla~it.s.
Thi.s invention includes the fir~st detailed characterization of the propertie.i of mononuclear cell.~ which can generate multinucleted osteoclast~; within 24 hours in culture. While not wishing to be bound by theory. ~ince the cells of thi~ invention have all the propertie.~; te.~ited so far which are attributed to highly differentiated o.steocla~t.s. it i~
believed that precur.~or o.steocla~t cell.s represent a itage of differentiation in the o.~teocla.st lineage cell~ just prior to fu~;ion, which is the last .step in o.steocla.st differentiation.
In ~ccordance with thi.s invention, bone marrow ~nd o.steoblast cell~i are co-cultured according to known techni~ue.s, and using known and available cell line~. For example. one mou.se o~teobla~t cell line which may be u~ed i.~ MB l.~s (a mou~e c~lvaria-derived o.steobla.~tic cell line), but any other cell line which ~;upport.i o~teoclaitogene~i~i may al~io be employed, including ST-2 (a publicly available line). other calvaria-derived o.~teobla~tic stromal cell line~, or primary o.~teoblaxt cell line~.
W 096/32469 PCTrUS~
Co-culture of bone marrow and osteoblast cells should continue for approximately 6 days (depending on the particular cell lines used) or until the cell lines have yielded the maximum amount of pre-fusion osteoclast cells. However, if cells are cultured too long, 5 multinucleated osteoclasts will forrn which, due to their attachment to the cell culture plate, cannot be used in this invention. Prior to the formation of giant multi-nucleated osteoclasts, the majority of cells of the osteoblast cell line should be detached from the co-cultured bone marrow cells using an enzyme system which disrupts the co-culture, 10 such as dispase/collagenase. The amount,s of enzymes may vary with the specifc enzyme used and the specific activity, but should be sufficient to break up the attachrnent of osteoblasts to precursor osteoclasts.
Generally, approximately 1 mg/ml of each enzyme is sufficient.
Next, the rem~ining cells are treated with the (xv~33 receptor ligand for a time and an amount sufficent to inhibit the formation of multinucleated osteoclastic cells in the co-culture sy~stem a~
well as to inhibit the attachment and function of multi-nucleated o~steoclasts themselves. In a preferred embodiment of this step, the 20 remaining cells were treated with 10 to 50 nM echistatin for at least about 10 minute~s. In a particularly preferred embodiment, approximately 30 nM of echistatin is used for approximately 20 minutes.
Cells isolated from bone marrow co-culture~s by treatment 25 with the oCV~33 receptor ligand attach to coverslips or tissue culture well~
within one hour. Thi~s ilsolated population of cells i~ well-defined:
based on their TRAP+ st;lining and their ability to form mature osteoclasts when ,seeded back on osteobla.sts, the.se cell.s were identified as precursor osteoclasts (pOC cell.~). Seventy-five to ninety per cent of 30 the cells are mononuclear; the rest are multinucleated cells with two to four nuclei. Almo.st all the cells (~-95%) are TRAP+. A few TRAP+
cells, (approximately 2-3%) may still be attached to alkaline CA 022l78l6 l997-l0-08 W 096132469 PCTnU~I0 phosphatase-positive mononuclear cells, presumably osteoblastic cells.
A small number of cells which (approximately 3-4%) do not stain for " either TRAP or ~lk~line phosphatase may also be present.
Another feature of this invention is that a high yield of cell.s S is possible. For example, yields of about 150,000 cells per 150 cm2 tissue culture dish, or approximately 1000 cells/cm2 are common.
To further characterize the precursor o,steoclast cells so produced, various assay,s were performed on the highly enriched population. Since the presence of abundant calcitonin receptors i,s regarded as a distinctive marker of the osteoclast phenotype, the pOC
cells were examined for thi~s property according to known techniques.
The osteocla.st precursors bound a large number of salmon calcitonin (125I-sCT) molecules, a.s visualized by autoradiography, and binding was displaced by a 100 fold excess of unlabeled sCT. However, ,some TRAP+ cell,s did not bind calcitonin.
Exposure to salmon calcitonin (sCT) for 10 minute,s increa,sed cAMP in pOC cells in a do,se-dependent manner, up to 250 fold at 10 nM sCT, while PGE2 had a small ,stimulatory effect and PTH
wa,~ without effect.
