Note: Descriptions are shown in the official language in which they were submitted.
W095/06063 PCT/GB94/01868
2169945
FACTOR FOR FIBROBLAST COLONY FORMING UNIT
This invention relates to colony-forming unit
fibroblast (CFU-F), and the factor which is responsible for
its activity.
The definition of the haemopoietic (blood-forming)
tissues of the bone marrow is relatively thorough and many
of the factors which influence their performance are known
and have been produced in recombinant form over the last 5-
6 years. In the intact animal, and in culture systems
which mimic the whole animal tlong-term bone marrow
cultures), the processes of blood cell formation are
dependent not only upon the blood-forming cells themselves,
but also upon the microenvironment (or stroma) within which
they reside. One cellular component of this complex is a
fibroblast cell, which for the last 20 years has been
studied in primary culture systems in the assay known as
the CFU-F assay. Cells derived from CFU-F may have
multiple roles to play, including (but not limited to) bone
formation. Some fibroblasts may also play a part in tissue
homeostasis and regeneration. Factors influencing their
growth may well also have a role to play not only in bone
formation and remodelling but also in repair and tissue
healing.
The CFU-F assay was first described by Alexander
Friedenstein in the early 1970s and at that time was
applicable only to Guinea pig bone marrow cells. In the
following years the assay has been adapted to suit many
species including rat, mouse, rabbit and human, and remains
the only quantitative assay for any component of the
haemopoietic microenvironment.
The CFU-F assay comprises simply the addition of
freshly isolated bone marrow cells to a tissue culture
flask containing tissue culture medium and (carefully pre-
selected) foetal calf serum (FCS). The number of cells
explanted shows, within certain limits, a linear
correlation with the number of CFU-F colonies formed.
W095/0~63 PCT/GB94/01868
~lGg~4~
Should the number of cells explanted fall below a certain
critical level (and the exact level varies from one species
to another) then no colonies are formed. To regain
linearity the addition of excess sterilized (by
irradiation) bone marrow cells was required. Under these
conditions, which we refer to as "feeder supplemented"
cultures, the assay shows precise and reproducible
- linearity.
A modification introduced by Prof. A.J. Friedenstein
took this observation to its extreme. ("Bone marrow
s~ colony formation requires stimulation by
haemopoietic cells", Friedenstein, A.J., Latzinik, N.V.,
Gorshaya, Y.F., Luria, E.A. and Moskvina, I.L.; Bone and
Mineral 18, 199-213, 1992 and "Haemopoiesis - A Practical
Approach", Editors: Testa, N.G. and Molineux, G., Oxford
University Press, 1992). Bone marrow target cells were
explanted at low numbers, allowed to adhere to the flask
for 2 hours, and then all other cells were removed by
washing. This had the effect of;
- 20 (i) Allowing the adherence of 100% CFU-F.
(ii) No colonies formed since no feeder activity
was present.
(iii) Complete colony formation could be regained
by adding sufficient sterilised bone marrow cells as
feeders.
The present invention describes the purification and
characterisation of CFU-F factor.
Brief descriPtion of the drawinqs
Figure 1: The graph shows the results of an experiment to
investigate the effect of b-FGF on colony formation at low
and high serum concentrations. Adherent cells obtained
from aliquots of 3 X 105 rat femur bone marrow suspension
cells were used as targets. The total culture volume was
5ml and b-FGF was added at concentrations of 5, 10, 15 and
20ng/ml. Flasks were incubated at 37C for 7 days and then
~ wo 9~,0~0-~ ~ 1 B ~ 9 4 5 PCT/GB94/01868
fixed, stained and colonies counted.
Figure 2: The graph shows the results of an experiment to
investigate the effect of PDGF on colony formation at low
and high serum concentrations. Adherent cells obtained
from aliquots of 3 X 105 rat femur bone marrow suspension
cells were used as targets. The total culture volume was
Sml and PDGF was added at concentrations of 5, 10, 15 and
20ng/ml. Flasks were incubated at 37C for 7 days and then
fixed, stained and colonies counted.
Figure 3: The graph shows the effect of adding heparin
(l~g/ml and b-FGF (lOng/ml) to adherent cells in the
presence of 10' irradiated (lSOOcGy,yl3'Cs) rat femur bone
marrow suspension cells. Adherent cells obtained from
aliquots of 105 rat femur bone marrow suspension cells were
used as targets and total assay volume was Sml. Flasks
were incubated at 37C for 7 days and then fixed, stained
and colonies counted. The data are compared to the
positive control (ADS+) i.e. the number of colonies formed
from the adherent cells in the presence of irradiated
feeders only. ADS-refers to the negative control i.e. the
number of colonies formed from adherent cells with no
feeders added.
