Note: Descriptions are shown in the official language in which they were submitted.
~'0 9~/06178 2 ~ 7 9 Pcrt~lsg1/07l59
~EGA~ARYOCYTE AND PI~l'ELET
GROWTH, PRODUCTION AND COMPOSITION
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of
application serial number 593,688, filed
October 5, 1990.
INTRODUCTION
Technical Field
The field of this invention is growth and
production of hematopoietic cells, more particularly
megakaryocytes and platelets.
Background
Thrombopoiesis is the differentiation and
maturation of megakaryocytes and platelets. Despite
enormous efforts that have been made to understand the
process of thrombopoiesis and the factors involved with
thrombopoiesis, there remains an extraordinary amount of
confusion and uncertainty about the process. Factors
that appear to have effects in vitro seem to have little
or no effect in vivo. An activity called thrombopoietin
has been elusive and has yet to be isolated in pure form
and characterized. Efforts to provide for long-term
culture of megakaryocyte progenitors providing for their
maturation and form~tion of platelets has also been
problematical.
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Thrombopoie~.is is an extremely important process
for the health of individuals. Platelets play a vital
role in a number of protective processes, providing
various factors, participating in clotting, and the
like. There is, therefore, substantial interest in
providing procedures which will aid in the understanding
of thrombopoiesis, as well as providing for long-term
production of megakaryocytes and platelets which may
find application as therapeutic procedures.
Relevant Literature
Volume 15, number 1, 1989 of Blood Cells is
dedicated to megakaryopoiesis. Hill and Levin, Blood
Cells (1989) 15:141_166 describe regulators of
thrombopoiesis. A discussion of the role of heparin
with endothelial cells may be found in Thornton et al.,
Science (1983) 222:623-5. WO 90 US1725 reports a
megakaryocyte growth promoting activator factor protein.
Regulation of megakaryocyte differentiation by thrombin
glycosaminoglycans has been reported.
SUMMARY OF THE INVENTION
Methods are provided for growing megakaryocytes and
platelets in culture for extended periods of time, by
employing media which encourage megakaryocyte progenitor
and megakaryocyte growth. Various sources of
megakaryocyte progenitor cells are employed. A
megakaryocyte growth factor is provided for enhancing
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megakaryocyte progenitor and megakaryocyte growth.
Assay systems are provided for assaying for the effect
of factors on the proliferation and maturation of
megakaryocyte progenitors and megakaryocytes, as well as
the formation of platelets. The megakaryocytes and
platelets find use in the treatment of thrombocytopenia,
identification of megakaryopoiesis activity of compounds
or compositions, and for an understanding of the
mechanism of megakaryopoiesis.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for the
growth of megaXaryocyte progenitors, megakaryocyt s and
platelets, in culture and in vivo. Megakaryocytes can
be grown in the presence of heparin and a megakaryocytic
growth factor or human plasma. In culture, the
megakaryocytes may be maintained for extended periods of
time without replenishment of the progenitor cells from
an exogenous source.
Bioassays are provided for monitoring
megakaryopoiesis and factors influencing
megakaryopoiesis. The megakaryocyte prog~nitors,
megakaryocytes and platelets can be produced by growing
in culture in an appropriate nutrient medium human
progenitor cells under conditions which are not
supportive, preferably are inhibitory, of fibroblast
growth, while inactivating, as appropriate, inhibitors
of megakaryopoiesis, particularly those produced by
~!0 92/0617~ 7 ~ PCT/~IS9l/07l59
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platelets. The source of progenitor cells may be any
convenient source, including bone marrow, fetal liver,
non-adherent cells from bone marrow, fractionated cells,
ieukocytes, e.g., buffy coat free of erythrocytes, CD34+
fraction from bone marrow, low density cells, Class II
HLA positive cells, or the like, so long as early
progenitors are present which are capable of maintaining
production of megakaryocytes.
For any long-term culture, the cell composition
which is employed will desirably include hematopoietic
stem cells. ~he cellular composition may be fetal,
neonate, or adult. While the subject methodology may be
used with any primate hematopoietic cellular composition
comprising stem cells, the methodology desirably finds
use with human cell compositions.