The isolated pOC cells, when plated onto 1,25-dihydroxy D3-treated osteobla,st cells fu~;e rapidly (within 24 hour,s) to form multinucleated TRAP+ osteoclast-like cell,s. The pOC cells which are kept frozen in liquid nitrogen and thawed~ retain the capability to form multinucleated TRAP+cells.
The pOC cell,s pos,ses,s other featurels of multinucleated osteoclasts. On slice,s of steer cortical bone, and in the presence of osteoblast,s and Vitamin D3 or a biologically active derivative of Vitamin D3, the pOC cells formed pits after 24 hour.s of incubation and this activity wa,s inhibited by calcitonin. The resorption pits were heterogenou,s in size, varying from single to compo,~ite pit~,. The re~iorption pit,s coincided with inten.se F-actin st;lining. The actin ring.
W 096/32469 PCTrUS~6/01'~1 could be visualized as early as 2 hours after the pOC cells are plated on r bone slices.
In accordance with this invention, the pOC cell.s resorb bone only in the presence of osteoblastic cells and 1,25-dihydroxy vitamin D3. It was also found that the number of pits as well as the number of F-action rings not only correlate with each other, but both increase proportionately when pOC cells are co-cultured with an increasing number of osteoblast cells. There were no F-action rings when pOC cells were cultured in the absence of o,steoblastic cells or a Vitamin D3 derivative.
The pOC cells expre.ss mRNAs for proteins found in multinucleated osteoclasts. For example, high mRNA levels for cxv (7.0 kb) and ,133 (6.5) integrin subunits, calcitonin receptor (4.2 kb), carbonic anhydrase II (1.8 kb) and osteopontin (l.~ kb) can be observed. In addition, they express mRNA for protein tyrosine phosphatase ~ (a major tran,script of about 5 kb and a minor transcript of about 3 kb), OC-2 (l.~ kb), a possible cy,steine proteina.se, and matrix metallo-proteinase 9 or 92 kD type rv collagenase (3.0 kb). All of these mRNAs are known to be highly or preferentially expressed in osteoclast.s. These finding.s illustrate the use of pOC cell.s produced according to thi~ invention for identifying and studying osteoclast-associated proteins.
Isolated osteocla.sts a.s well as multinucleated osteocla~sts derived from the i~1 vitro co-culture.s express high level.s of pp60C-~srC.
pOC cell Iysates immunoprecipitated with the monoclonal antibody mAB 327, which recognize.s C-.SFC 60 kDa protein, and immunoblotted with phosphotyro.sine antibodie.s, show an abundance of phosphorylated pp60C-src The highly enriched population of pOC cell.s can then be re.seeded onto o.steoblalst cell.s in order for the pOC cells to differentiate into multinucleated o.steocla.sts. The osteobla.stic cells may be the same a.s u.sed in the co-culture proces.s, or may be a different o.steoblast line.
CA 022l78l6 l997-l0-08 W O 96/32469 PCTrUS~6/C163 An example of a suitable osteoblast line is the murine MB 1.~ line which has been treated with 1,25-dihydroxy-vitamin D3. Other appropriate lines include: bone marrow stromal lines, primary osteobla.st cells lines from calvaria or any other cell line which supports osteoclast formation. Upon plating, the pOC cells fuse within a short period of time, generally within 24 hours, to form multinucleated, TRAP+
osteoclasts.
Another aspect of this invention is an assay for the bone resorption inhibitory or stimulatory effect of a test substance comprising placing an osteoclast-enriched population of cell,s which al.so comprises osteoblasts on a bone slice, in the presence of Vitamin D3 or a biologically active derivative thereof, and a test substance (which m~y contain a putative inhibitory substance or a putative ,stimulatory substance) and measuring the amount of bone resorption, by, e.g., determining the number of pits formed. The resulting activity can be compared to the activity of a control where no test substance was present. Thus a further aspect of this invention is a method of identifying a drug which inhibits bone resorption comprising placing ;ln osteoclast-enriched population of cells which al.so comprises o~steobla.st~
on a bone slice in the pre.sence of Vitamin D3 or a biologically active derivative thereof, and a putative drug having bone resorption inhibitory activity, and determining whether re~sorption occur~s. Thi~
invention also includes bone resorption-inhibiting drug~; identified by this method.
The following non-limiting Example~s are presented to better illu~strate the invention.