Figure 4-11: These graphs depict the assay of cell lines
and their conditioned medium (CM) for feeder activity.
Adherent cells obtained for aliquots of 105 rat femur bone
marrow suspension cells were used as targets and total
assay volume was 5ml. All feeder cells were irradiated
with l500cGy (y,13'Cs) and cell numbers quoted on the graphs
refer to total feeder cells added per assay. Conditioned
medium was filtered through 0.2,um Acrodisc filters prior to
use and was added to the assays to the final % quoted.
Flasks were incubated at 37C for 7 days and then fixed,
stained and colonies counted. The data are compared to the
positive control (ADS+) i.e. the number of colonies formed
W095/06063 PCT/GB94/01868
2169945
from the adherent cells in the presence of 10' irradiated
rat femur bone marrow suspension cells. ADS-refers to the
negative control i.e. the number of colonies formed from
adherent cells with no feeders added. The * on certain
graphs indicates that the initial concentration of
irradiated feeders added to the assay was too high causing
a decrease in pH of the growth medium, hence the
concentration of feeders was lowered 10 fold on the second
day of the assay.
Figure 12: This shows the Sephadex G-50 Elution
Profile. A dialysed 416B cell lysate was fractionated by
anion-exchange chromatography on DE-52. The fraction
eluting at 0.3 M sodium chloride was subsequently applied
to a 700 X 26mm column of G-50 pre-equilibrated with 50mM
HEPES, 150mM sodium chloride pH 7.2. The column was then
eluted with the same buffer at 30ml/hour and 85 X 5ml
fractions were collected. These fractions were filtered
through 0.2~um Acrodisc filters and then assayed for feeder
activity. Adherent cells obtained from aliquots of 105 rat
femur bone marrow suspension cells were used as targets and
total assay volume was 5ml. Flasks were incubated at 37C
for 7 days and then fixed, stained and colonies counted.
The data are compared to the positive control (ADS+) i.e.
the number of colonies formed from the adherent cells in
the presence of irradiated feeders only. ADS-refers to the
negative control i.e. the number of colonies formed from
adherent cells with no feeders added. The column had been
calibrated with molecular mass markers to allow an
estimation of molecular mass of eluting components.
Figure 13: This shows the elution profile from heparin-
agarose chromatography of cell lysate or conditioned medium
(CM) obtained from 416B cells.
Figure 14: This shows the 15~ SDS-PAGE gel
fractionation of the 416B cell conditioned medium after
.~ WO 9J/~0~3 PCT/GB94/01868
~1~994~
heparin-agarose fractionation, eluting with 0.4-0.8M NaCl.
Figure 15: This shows the results of the assay involved
in eliminating TGF-B as the CFU-F factor.
Detailed Description of the invention
The assay procedure used in our work was based on the
above described procedure of Friedenstein, as follows:
l. Working quickly to avoid degeneration of the cells,
obtain single cell suspension of rat bone marrow cells.
Using male OBW rats of any age sacrifice and aseptically
remove the femur. Flush out the contents of the femur with
sterile Iscoves medium using gentle passage through syringe
and 21G needle and count the nucleated cells. Use cells
within one hour to ensure a high percentage of viable
cells.
2. Prepare 25cm2 tissue culture flasks (of high quality,
Falcon 3013E or equivalent) containing 105 marrow cells
(sufficient to give rise to approximately 50 colonies) in
Iscove's medium (320 mOsM/kg, prepared using water of high
purity i.e. MilliQ or better) supplemented with 15% FCS.
FCS of suitable quality is difficult to obtain. Around l
in 15-20 batches works. To obtain a batch of suitable
quality screen samples at between 10 and 25% v/v in CFU-F
cultures prepared using unseparated bone marrow cells.
Maintain a concentration of at least 106 bone marrow cells
per ml in these cultures. Gas the flasks with 5% CO2, 5% 2
in N2, place at 37C for 2 hours and leave undisturbed.
This gas mixture is essential if high plating efficiency is
to be attained. Flasks must also be tightly closed to
maintain pH.
3. Pour off supernatant liquid and aspirate residuum (in
definitive experiments wash the flasks gently at this
stage, this is not necessary for routine assays).