For many purposes, the growth of megakaryocytes,
megakaryocyte progenitors, and platelets is desirable.
Thus, having a system whereby one can grow cells of the
megakaryocytic lineage is extremely important in
providing a source of megakaryocytic cells and
platelets. The following describes the method for
growing cells of the megakaryocytic lineage and
platelets in culture.
For the most part, containers which are used for
the growth of cultures are coated to enhance fibroblast
formation. In accordance with the subject invention,
containers are employed which remain uncoated or have
been treated to avoid encouraging fibroblast formation.
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Containers can be obtained where the containers are free
of coating, either by being commercially available or by
requesting such containers from sources such as Falcon,
Cornins, Ol- ~he like. The containers may include
multiple well plates, roller bottles, fermentors, Petri
dishes or the like.
The medium which is employed may be any convenient
medium providing the required salts, minerals and
nutritional supplements, such as amino acids, glucose
and vitamins. Also, a small amount of a physiologically
acceptable reductant is used such as 2-mercaptoethanol.
The mercaptoethanol will generally be present in from
about 10-4 - 10-6 M, preferably about 10-5 M.
Various additives may be employed to discourage or
inhibit fibroblast formation and proliferation or
preferentially encourage megakaryopoiesis.
Additives which may be employed to discourage
fibroblast formation in the presence of fetal calf serum
include heparin, citrate, or other additives that
prevent aggregation and degranulation of platelets. The
amount of heparin will be at least about 50 ~g/ml,
preferably at least about 100 ~g/ml and usually not
exceed 4 mg/ml, more usually not exceed 2 mg/ml,
preferably being in the range of about 0.5 to 1.5 mg/ml.
The amount of citrate will vary, when present, generally
in the range of about 0.1 to 1%. Other additives will
be used in accordance with their activity and any
adverse effects on the system. The medium generally
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will also have a low level of plasma or serum, human
pla6ma or serum, fetal calf serum or fetal bovine serum,
preferably being about lO~ or less, more preferably
be ~ ny- abOut 5% ~ there generally being at least about l~
in the absence of specific cytokines in a defined
medium. The amount of serum, as well as the other
additives may be optimized, where cells of the
megakaryocytic lineage are encouraged to grow, while
fibroblast growth and proliferation may be inhibited.
Any complex eukaryotic growth media may be used,
which includes MCD3107, IMDM, RPMI, EX-CEL, etc.,
normally enhanced with a low level of serum.
Desirably, factors which may be produced by
platelets and inhibit megakaryopoiesis will be
maintained at a low level. Factors of particular
concern include platelet factor 4 (PF-4) and its
de~radation product thrombospondin. It is found that
heparin is able to bind to pr._4 and diminish its
inhibitory activity against megakaryopoiesis. Thus,
when heparin is maintained in the ~edium, the heparin
prevents the PF-4 and its degradation product from
exerting their inhibitory influence. In the absence of
heparin, other methods may be used for minimizing the
level of PF-4. This can be achieved by continuous
changing of the medium and removing PF-4 and its
degradation products from the medium, the addition of
antibodies to PF-4, dialysis for removal of PF-4, and
the like.
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one may contxol the calcium level and the
calcium/phosphate ratio to enhance the preservation of
platelets which are formed in the culture. Generally
the calcium level should be in the range of about o to
lOmM, whil~ the phosphate concentration will be in the
range of about 0 to lOmM. The level of ADP may also be
controlled by providing for a system to convert ADP to
ATP. A convenient system includes creatinine phosphate
and creatinine phosphate kinase, although other systems
may find use. The concentration of the creatinine
kinase and creatinine kinase phosphate will be
maintained in a range to provide the desired ADP/ATP
levels. These concentrations are not critical and are
intended to be primarily of convenience, enhancing the
productivity of the system. Platelet preservation may
also be achieved with dextrose acid citrate.