W 096/32469 PCT~US9.r~1C~
Co-Culture And Isolation of Pre-Fusion Cells Mouse bone marrow-osteoblast co-culture system was 5 performed essentially as described by T~k~h~hi et al., 198~
Endocrinology 123:2600-2602, which is hereby incorporated by reference. A cell line (MB 1.8), (Tanaka et al., 1992 J. Bon~ Min. Res.
7:S307) established from neonatal mouse calvaria was found to support bone marrow cell differentiation into osteoclasts. MBl.~ cell~ were plated at 10,000/cm2 in Alpha Modification of ~inim~l Eagle's Medium (a-MEM) con~ining 10% fetal bovine serum andlO nM 1,25(0H)2D3.
(Fetal Bovine Serum was from JRH Biosciences, Lenexa, KS. Other tissue culture reagents, were from Gibco/BRL Life Technologie.s, Inc., Grand I.sland, NY.) Balb/C male mice (six weeks old) were sacrificed under C02; and tibiae and femora were aseptically removed. The bone end~i were cut off with scissors and the marrow cavity was flushed with l ml o~-MEM by using a 27G needle. The bone marrow cell.s were then filtered through 70 mm nylon mesh. Cellls were centrifuged for 7 20 minute~ at 300 x g and washed once with a-MEM and finally re.su~ipended and aliL~uoted at 25,000 cell.s/cm2 onto the MB l .~ culture.~.
Medium wa~ replaced every two day.s.
After 6 days, many mononuclear and some multinuclear TRAP+ cell.s were pre.sent in the,se cultures. Cultures were wa.shed 25 twice with PBS and treated with collagena~;e/dispase (1 mg/ml each in PBS) at 37~C for 20 minutes. (Collagena.se Cat. # 034-10533 wa.~ from Wako Chemical.s USA, Inc., Dalla~;, TX. Dispase Cat. # 165~59 wa~
from Boehringer Mannheim Corp., Indianapoli,s, IN.) Relea.sed cell.~, mo.~tly o~teoblast~ were removed with a pipet and the plate~s were 30 wa.~hed three time~ with PBS. The remaining cell~ were incubated with ~0 nM echi~tatin in a-MEM cont~ining l % BSA for 20 minute.s at ~7~C.
The~e cell.s were collected, pelleted and wa.shed once with cx-MEM
W O 96/32469 PCTrUS96/04634 cont~inin~; 10% FBS and were plated either on bone, coverslips or in wells, as indicated below.
J
Table 1 s Abundance of TRAP+ cells in enriched populations of pOC cells Experiment number TRAP+ cell,s TRAP- Cell~
~29 (94%) 4g (6%) 2 614 (g8%) ~1 (12%) 3 1006 (91%) 102 (9%) Freshly isolated pOCs were plated at 12,500 cell~s/cm2 on 10 .serum coated wells and allowed to attach for 2 hrs. Cells were fixed and stained for TRAP and in some experiments counterstained for alkaline phosphatase as described. Approximately 1,000 cell.~i were counted per well.
Characterization of pOC Cells Measurement of cAMP: O,~;teoclast precur~;or~ were i~iolated as de.scribed in Example 1 and plated at 240,000 cell~ per well in 24 well di~he~ that were precoated with FBS. Cell~; were allowed to attach for 90 minute~i, wa~shed twice with o~-MEM and treated with I
mM i~obutylmethylxanthine (IBMX) for 10 minute.~; at 37~C. Bovine parathyroid hormone (PTH), ~almon calcitonin (~CT), or PGE2 were added at the indicated concentration~; and the culture.~ were incubated for 10 minute~; at 37~C. (Bovine PTH 1-34 wa~; from Bachem, Inc Torrance, CA. Salmon calcitonin wa~ from Penin~;ula Laboratorie~, W 096132469 PCTrUS96/04634 Belmont, CA. The IBMX and TRAP kit were from Sigma, St. Louis, MO).
The medium was aspirated and the cells were extracted three times with absolute ethanol. The extracts were evaporated to 5 dryness and analyzed for cAMP by radioimrnunoassay according to the manufacturer's instructions (Arnersham Corp.).
Table 2 10 Effects of CT, PTH and PGE2 on cAMP accumulation in pOC cell~s.
Additions cAMP (pmol/l 05 cell.s) Experiment 1 none 0.29 + 0.026 .sCT (10- 1 1 M) 6.~i 1 + 1 . 1 4*
sCT(10-~ M) 70.~ + S.l*
Experiment 2 none 0.15 + 0.025 sCT (10-~ M) 36.9 + 2.5*
PTH (10-7 M) 0.19 + 0.027 PGE2 (10-6 M) 0.27 + 0.00~*
Fre.shly isolated pOCs were plated on serum coated 24-well culture dishes at 1'~0,000 cells/cm2 (Exp. 1) or 65.000 cells/cm2 (Exp.