4. Replace supernatant with fresh medium and add "feeder"
activity.
5. Normal experimental design includes
WO95/06063 ~16 9 9 4 S PCT/GB94/01868
a) Adherent cells only (zero colonies)
b) Adherent cells plus irradiated marrow cells as
positive control (100% for each specific assay)
c) Complete cells (i.e. non-adherent cells not
removed)
d) Complete cells plus irradiated marrow feeder
cells (to confirm that no potential CFU-F colony formers
are removed in the adherence procedure).
Following standardisation of the assay system as
indicated above, we then set about looking for a suitable
source of the feeder activity. We also had to eliminate
known growth factors as the source of the activity.
Elimination of known qrowth factors
We found that, no known growth factor including FGF,
EGF, PDGF, Interleukins 1-7, G-CSF, GM-CSF, SCF, HGF, MK
and TGF-B show this type of activity.
For example; basic fibroblast growth factor (b-FGF)
was tested at high and low serum concentrations and these
data are shown in Figure l.
Platelet-derived growth factor (PDGF) is synthesised
by both platelets and megakaryocytes and is the most active
known growth stimulating factor for fibroblasts. PDGF was
assayed at low (2%) and high (15%) FCS concentrations.
These data are depicted in Figure 2.
The graphs clearly show that neither growth factor at
either serum co~cPntration can reproduce the activity
observed in the positive controls.
However, it is possible that FGF requires the presence
of heparin to exert its effects. It is known that the
b; nA; ~g of heparin to FGF facilitates the steric
presentation of the facto~ to the cell surface receptor.
This hypothesis was investigated under the conditions of
our assay these data are presented in Figure 3. The graph
shows that when heparin and b-FGF are added to adherent
cells and feeders, the efficiency of colony formation is
lowered, hence the formation of CFU-F under our conditions
W09~,~C~ 3 PCT/GB94/01868
21~3915
is not an FGF/heparin dependent event.
Identification of a Cell Line as a Feeder Source
To facilitate identification and purification of the
factor it was necessary to find a simple and readily
amplified source. Obviously the irradiated bone marrow,
used as our positive control was far too complex a mixture
to ascertain which cells were producing the factor.
The ideal solution to this problem was to find a cell
line which could produce the factor, that was also easily
maintained and could be relied upon as a constant renewable
source.
A number of cell lines available to us were assayed as
feeder sources.
Assays were set up as previously described, using
targets derived from the aliquots of 105 rat bone marrow
suspension, Iscove's medium containing 15~ FCS in Falcon T-
25 flasks containing a total of 5ml. Cells were spun down
at 800g for 8 minutes at 20C, resuspended at suitable
concentrations in Iscove's medium and then irradiated at
1500 cGy (y, l3'Cs).
The conditioned medium from these cells was also
assayed for feeder activity after filtering through 0.2~m
Acrodisc filters.
Cell lines assayed: L1210, TK6, RAJI, KYM, K562, P388,
416B, HOC-8 (data not presented)
These data are shown on Figures 4-11; the positive
control was counted as 100~ and all other results as
fractions thereof. It is clear from figures 4-8, that none
of the irradiated cells or their conditioned medium were a
positive source of the factor.
Figure 9 shows that irradiated P388 cells were found
to be a good source of the factor, giving up to 50% of the
activity of the positive control at a concentration of
2.5xlO5/ml and the CM from these cells a maximum of 4~
activity of the positive control when CM was added to the
assay to a final concentration of 10%.
W095/06063 PCT/GB94/01868
~ 1 6 99 ~ ~
P388
The experiment using irradiated P388 cells (a publicly
available cell line) was repeated and Figure 10 depicts
these data. This suggests that a concentration of lxlOs/ml
feeder cells was most efficient at stimulating CFU-F
formation and these data were found to be reproducible. In
addition CM was concentrated 10 fold by Amicon
Ultrafiltration, in an attempt to concentrate the low
activity observed in the CM, this was unsuccessful.
P388 was easily cultured in Iscove's medium and 10%
horse serum (HS) and hence work began on further
characterization of the factor. Microscopic observation of
the irradiated feeder cells in the assay system suggested
that cell-cell contact between targets and feeders may be
necessary. Feeders were seen to be closely associated with
the CFU-F.