Megakaryocytic cell cultures may also be grown in
the substantial absence of serum (s 1%) by employing at
least one growth factor in combination with the subject
megakaryocytic growth factor. Growth factors include
the interleukins IL~ 3, -6 and erythropoietin,
particularly IL-6 and erythropoietin at relatively high
concentrations, usually greater than about 25 ng/ml,
preferably greater than about 50 ng/ml, and may be 100
ng/ml or higher. A rich defined medium should be
employed, having high levels of cofactors, e.g.,
vitamins, and other components, e.g., ~-carotene,
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insulin, etc., such as X-VIV0 and EX-CEL (J.R.
Scientific).
The cell composition obtained in the medium is
nighly enriched for megakaryocyte progenitors,
megakaryocytes and platelets, greater than 10% by
number, usually greater than 50%, of the liquid culture
medium being in the megakaryocyte/platelet dedicated
lineage. Cell compositions in liquid culture can be
obtained having at least 90 weight percent of
megakaryoblastoid cells and megakaryocytes (excluding
platelets in the weight count), particularly
megakaryocytes. Thus, a culture is obtained comprising
cells committed to the megakaryocyte/platelet lineage,
an adherent population of cells which may provide
factors and a conditioned medium supportive of
megakaryopoiesis by virtue of comprising the appropriate
growth factors, agent(s) for stabilizing platelets and,
as appropriate, agent(s) for inhibiting fibroblast
growth and proliferation.
The cells of interest may be isolated from the
culture medium or bone marrow. Cells of interest may
also be available from buffy coat isolated from blood by
using separation techniques associated with surface
membrane protein markers. Cells of interest will
fractionate as CD34+, -41+, -14-, and usually -33+.
These cells will be diploid, as distinguished from the
polyploid mature megakaryocytes. Thus, one can obtain a
highly enriched cellular composition of megakaryocyte
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progenitors by separation using magnetic beads,
employing positive and negative selection, FACS,
affinity columns, and the like. By employlng an
enriched population of megakaryocyte progenitors, more
rapid maturation and production of platelets can be
achieved.
Factor(s) present in the conditioned medium
associated with megakaryopoiesis are found to be stable
for greater than two weeks at 41-C, usually at ambient
temperatures or higher.
In carrying out the production of megakaryocytes
and platelets, usually from about lx105 to lx107 human
bone marrow cells per ml will be seeded, preferably lxlO5
cells per ml, or a unit of leukocytes (buffy coat) from
an adult, containing about 2 x lo8 white cells and freed
of erythrocytes. Conventional physiologic conditions
will be employed for growing human cells in culture,
namely about 37 C, in a humidified chamber, with air
having about 5~ carbon dioxide, where the oxygen will be
at least about 5% usually at least about 20%. Since
higher oxygen levels are reported to inhibit fibroblast
growth, high oxygen levels, usually not exceeding 90%
may be employed, depending on the other conditions used
for the growth of the cells. Conveniently, wells of 6 -
lO cm or 96 well plates may be employed, which allowsfor the growth Oc the adherent cells to the surface.
Within about 7 days, megakaryocytic cells can `oe
observed and the culture may be continued for six months
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W092/06178 2 ~ ~ 9 ~ 7 ~ PCT/~S91/071~9 ~
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or more. Unless dialysis is used, the media is changed
on a regular basis, generally at least about twice
weekly, preferably at least about every 2 days. One
observes islands of cells, with adherent cells set down,
which differentiate to mature megakaryocytes, observed
as multi-lobular cells. An adherent layer is observed
which is a combination of megakaryocytes and adherent
(stromal) cells. Islands of cells appear on the
adherent layer which have the characteristic CD4l marker
for megakaryocytes but are mononucleated, supporting the
cells being megakaryocyte progenitors (megakaryoblasts).
The progenitor cells will also carry the CD34 marker.
In the medium and associated with the adherent layer
will be platelets. The megakaryocyte cells are shown to
have the surface protein markers CD4l (IIb/IIIa)
(integrin) and CD42 (Ib), and when the cells are stained
with Wright-Giemsa, show the multi-lobular or polyploid
characteristic of megakaryocytes. The progenitor cells,
by comparison, will carry the CD41 and CD~4 markers and
be mononucleated.