15 2) and allowed to attach for 2 hours. Cultures were washed and pretreated for 10 mins at 37~C with 1 mM IBMX. Cells were then incubated for 10 min.s with sCT, PTH or PGE2 at 37~C. The medium w~s a.spirated and cAMP wa~ extracted 3x with ethanol. The extracts were analyzed for cAMP by radioimmunoa~ssay as de~icribed. Re,sults 20 given in Table 2~ above are the mean + s.d. for triplicate wells. The asterisk signifies that the amount was significantly greater than control group, p<0.001.
~ = ~
W O 96/32469 PCT~US96104634 Calcitonin Autoradio~raphy Isolated osteoclast precursors from Example 1 were plated on coverslips that were precoated with F13S for two hours at 37~C. The cells were incubated for one hour at room temperature in a-MEM
containing 0.1% BSA and 0.3 nM 125I-sCT with or without 100 fold excess unlabeled sCT. (125I-salmon calcitonin (2000 Ci/mmol) was purchased from Amersharn Corp., Arlington Heights, IL.) Cultures were washed five times with ice cold a-MEM, fixed in 10% folmalin for 10 ~ninutes at room temperature and permeabilized with ethanol/acetone (1:1) for 2 minutes and ~tained for TRAP. Coverslips were mounted on slides, dipped in ILFORD K.2 emulsion (Polysciences, Warrington, PA) diluted 2 parts emulsion:l part 6% glycerol, dried and stored at 4~C for 2 week.s. Slide,s were developed in Kodak D-l9 developer diluted 1:1 with water for 5 minutes followed by fixer for 5 minutes, washed with deionized water for 15 minutes and air dried.
RNA I.solation and Northern Blot Analysi.s Total cellular RNA was isolated by guanidinium isothio-cyanate and phenol extraction as described in Chomczynski, P ~t ~
19~7 An~ll. Bioc~h~m. 162:156-1~9, which i~i hereby incorporated by reference. Total RNA (25 ,ug) wa~; electrophore~ied through 1 agaro.se-formaldehyde gels and electroblotted onto nylon filter.s (Hybond-N. Amersham Corp., Arlington Height~i, IL). Blot.~ were prehybridized in buffer cont~ining 50% formamide, 5X SSC (I X SSC =
0.15 M NaCI. 0.015 M sodium citrate), 5X Denhardt'.s .solution and 100 rL~/ml ~onicated .s;al.m.oP. spe.rm~ DNA and hybri~d a~42~~ i]~l fre.~h buffer containing the indicated cDNA~; which were labeled with ~
random primer DNA labeling kit (Pharmacia Biotech Inc., Pi~icat~way, NJ) u.sing [o~-32P-dCTP] (Amer.sham Corp.). Blot~i were wa.shed with 0.1 X SSC/O. 1% SDS at 65~C for 30 minute~ ~nd expo.sed to Kod~k XAR
film. The cDNA clone.~ were generated for MMP-9, OC-2, ~nd W 096/32469 PCTnUS~ q osteopontin by using cDNA libraries of rabbit osteoclasts (Tezuka et al., 1992, Biochem. Biophys. Res. Comm. 186: 911-917, 1994 J. Biol.
Chem 269: 15006-15009; and 1994 J. Biol. Chem. 269: 1106-1109 each of which is hereby incorporated by reference). PTP ~ was cloned from S mouse osteoclastic cells (Schmidt ef al., 1993,J. BoneMin. Res. 8:S144, which is hereby incorporated by reference). The following cDNA
probes were cloned by PCR based on the reported sequences: human av (Suzuki et al., 1987 J. Biol. Chem. 262:14080-14085, which is hereby incorporated by reference; mouse calcitonin receptor (Lin et al., 1991 Science 254:1022-1024, which is hereby incorporated by reference);
and human ~3 (Frachet et al., 1990 Molec. Bio. Rep. 14:27-33, which is hereby incorporated by reference). mAB 327 which recognizes pp60C-~rc was a gift of Dr. J. Brugge (Lipsich, et al., 1983 J. Vi~-ology 4~:352-360, which is hereby incorporated by reference).