It therefore seemed possible that the factor could be
a peripheral or integral membrane protein. Various buffers
were used to disrupt ionic interactions to remove
peripheral proteins from the P388 cell surface and buffers
containing detergent were used to release the integral
membrane proteins from the lipid bilayer. Subsequent
fractions were assayed, after dialysis to remove the
buffers. Very little activity was observed with the
peripheral proteins fractions, although there was some
activity in the whole membrane and digested membrane
fractions. However, our failure to completely remove the
detergents from the fractions prior to assay lead to
inconsistent data. Solubilized samples of P388 membranes
were also subjected to ion-exchange chromatography (IEC)
and size-exclusion chromato~raphy (SEC). These experiments
were limited in success, as the sensitivity of the assay to
the reagents involved was always a problem.
416B
The cell line 416B was tested as a feeder source in
our assay system, after irradiation at 1500 Rads. The data
_ WO95/06063 ~ 16 ~ 9 ~ S PCT/GB94/01868
for this initial experiment are shown in Figure 11. The
irradiated cells and CM were shown to be equally good
sources, giving 32% of the activity of the positive control
where irradiated cells were added at a concentration of 5
x 105/ml and 10% CM respectively. At a concentration of 5
x 106/ml, irradiated 416B cells were found to give up to 80%
of the activity of the positive control.
The 416B cell line was derived from long term bone
marrow cultures (LTBMC) infected with friend leukaemia
virus (FLV)(Dexter, T.M., Allen T.D. & Scott D., "Isolation
and characterisation of a bipotential haematopoietic cell
line", Nature (1979) 277, 471-474.) The cell line is
growth factor independent and has an activated ras gene.
There is also some evidence for the cells having the
ability to differentiate into megakaryocytes which is of
interest, in view of the data indicating that platelets are
a positive source of the factor. The 416B cell line has
been deposited under the Budapest Treaty provisions with
the ECACC, Porton Down, England, on 24 August 1994, under
the provisional accession number 94082401.
Conditioned Medium
Logistically speaking, it should be much easier to
purify a factor from CM than from a complex mixture of
components e.g. a cell lysate, however we had the
complication of growing the feeder cells in Iscove's medium
and 10% HS. A time course was set up to ~X~m; ne the
production of factor over time in serum free conditions
i.e. Iscove's medium alone and also in phosphate buffered
saline (PBS) - the simplest culture conditions we could
maintain the integrity of the cells in. Fortunately, the
production of factor varied very little between the
Iscove's medium and the PBS. The optimum time for
production of factor was 24 hours. Native polyacrylamide
gel electrophoresis (PAGE) indicated the presence of less
than 25 proteins smaller than lOOkDa, suggesting there
would be relatively few contaminating proteins.
WO95/06063 PCT/GB94/01868
3 ~ $
It was found possible to concentrate the CM by freeze
drying:
Added to assay: lml 0.5ml 0.25ml
PBS CM 50% 50% 13%
X2 conc. PBS CM
~ of positive control 50% 66% 100%
___________________________________________________________
However, freeze drying the PBS CM was found to be a
rate limiting step in the purification procedure and hence
further progress was made with 416B cell lysates.
Attempts `to stimulate the cells to synthesize more
factor into their growth medium using the lectin;
phytohaemaglutinin (PHA) and the phorbol ester; 12-0
tetradecanoylphorbol-13-acetate (TPA) were unsuccessful,
however the CM was still considered to be an important
source of activity.
416B Cell LYsates
Crude cell lysates prepared by osmotic shock were
found to be a positive source of activity. However, the
lysates were seen to display only very low levels of
activity unless fractionated further, probably due to the
presence of some kind of inhibitor.
Lysis Method
i) Cells grown up in Iscove's medium + 10% HS
ii) Spun down at 800g 8 minutes 20C
iii) Cells washed twice in PBS to remove residual HS.
iv) Cells resuspended in lysis buffer A ; lOmM HEPES,
2mM PMSF pH 7.2 and incubated on ice for 1 hour.
v) Cell debris spun down at 4000g 15 minutes 4C.
vi) An equal volume of lysis buffer B: 250mM HEPES,
5mM DTT, 5mM PMSF, 250mM sodium chloride pH 7.2,
was then added to the supernatant.
Cells may be checked for lysis using trypan blue.