Megakaryocytic cells may be further distinguished
by staining with Megacolor (Cytocolor, Inc., Hinckley,
OH). In megakaryocytes, the cytoplasm stains intensely
purple. Also, as evidenced by scanning electron
microscope, a majority of the cells in the culture are
actively budding platelets.
Alternatively, the level of src protein expression
and its kinase activity may be used as a measure of
W0 92/()617~ 3 ~ ~ ~ 9 PCI/US91/071~9
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megakaryocytic cell growth, measuring megakaryocyte
progenitors, megakaryocytes and platelets. The assay
can be carried out with cellular lysates of relatively
homogeneous or heterogeneous phenotype or populations
selected by one or more markers, isolating the src
~inase, e.g. by means of specific antibodies, and then
measuring src kinase activity under conditions
supportive of src kinase catalyzed phosphorylation. To
remove non-specific proteins from the isolated src
lo kinase, one or more washes may be employed. 8y using a
labeled nucleotide triphosphate, e.g. radioisotope
labeled, one can determine the level of phosphorylation
as indicative of the presence of cells associated with
megakaryopoiesis. Alternatively, Western blotting or
SRC ELISA can be used to ~uantitzte src protein.
As the cultures mature, the concentration of
platelets continues to increase, 60 that after about one
week, the concentration of platelets is ~105 platelets
per ml, and may be maintained at about 105-107
platelates/ml. The level of platelet production may be
maintained by the continuous growth of the culture and
maintenance of the medium at the appropriate component
composition, and may be increased by concentration of
the platelets using various techniques for removing
fluid.
The cells may be harvested by separating the
adherent layer mechanically, using chelating agents,
e.g., EDTA, sonication, enzymes, e.g., proteases, such
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as trypsin, collagenase, plasmin, etc., or the like.
The particular manner employed is not critical to this
invention. The megakaryoblasts and megakaryocytes may
then be isoiated using various techniques, such as
cytospin, FACS, affinity separation, density separation,
magnetic bead separation, and the like. As a result, a
substantially pure cellular composition can be obtained
of cells dedicated to megakaryopoiesis, namely dedicated
megakaryocyte progenitors, megakaryocytes and platelets.
The composition will be free of components of peripheral
blood, which might be associated with isolation of such
cellular composition from bone marrow or blood. In
addition, each of the types of cells may be isolated in
substantially pure form, namely, megakaryoblasts,
megakaryocytes and platelets, generally comprising at
least 75~ by number of the same cel~ular type, usually
at least about 90% of the same cellular type and up to
and including about 100%.
The conditioned medium, or human plasma, may be
used for isolation of the factors associated with
megakaryopoiesis. Fractionating ammonium sulfate
precipitation is used to enrich protein fractions for
growth factors. Further enrichment is achieved using
gel permeation to separate different size fractions,
followed by assaying for the enriched fractions. The
enriched fractions are then further enriched using rp-
HPLC, eluting with an agueous acidic medium against n-
propanol or acetonitrile, e.g., 0.1% trifluoroacetic
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acid, or lM acetic acid.
Purification of the megakaryocyte activity may be
further achieved by HPLC using a cationic column, where
the column buffer is at a Ph o~ about 7.5 to 9, and the
sample is eluted with a high ionic strength eluent, e.g.
2 ~ inorganic salt, e.g. NaCl, using a linear gradient
in the column buffer. The activity is obser~ed in the
fractions of the tail end of the highest peak when using
a linear gradient, yenerally eluting in the ~ractions of
about 1.25 to 1.6 M salt.
The fractions are bioassayed and enriched fractions
may be further purified by gel electrophoresis or other
separation technique.