Measurement of Bone Resorption Bone ,slices (20 mm2) were prepared from bovine cortical bone by a low speed diamond s~w (Buehler, Lake Bluff, IL). Slices were cleaned by ultrasonication (Branson, Shelton, CT) in distilled water (15 mins) three times, rin,sed in distilled water and placed in 96-well culture plates (Costar Co., Cambridge, MA). The bone ,slices were .sterilized under UV light. Bone .slice~; were rehydrated with medium 199 containing 10% FBS and penicillin/streptomycin solution. A
.suspension (20,000 cells) of the TRAP+ bone marrow cell~s were added to each well with or without MB l .~S cells in the presence or absence lOnM 1,25-dihydroxy-Vitamin D3, or in the presence or absence of MBl .~ cells pre-treated for 4g hours or not with 1,25-dihydroxy Vitamin D3 (1,25(0H2)D3). After 24 hr.s, the bone slices are fixed and st~ined with 1 % toluidine blue as described previou~ly (Dem.ster, et ~
19g7 J. Bone Min. Re~. 2:443-44~; 19~7.). Re~;ult.~ are given in TABLE
3, below.
W O 96/32469 PCTrU~ 631 Table 3 Effects of osteoblastic cells and 1,25-dihydroxy - Vitamin D3 on bone resorption by pOC cells s Culture Conditions Number of pits/bone slice Experiment 1Experiment 2 pOCcells O O
+ 1,25 (0H)2D3 0 + MB 1 .g cells ND
+ MB1.8 cells +1,25(0H)2D3 ND 56 + 20 Pretreated with 1,25(0H)2D3:
+ MB1.8 cells 17g + 59 ==57 + 40 + MB1.~ cells + 1,25(0H)2D3 3~7 + 60 196 + 6 ND = not determined Immunofluore,scence Microscopy Bone .~lices containing the pOC cell~i in the pre.sen-ce or absence of MB 1.~ cells or 1 ,25-dihydroxy Vitamin D3 (under the ~ame 10 conditions as described above for measurement of bone re~orption) were fixed in 3% paraformaldehyde, 2% .~iucrose in PBS for 5 min at room temperature. The cells were permeabilized in 0.5% Triton X-100 for 5 min on ice. F-actin was stained with rhod~mine-conjugated phalloidin (Molecular Probes, Inc.), 5U/ml for 15 min at room 1 5 temperature.
Immunoprecipitation and Immunoblot~i The pOC cells from Example 1 were allowed to attach to dishes for 3 hour.~; and Iysed in RIPA buffer (150 mM NaCl, 10 mM
Tri.<" 1% Triton X-100, 1% deoxycholate, I mM EDTA, I mM ~odium W 096/32469 PCTnUS~J'~
orthov~n~ te, 10 mg/ml leupeptin, 1 TIU/ml aprotinin, and 1 mM
PMSF, pH 7.3) 100 ,uV100,000 cells. The lysate was preincubated with sepharose 4B-200 for 1 hr at 4~C, then divided into 2 aliquots. One was incubated with 2 ,ul pp60C-srC antibody (mAb327) overnight at 4~C, the 5 other was left untreated. The lysates were reacted with goat-anti-mouse sepharose for 1 hr at 4~C, the beads were washed 5 times with RIPA
buffer, then treated with Laemmli sample buffer for 3 minutes at 95~C
and run on a 10% SDS polyacrylamide gel (Laemmli, 1970 Nature 227:680-685, which is hereby incorporated by reference). The proteins 10 were electro-transferred to I~lmobilon P overnight, the membrane was blocked in 100 mM NaCl, 10 mM Tris, 0.1% Tween, 1% BSA, incubated with anti-phophotyrosine antibody hor.seradish peroxidase conjugate RC20H (Transduction Laboratories, Lexington, KY) diluted 1:10,000, washed exten.sively, reacted with ECL reagent.s (Amer.sham) 15 and exposed to XAR5 film (Kodak). Each lane contained protein from 100,000 cells.