WO9~lOGOC~ ~ 16 3 9 4 5 PCT/GB94101868
ChromatoqraPhy
(i) Anion-Exchange (Whatman DE-52)
To lower the salt concentration the cell lysate was
dialysed for 2 hours against 5 litres deionised water, the
water being replaced after the first hour. The dialysed
extract was then pumped onto a pre-equilibrated column (150
x 16mm) of DE-52. The equilibration buffer was lOmM HEPES
pH 7.2. the column was then washed until all unbound
material had been eluted (tested by measuring absorbance at
280mm). Initially the column was eluted with increasing
levels of salt (O.lM, 0.2M, 0.3M, 0.4M, 0.5M, l.OM in the
equilibration buffer). Assay of these fractions after
dialysis (to reduce salt concentration) indicated the
highest level of activity to be in the 0.3M sodium chloride
wash.
Added to assay: lml 0.5ml 0.25ml
___________________________________________________________
0.3M wash 50% 100% 73%
% of positive control
___________________________________________________________
Anion-exchange chromatography proved to a successful
first purification step, as the matrix has a high binding
capacity and large volumes of material can be pumped onto
the column without any detrimental effect on the
separation. This step also serves to concentrate activity,
as the desired fraction can be eluted in a small volume,
hence suitable for a second step such as SEC, where sample
size is important and affects resolution of components.
To PX~i ne whether the activity of the fractions at
this stage could be ~ oved, they were exposed to heat
(100C for 10 mins) or trypsin (2% for 1 hour at 37C)
treatment. Preliminary data at this stage suggest the
factor to be a peptide.
(ii) SEC (Pharmacia, Sephadex G-50)
Sephadex G-50 is a SE matrix which can separate
WO95/06063 ~ 5 PCT/GB94/01868
molecules with masses between 1.5-30kDa i.e. the mass range
that most growth factors fit into. The dialysed 0.3M wash
from the DE-52 column was applied to a pre-equilibrated
column of G-50 (700 x 26mm) and 80 x 5ml fractions were
collected. The equilibration and elution buffer was 50mM
HEPES, 150mM, sodium chloride (to prevent ionic
interactions) pH 7.2.
As this buffer was compatible with the assay system
these fractions could be assayed directly after filter
sterilisation using 0.2,um Acrodisc filters.
The elution profile for this experiment can be seen in
Figure 12. Clearly, most of the activity eluted at
approximately 245ml. This elution volume corresponds to a
molecular mass of less than 15kDa, which is typical of many
growth factors.
(iii) Heparin-agarose
Conditioned medium (CM) was prepared by incubation of
5xlOs/ml 416B in phosphate buffered saline (PBS) for 24
hours at 37C. The CM was filtered (0.2~m) prior to use
and stored at 4C. A cell lysate was prepared as
previously described.
A 2ml column of heparin agarose was prepared from type
I heparin immobilised on agarose (Sigma) and equilibrated
with lOmM HEPES pH 7.2. A 50ml aliquot of the CM was then
applied to the column which was then washed with the
equilibrating buffer. The column was then eluted with
increasing concentrations of NaCl in the HEPES buffer. The
cell lysate was dialysed and chromatographed by the same
method. The resultant fractions were then desalted by
dialysis against dH20 for 2 hours, the water being replaced
after the first hour. All the above procedures were
performed at 4C. The dialysed fractions were then filter
sterilised (0.21um).
The fractions were assayed for stimulatory activity in
the CFU-F assay, using the adherent cell depletion assay
described above. Adherent cells from 105 rat bone marrow
WO 95/06063 ~ 1 6 3 9 4 ~ PCT/GB94/01868
13
( BM ) cells were used as targets and the fractions tested
were added to a final concentration of 20% ( i.e. lml in a
total of 5ml). After 7 days incubation at 37C, the medium
was removed from the flasks, the adherent cells were then
washed with PBS, fixed with methanol and stained with 1%
crystal violet. Colonies of fibroblasts (>50cells) were
then counted.
The heparin agarose elution profile is shown in Figure
13. The number of colonies counted in the positive control
for the assay was counted as 100%.
For PBS CM, the active fractions eluted from the
column between 0. 4M and 1. OM NaCl and 1. 6M. For cell
lysate, the active fractions eluted from the column between
0.4M and l.OM NaCl, 1.2-1.4 and 1.8-2Ø
It appears therefore that there is at least one
heparin binding molecule, secreted into the PBS CM by 416B,
that is capable of stimulating CFU-F colony formation.