A megakaryocytic growth factor can be obtained by
fractionating medium from the culture or human plasma
through a heparin column. The factor is characterized
by binding to heparin, capable of being transferred from
heparin-Sepharose to heparin in a conventional co~plex
eukaryotic growth media, with heparin at a concentration
of at least about 1 mg/ml, preferably at least about 3
mg/ml, and at 4-C. The ratio of heparin-Sepharose beads
to medium will generally be at least about 1 g:2 ml,
preferably 5 ml, and not more than about 1 g:lO ~1. The
factor is further characterized by supporting cell
growth and proliferation of cells of the megakaryocytic
lineage in the culture system as described above, while
its absence results in the substantial absence of
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prollferation and -maturation of cells of the
megakaryocytic lineage.
As measured in solution, the molecular weight of
the factor appears to be greater than about 40kD. It
also is stable at 56 C. in whole plasma. It also
appears to be species specific, not being effective with
~ouse cells under comparable conditions to being
effective with human cells. Finally, it does not bind
Affi-gel blue CM.
The subject factor may be further purified by
conventional techniques as described above to provide
for l,000-fold or greater enhancement in concentration
and specific activity.
In order to evaluate the response of megakaryocyte
progenitors to various factors, the factors may be
evaluated using a standard semi-solid medium assay
composition, e.g., methylcellulose assay composition
comprising factors which support megakaryocyte growth.
The methylcellulose which is employed will generally be
at least about 1.5% and not more than about 3% v/v in
water. The cellular source may be the same as that used
for the culture. Also included in the culture will be
at least about 15% fetal calf serum, fetal bovine serum,
or human plasma, preferably about 20~. A convenient
volume for carrying out the assay is to employ about 200
~1 in a 24-well plate.
To support growth of the megakaryocytes, high
concentrations of the cytokines I~ 3 and -6, as well
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WO92/06178 ~ ~ ~fJ~ 7 9 PCT/~S91/07159
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as erythropoietin are included, generally at least at
about 0.5 times saturation or higher. Other factors may
be included such as sl or steel factor at comparable
concentration. By saturation it is intended that a
further increase in concentration does not have a
significant effect on the number of colonies of cells
which are produced. Conveniently, about 100 ng/ml of
each of the factors may be employed.
The cells which are seeded will include CD41+,
CD34+ cells, which cells serve as megakaryocyte
progenitors. The cells may be whole bone marrow or an
enriched fraction comprising CD41+, CD34+ cell6. The
mature cells in the colonies which form will, for the
most part, be free of the CD34 marker and will normally
have the CD33 marker, as well as having the morphology
of the mature megakaryocyte.
A factor or composition of interest may be studied
by adding such factor or composition to the medium with
all of the above-indicated factors or where one or more
of the above-indicated factors are lacking. Thus, one
may determine the inherent capability of a composition
in conjunction with fetal calf serum to stimulate
megakaryocyte growth or its ability to interact with
other factors or cytokines to provide for megakaryocyte
growth. The assay is carried out for at least about 5,
usually at least 7 days, usually not more tha~ about 21
days, preferably from about 7 to 14 days. The
conditions for the assay~are that the liquid medium or
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methylcellulose is maintained at 37-C, 5% C02 in a
humidified chamber.
By employing various dosages of a composition or
factor, one can determine the activity of such
composition or factor. In this manner, rapid screening
can be carried out to determine whether a particular
composition or factor is useful for the growth of
megakaryocytes or the production of platelets.
The megakaryocyte progenitors and/or megakaryocytes
may be used for the treatment of thrombocytopenia by
themselves or in conjunction with the infusion of
platelets. In many instances, such as surgery,
chemotherapy, etc., autologous megakaryocytic cells may
be expanded in culture and returned *o the host to
minimize bleeding or other e~fect of the treatment which
megakaryocytes and/or platelsts may address. The cells
may be infused in a Faysiologically acceptable medium,
e.g., saline, generally being present at a concentration
of at least about 105 cells/ml, usually in the range of
about l05 - lo8 cells/ml. The dosage will generally vary
in the range of about l06 _ l08 cells/kg of host.
Usually, the composition will be at least 25%, ~ore
usually at least 50% and frequently at least 90% by
weight of megakaryocyte progenitors and megakaryocytes.
One or more infusions may be necessary, as required.