Claims (22)
1. A suspension culture comprising a highly enriched population of osteoclast precursor cells.
2. A culture according to Claim 1 which is mammalian.
3. A culture according to Claim 2 which is selected from the group consisting of human, mouse and rat.
4. A suspension culture comprising a population enriched with mature osteoclast cells.
5. A culture according to Claim 4 which is mammalian.
6. A culture according to Claim 5 which is selected from the group consisting of human, mouse and rat.
7. A method of producing a cell population which is enriched in osteoclast precursor cells comprising:
a) co-cultivating bone marrow cells and osteoblastic cells; and b) treating the co-cultivated cells with an av.beta.3 receptor ligand; and recovering the population enriched in osteoclast precursor cells.
a) co-cultivating bone marrow cells and osteoblastic cells; and b) treating the co-cultivated cells with an av.beta.3 receptor ligand; and recovering the population enriched in osteoclast precursor cells.
8. A method according to Claim 7 wherein the cells are mammalian cells.
9. A method according to Claim 8 wherein the .alpha.v.beta.3 receptor ligand is echistatin.
10. A population of cells enriched in osteoclast precursor cells produced by the process comprising:
a) co-cultivating bone marrow cells and osteoblastic cells; and b) treating the co-cultivated cells with an .alpha.v.beta.3 receptor ligand; and recovering the population enriched in osteoclast precursor cells.
a) co-cultivating bone marrow cells and osteoblastic cells; and b) treating the co-cultivated cells with an .alpha.v.beta.3 receptor ligand; and recovering the population enriched in osteoclast precursor cells.
11. A population of cells according to Claim 10 which are mammalian cells.
12. A population of cells according to Claim 11 which are selected from the group consisting of human, mouse and rat cells.
13. A method of producing a cell population which is enriched with mature osteoclast cells comprising:
a) co-cultivating bone marrow cells and osteoblast cells;
b) treating the co-cultivated cells with an .alpha.v.beta.3 receptor ligand to produce a precursor osteoclast enriched population; and c) reseeding the precursor osteoclast enriched population on osteoblastic cells for a time sufficent for mature osteoclast cells to form.
a) co-cultivating bone marrow cells and osteoblast cells;
b) treating the co-cultivated cells with an .alpha.v.beta.3 receptor ligand to produce a precursor osteoclast enriched population; and c) reseeding the precursor osteoclast enriched population on osteoblastic cells for a time sufficent for mature osteoclast cells to form.
14. A method according to Claim 13 wherein the cells are mammalian.
15. A method according to Claim 14 wherein the .alpha.v.beta.3 receptor antagonist is echistatin.
16. A population of cells enriched in osteoclasts produced by the process comprising:
a) co-cultivating bone marrow cells and osteoblast cells;
b) treating the co-cultivated cells with an .alpha.v.beta.3 receptor ligand to produce a precursor osteoclast enriched population; and c) reseeding the precursor osteoclast enriched population on osteoblastic cells for a time sufficent for mature osteoclast cells to form.
a) co-cultivating bone marrow cells and osteoblast cells;
b) treating the co-cultivated cells with an .alpha.v.beta.3 receptor ligand to produce a precursor osteoclast enriched population; and c) reseeding the precursor osteoclast enriched population on osteoblastic cells for a time sufficent for mature osteoclast cells to form.
17. A population of cells according to Claim 16 which are mammalian cells.
18. A population of cells according to Claim 17 which are selected from the group consisting of human, mouse and rat cells.
19. An assay for the bone resorption inhibitory or stimulatory effect of a test substance comprising: placing an osteoclast-enriched population of cells, said population also comprising osteoblasts, on a bone slice in the presence of Vitamin D3 or a biologically active derivative thereof and the test substance, and measuring the amount of bone resorption.
20. An assay according to Claim 19 wherein the amount of bone resorption measured is compared to a result obtained in the absence of a test substance.
21. A method for identifying a drug which inhibits bone resorption comprising: placing an osteoclast-enriched population of cells, said population also comprising osteoblasts on a bone slice in the presence of Vitamin D3 or a biologically active derivative thereof, and a putative drug having bone resorption-inhibitory activity, and determining whether resorption occurs.