(iv) SDS-PAGE
The active fraction eluting off heparin-agarose
between 0.4-0. 8M NaCl was freeze dried and desalted by
dialysis for 2 hours against dH20, the water being replaced
after the first hour. The freeze-dried sample was then
redissolved in SDS sample buffer with the addition of ~-
mercaptoethanol, as a reducing agent, to one half. The
samples were then heated to 95C for 4 minutes. The
samples were then applied to a 4% stacking, 15% resolving
acrylamide gel. Rainbow markers (Amersham) were used as an
estimation of molecular weight. The gel buffer, sample
buffer and electrode buffers were as defined in Schagger &
von Jagow, Analytical Biochemistry, (1987), 166, 368-379.
The gel was then run at 30V for 60 minutes to allow
stacking and then 30mA for separation. The gel was then
fixed and stained as described by Schagger & von Jagow
(supra).
As shown in Figure 14, four bands were observed in the
active~fraction off heparin-agarose, with approximate sizes
W095/06063 PCT/GB94/01868
216~945
14
of 12, 15, 16 and 17 kDa. The gel thus indicates that
there are four low molecular weight peptide components in
the heparin-binding fraction. In hindsight, it is possible
that the reducing agent in lane 5 could have contaminated
the non-reduced sample in lane 4. Therefore, it is
impossible to say whether or not the peptides are naturally
monomeric or subunits of a larger protein. These four
bands are routinely observed in the heparin-binding
fraction.
Heat StabilitY
PBS CM was prepared as described above and was filter
sterilised (0.2~m) into glass universal bottles and then
placed into a boiling water bath for 10 minutes.
CFU-F assays were performed as described above. The
untreated and heat treated CM was added to the CFU-F assay
at the percentages indicated.
%CM COLONY No. IN COLONY No. IN
ASSAYS WITH HEAT ASSAYS WITH
TREATED CM UNTREATED CM
10% 6.5 5
20% 5.0 5
50~ 2.0 3-5
refers to the amoun of CM added to the assay. Values in
the table refer to colony numbers (~50 cells). The
negative control for the assay had a mean number of 1.5
colonies and the positive control 27.5.
It can be concluded that the ability of the PBS CM to
stimulate CFU-F colony formation is not affected by heat
treatment.
Eliminatina Growth Factors
Hepatocyte Growth Factor (HGF)
HGF is a heparin-binding growth factor, eluting at
approximately 0.7-0.8M NaCl. It was necessary therefore to
eliminate it as a component of the 416B PBS CM. The cell
line MDCK is sensitive to HGF and in its absence grow as
_ W095/06063 ~ 1 6 3 9 ~ ~i. ` . PCT/GB94/01868
clumps and in its presence are seen to be much more
dispersed (scattered). The easiest way to eliminate HGF
was to see if the PBS CM could induce this scatter effect
on the MDCK cell line.
In 24 well plates, in a total volume of lml the
following experiments were set up. The growth medium used
was Iscoves and 10% horse serum.
(a) 103 MDCK Alone
(b) 103 MDCK HGF lng/ml
0 ( C ) 103 MDCK 10% PBS CM
(d) 103 MDCK 40~ PBS CM
(e) 103 MDCK 50% PBS CM
The only experiment resulting in the scatter effect
was (b), from which it can be concluded that the PBS CM
does not appear to contain HGF.
Midkine (MK)
MK protein is a pluripotential embryonic stem cell-
derived neuroregulatory factor. It was first described as
a retinoic acid-induced protein in HM-l embryonal carcinoma
cells (Kadomatsu et al, Biochem Biophys Res Commun, (1988),
151, 1312-1318). MK has a 65~ homology with the 18kDa
heparin-binding protein known as HB-GAM (Rauvala et al,
EMBO J, (1989), 8, 2933-2941), pleiotropin (Li et al,
Science, (1990), 250, 1690-1694) and heparin-binding
neurotrophic factor (Bohlen et al, Growth Factors, (1990),
41, 97-107). It too elutes from heparin-agarose at similar
salt concentrations to the active fraction from PBS CM and
has a similar molecular weight. The MK cDNA has been
cloned into the eucaryotic expression vector PXMT2 and this
plasmid was supplied to us (J Heath). Purified recombinant
factor was found to be inactive and therefore the factor
must be assayed in the presence of the producer cells.