Platelets may be harvested and administered in
accordance with conventional ways.
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The megakaryocytic cells may be used for gene
therapy, where the cells may serve to maintain function
under conditions where normal megakaryocytic cells may
be adverseiy affected or tO provide a function normally
unavailable from megakaryocytic cells. For example, the
multiple drug resistance (mdr) phenotype may be
introduced into the cells, which would make them
resistant to chemotherapeutic agents, allowing for the
continued normal megakaryocytic response during the
chemotherapy treatment. Genes encoding growth factors
may be introduced, which would provide an enhanced
source of growth factors in the bone marrow, where a
deficiency might exist during a particular episode or
one wished to enhance the growth of megakaryocytes or
other hematopoietic cells. Various methods are known
for providing transformation of cells, providing for
stable extrachromosomal elements or homologous or random
integration. Viral vectors incapable of replication
have been described in the literature, which can be used
for infection, transfection and integration. By being
able to grow megakaryocytic cells in culture, one has
the opportunity to transform such cells and expand and
clone desirable transformants.
The following examples are offered by way of
illustration and not by way of limitation.
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EXPERI~'ENTAL
Example 1: Heparin containing fetal bone marrow
cultures.
~. Cells: ~265 fetal bone marrow, 2x.
Media: IMDM + 5% Gibco FCS + 10-sM
2-mercaptoethanol. The FCS and 2-mercaptoethanol were
added fresh each media change.
Heparin: Sigma 178 U/ng, stock solution 200 mg/ml.
Plate: 100 mm Falcon plate.
Plate 1: No added heparin.
Plate 2: 1000 ~g/ml heparin sigma concentration.
Both plates were tended to by removal of 10 ml
medium and addition of media plus heparin approximately
every day, occasionally every other day. By day 10 a
noticeable difference in the predominant cell morphology
was evident. Photomicrographs were taken evidencing the
morphology. Plate 1 had sheets of fibroblast-like cells
which would form large balls of cells often dislodging
from the plate. Plate 2 had confluent layers of rounder
cells with a different morphology. No sheets of
fibroblasts were observed.
B. The above study was repeated except that 5% normal
human plasma was used in place of FCS. In both plates
egg-shaped morphology indicative of megakaryocyte type
cells was observed, indicating that normal human plasma
could be used in place of FCS and that the effect of the
heparin was related to the FCS.
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Example 2: High heparin concentration effect on
fetal bone marrow cultures.
Bones: K266 and ~267 samples were stripped of
protein by cutting along the long axis in one half,
incubating at 37C with collagenase or trypsin and every
0.5 hr, mixing for 3 hours followed by three washes with
media. Cell concentration was 2xlO~ per ml.
Plates: 100 mm Falcon plate.
Medium: IMDM plus 5% FCS plus 10-5M 2-ME.
Heparin: When used, 1000 ~g/ml (Sigma) diluted in
sterile water.
Four plates were prepared, two each of K266 and
K267, each pair including a plate with heparin and a
control.
As observed in the previous experiment, the plates
without heparin became overgrown in ten days to two
weeks with elongated spindle-shaped cells. The plates
with added heparin show the growth of the "egg-shaped"
cells seen previously. These cultures were tended as
previously described with change of ~edia approximately
QOD.
Example 3: The effect of different plates on
megakaryocyte and fibroblast growth.
The procedure described in Examples 1 and 2 was
employed, using the cell preparation K275. Four plates
were prepared, two plates from Corning and two plates
from Falcon, where each pair included a control and a
heparin-containing medium.
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The cultures were tended in the same way as
previously described.