22. A bone resorption inhibiting drug identified by the method of Claim 21.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/419,210 US5719058A (en) | 1995-04-10 | 1995-04-10 | Method for producing a highly enriched population of osteoclast cells |
| US08/419,210 | 1995-04-10 |
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| Publication Number | Publication Date |
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| CA2217816A1 true CA2217816A1 (en) | 1996-10-17 |
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| CA002217816A Abandoned CA2217816A1 (en) | 1995-04-10 | 1996-04-04 | Method for producing a highly enriched population of osteoclast cells |
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|---|---|
| US (3) | US5719058A (en) |
| EP (1) | EP0821727A4 (en) |
| CA (1) | CA2217816A1 (en) |
| WO (1) | WO1996032469A1 (en) |
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| US5719058A (en) * | 1995-04-10 | 1998-02-17 | Merck & Co., Inc. | Method for producing a highly enriched population of osteoclast cells |
| US6255112B1 (en) * | 1998-06-08 | 2001-07-03 | Osiris Therapeutics, Inc. | Regulation of hematopoietic stem cell differentiation by the use of human mesenchymal stem cells |
| US6428973B1 (en) * | 1999-02-01 | 2002-08-06 | The Trustees Of The University Of Pennsylvania | Compositions and methods for evaluating bone resorption |
| GB9906233D0 (en) * | 1999-03-19 | 1999-05-12 | Univ Newcastle | Bone formation |
| JP2003507035A (en) * | 1999-08-13 | 2003-02-25 | ユニバーシティー オブ ロチェスター | Ex vivo method for producing functional osteoclasts from bone marrow in a three-dimensional bioreactor |
| EP1259634A4 (en) | 2000-03-02 | 2004-07-07 | Univ Rochester | Ex vivo generation of functional leukemia cells in a three-dimensional bioreactor |
| WO2002097121A1 (en) * | 2001-05-28 | 2002-12-05 | Nordic Bioscience A/S | Method for assaying osteoclast recruitment and resorption |
| US20070065803A1 (en) * | 2003-06-11 | 2007-03-22 | Centre National De La Recherche Scientifique | Patterns for a skeletal system |
| WO2007130619A1 (en) * | 2006-05-03 | 2007-11-15 | The Trustees Of The University Of Pennsylvania | Compositions and methods for regulating osteoclast differentiation, activation and bone resorption |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5073114A (en) * | 1988-02-23 | 1991-12-17 | Detsch Steven G | Bone growing method and composition |
| US5258494A (en) * | 1988-04-08 | 1993-11-02 | Stryker Corporation | Osteogenic proteins |
| US5324819A (en) * | 1988-04-08 | 1994-06-28 | Stryker Corporation | Osteogenic proteins |
| US5399493A (en) * | 1989-06-15 | 1995-03-21 | The Regents Of The University Of Michigan | Methods and compositions for the optimization of human hematopoietic progenitor cell cultures |
| US5437994A (en) * | 1989-06-15 | 1995-08-01 | Regents Of The University Of Michigan | Method for the ex vivo replication of stem cells, for the optimization of hematopoietic progenitor cell cultures, and for increasing the metabolism, GM-CSF secretion and/or IL-6 secretion of human stromal cells |
| US5168050A (en) * | 1990-05-24 | 1992-12-01 | Genentech, Inc. | Mammalian expression of the bone morphogenetic protein-2b using bmp2a/bmp2b fusion |
| US5486359A (en) * | 1990-11-16 | 1996-01-23 | Osiris Therapeutics, Inc. | Human mesenchymal stem cells |
| US5169837A (en) * | 1991-03-28 | 1992-12-08 | Allelix Biopharmaceuticals Inc. | Isolated osteogenic factor |
| US5405772A (en) * | 1993-06-18 | 1995-04-11 | Amgen Inc. | Medium for long-term proliferation and development of cells |
| JPH09502614A (en) * | 1993-09-14 | 1997-03-18 | メルク エンド カンパニー インコーポレーテッド | CDNA encoding a novel human protein tyrosine phosphatase |
| US5972703A (en) * | 1994-08-12 | 1999-10-26 | The Regents Of The University Of Michigan | Bone precursor cells: compositions and methods |
| US5719058A (en) * | 1995-04-10 | 1998-02-17 | Merck & Co., Inc. | Method for producing a highly enriched population of osteoclast cells |
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1995
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1996
- 1996-04-04 US US08/913,988 patent/US6093533A/en not_active Expired - Fee Related
- 1996-04-04 CA CA002217816A patent/CA2217816A1/en not_active Abandoned
- 1996-04-04 EP EP96910739A patent/EP0821727A4/en not_active Withdrawn
- 1996-04-04 WO PCT/US1996/004634 patent/WO1996032469A1/en not_active Application Discontinuation
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| US6093533A (en) | 2000-07-25 |
| WO1996032469A1 (en) | 1996-10-17 |
| US5719058A (en) | 1998-02-17 |
| EP0821727A4 (en) | 2002-08-28 |
| US5856186A (en) | 1999-01-05 |
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