The plasmid DNA was electroporated into Cos-7 cells
which were then incubated for 48 hours at 37C. Efficiency
was monitored by a positive control where RSV LUC was
electroporated into the cells and a negative control where
W095/06063 ~ 16 9 9 4 3 PCT/GB94/01868
16
PGL 2 was used. Luciferase levels were estimated after 48
hours. The Cos-7 cells were lysed and 105 rat bone marrow
suspension cells added to the flasks. It was not possible
to transfer adherent cells to the flasks after trypsin
treatment. Iscove's medium containing 15~ foetal calf
serum was then added, the flasks gassed and incubated for
7 days at 37C.
There was no stimulation of CFU-F colony formation
above background level in the presence of MK. We therefore
conclude that MK is not responsible for the stimulation of
CFU-F colony formation.
TGF-~l, TGF-~2 and TGF-~3
TGF-~ is a member of a family of related
multifunctional cytokines abundant in platelets and bone
marrow. TGF-~ is a potent inhibitor of many haemopoietic
cells, with a decreasing effect as cells mature. However,
its presence in both platelets and bone marrow necessitate
its elimination as stimulatory molecule in the CFU-F assay.
The three growth factors were added to adherent cells
in the ansence of irradiated feeders at the following
concentrations; 25, 250, 2500, 25000 pg/ml. Assay
conditions and times were as described above.
The results are shown in Figure 15. In the assay the
negative control had an unusually high number of colonies,
owing to a higher number of cells being added to each
flask; however this was fortuitous as the negative effect
on the colony number could be seen. The dose response seen
was bell-shaped but a consistent inhibition of colony
formation was observed with all three factors at 250pg/ml.
Concludinq comments
The heparin-agarose separation procedure proved to be
reliable and reproducible on CM or cell lysate. Active
fractions from heparin-agarose chromatography can be
further separated by SEC, as previously noted, and if
desired further separated by HPLC and/or SDS-PAGE.
W095/06063 2 ~ 6 9 3 ~ ~ PCT/GB94/01868
Thus, the two-stage purification (anion exchange/SEC
or heparin-agarose/SEC) can be applied to CM or cell lysate
to provide sufficient quantities of factor for further
purification if desired, e.g. using HPLC and/or SDS-PAGE.
The SEC step is optional, but may be preferred in order to
provide an additional purification step to the initial two-
step procedure or to reduce the number of contaminating
proteins going into later purification steps such as SDS-
PAGE or HPLC.
The isolated polypeptides thus obtained can be further
analysed, for example amino acid sequencing. This can be
accomplished in conventional manner, eg after blotting the
SDS-PAGE bands onto nitrocellulose.
The amino acid sequences obtained from the bands can
be compared with databases of protein sequences with a view
to eliminating any irrelevant proteins before investigating
the CFUF properties associated with the protein bands.
The latter investigation may typically involve the
cloning and expression of DNA encoding the polypeptides.
A cDNA library can be prepared from 416B for use in cloning
and expressing DNA encoding the factor.
WO 95/06063 PCT/GB94101868
18
INDICATIONS REIATING TO A DEPOSITED MICROORGANISM
(PCI' Rule 13bis)
A. The i~ a~ionC made below relate to the microorganism referred ~o in the description
on page 9 , line 17 -- ~n
1~. IDENTIFICATION OF DEPOSIT Further deposits are identified on an a~ inn~l sheet O
Name of depositary ti~ - an
EUROPEAN COLLECTION OF ANIMAL CELL CULTURES
Address of depositary ;~ vtin~ (inclulin~ postal code and col~ntry)
E~ ed,- Collection of Animal Cell Cultures
Centre for Applied Microbiolo~y ~ Research
S~lisbl-ry
Wiltshire
SP4 OJG
GB
Date of deposit Accession Number
24 August 1994 416B 9408Z401
C. ADDITIONAL lNDICATIONS (leavt blank if not applicable) This infnrm~jon is continued on an ~ it; - I sheet O
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if~heindicationsarcnotforaadesigna~cdStatcs)
ALL DESIGNATED STATES
E. SEPARATE FURNISIIING OF INDICATIONS (leave blank if not applicable)
Ibe; ~ir-~;n~clistedbelowwillbes~bmit~Pdtothelntema~ionalBureaulater(specifythegcncral,,alurcofthcin~ca~ionscg.~Acccssion
Nvm~r of Dcposit')
For receiving Offce use only For International Bureau use only
E~i lbis sheet was received with tbe international application [~ n~iS sheet was received by the International Bureau on:
Autbonzed officer Authonzed offcer
NATHAN GJNTER
ROOM:- ~'3 E)(T~
Fomm PCIIRO/134 (luly 1992)