The Corning control plate showed a rapid growth of
adherent cells with a fibroblast morphology. Within 7
to l0 days, the culture was overgrown in areas and the
cells peeled away from the plate. Often, balls of these
cells were found floating in the medium. The medium
became yellow rapidly despite frequent changes. In the
corning heparin-containing medium, a mixed pattern of
growth was observed with both fibroblast-like cells and
cells with the "egg-shaped" morphology. The growth of
the fibroblast-like cells was very significantly reduced
and the overall cellularity of the culture was reduced
compared to the control plate. At about 2-3 weeks, the
fibroblast-like cells predominated in the culture and
the number of egg-shaped cells was reduced. Overall,
the non-adherent cell number was minimal. --
In the Falcon plates, in the control plate, there
was a rapid overgrowth of fibroblast-like cells. These
cells did not come to confluence as rapidly as in the ~ -
Corning control plate and there was a greater number of
"egg-shaped" cells than in either of the Corning plates.
These cells form clusters in an area which seemed to
exclude the fibroblast-like cells. Finally, in the
Falcon plate with heparin-containing medium, growth to
confluence of the egg-shaped celIs was observed with
eventual formation of multinucleated cells. An adherent
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WO92/0617~ ~ ~/3~ 9 PCT/US91/07159
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layer of megakaryocytes and stromal cells formed
supporting colonies with megablastoid cells.
~xample 4: Fluorescence-activated cell sorter
scan.
Cells from the K266 and K267 cultures were
collected by gentle pipetting, containing about 105
cells/ml. The antibodies used were anti-CD7 and anti-
CD42 in 0.1 PBS plus 2% FCS. The cell~ were incubated
for 20 min on ice with 20 ~1 of anti~ody and then washed
lo through FCS. FITC labeled goat anti-mouse Ig diluted
1:100 (20 ~l/sample) was incubated on ice with the
mixture described above for 20 min, followed by washing
the cells through FCS. The cells were then analyzed on
a FACScan. The scan was ungated, standard threshold 52
(arbitrary unites) and the amplification was 1. Culture
K266 showed 2 sharp peaks where the CD7 peak was
substantially displaced from the CD42 peak. A similar
result was obtained with K267. By comparison, a control
showed no significant increase in fluorescence intensity
obtained over no added antibody (autofluorescence).
_xamPle 5: Methylcellulose assay.
In the next study, a methylcellulose assay was
carried out with conventional conditions, employing 20%
fetal calf serum and 100 ng/ml each of IL-l, -3 and -6
and erythropoietin. Whole bone marrow was employed at
different seeding levels as well as selected cells based
on CD34 and CD41. The CD34+ CD41+ is between 0.5 and
0.9% of the whole bone marrow population.
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WO 92/0617X 2 ~ ) 7~ PCr/US91/071~9
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I
The followin~ table indicates the results:
CELLS CELL COLONIES CLllSTERS
WBM CD34 CD41 CONC.NO.* /105
- - lxlO4ml 0-1-3
- + - lxlO4ml 1-2-0
- + + lxlO4ml 10-7-19 4-5-7
- - - lxlO4ml 0-0-1
-- -- + lx104~n1 0--1--O
+ - - 2xlO4ml 0-1-5 2-3-2
- + - 2xlO4ml 1-2-1 1-1-1
- ~ + 2xlO4ml 34-40-40 9-8-12
- - - 2xlO4ml 0-0-0
- - + 2xlO4ml 0-0-3 .
+ - - lxlO5ml 15-16-15 3-9_7
15 * These replicate colonies comprised 2 50 cells, while
clusters comprise 220 cells.
The colonies comprised of morphologically
identif iable megakaryocytes . Thus, colonies were
observed at the lower concentration from the CD34+ CD41+
2 0 cells which matured to megakaryocytes . In addition,
when whole bone marrow was employed at a seeding of 105
per ml of cells, a significant number of cc~lonies was
observed which provided megakaryocytes. In addition,
the CD34+ CD41~ population was found to be sensitive to
25 the indicated factors, so that by removing one faotor
and replacing such factor with a test composition, one
can determine the effect of such test factor on the
formation of megakaryocytes.
ExamPle ~: Growth of megakaryocytes from buffy
3 0 coat .
WO92/0617R ~ g ~ PCT/US91/07159
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One unit of leukocytes (buffy coat) from an adult
human contains about 2 x l0~ white cells. The red cells
in the unit are lysed by a l0 min incubation at 37C in
a solution of ammonium chloride buffered with Tris-HCl.
The leukocytes were then plated under standard heparin
culture conditions as described in Example l at 107
cells/l0 cm plate, 5 x l0~ cells/6 cm plate, or 105
cells/well in a microtiter plate in a volume of l0 ml, 5
ml, or l00 ~l, approximately 20 l0 cm plates can be
made.
After 1-2 weeks following the regimen described in
Example l, a confluent monolayer of megakaryocytes and
precursors is obtained. The majority of other cell
types are no longer present. If one assumes that the
original inoculum contains 0.1% megakaryocyte
precursors, then this preparation has been expanded 103-
fold, since a confluent l0 cm dish contains 107 cells.
Alternati~ely, aliquots of 1-2 x 107 fresh
leukocytes can be frozen in liquid nitrogen in 10% DMSO
and 20% FCS. These vials can be thawed at a later time
for use in heparin cultures as described above.
~ xample 7: Megakaryocyte assay by measuring src
protein by ELISA.
Megakaryocyte cultures are est blished with 8x104
cells in 96 well plates. Each growth factor source is
tested in quadruplicate ~4 wells). After l0 days o~
growth the medium is removed from the wells and the
cultures are air dried overnight.
.
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WO92/0617~ 2 ~ ~ ~ 3 7 ~ PCT/US91/071~ ~
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The src ELISA is performed by rehydrating the cells
in PBS for s minutes. The src antibody (~-17) (mouse
monoclonal) is diluted 1:800 in PBS + 1% BSA. 40 ~l are
added ~o each well and incubated 30 minutes. Tne wells
are washed x 3 with PBS. The second antibody, goat
mouse Ig (alkaline phosphatase conjugated) (Caltag) is
diluted l:250 in PBS + 1% BSA. 40 ~l are added to each
well and incubated 30 minutes. The wells are washed x 3
with PBS. One tablet of p-nitrophenylphosphate is
dissolved in 5 ml of diethavolamine buffer (Sigma) and
l00 ~l are added to each well. The plate is developed
for 30 minutes in the dark and read at 405 nm.
Exam~le 8: Isolation of megakaryocyte growth
factor.
5 g of heparin-Sepharose CL-6B (Pharmacia) were
swollen in PBS to a final volume of 25 ml, followed by
washing in l liter of P~S. Fresh human plasma (25 ml)
pooled from three donors was added to the swollen beads
in a 50 ml tube and mixed at 4-C overnight. The plasma
was removed and filtered (referred to as "depleted").
Beads were then mixed for 5 h with ~5 ml of IMDM
containing 3 mg/ml heparin at 4~C. The medium was
decanted and filtered (referred to as "eluate").
Heparin cultures were then set up as described in
Examples l and 6 with either bone marrow or buffy coat,
where the various forms of plasma: (l) plasma (10%) used
without treatment; (2) depleted plasma; or (3) depleted
plasma plus the eluate (30% final volume), were used in
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WO92/0~17X ~ PCT/US91/071~9
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place of the fetal calf serum described in Example 1.
Src ~inase assay was performed on cultures grow~ 2 wks.
Total protein in the dish was measured: CPM in src band
was determlned after excising band rrom the gei anà
5 scintillation counting. The results are as follows:
Lysate
CPM in src Protein ~E~
Source* band mq/ml ~rotein
1) HL-60 60 0.80 1%
2) HE~ 837 1.00 13%
3) HC, 10% 4,860 0.73 100%
HP
4) HC, 10% 316 0.25 19%
HeSE
5) ~C, 10% 3,090 0.50 93%
HeSe+F
* HL-60 control line; myeloid leukemia
HEL human erythroleukemia line
HC heparin culture
HP human plasma
HeSE depleted plasma from heparin-Sepharose column
F megakaryocyte growth factor eluted from heparin-
Sepharose column (eluate)
+ Expressed as percentage of culture #3.
Example 9: Purification of megakaryocyte growth factor.
25 Human plasma protein (0.65g) was placed on a 50mm x
150mm DEA~-cellofine column prepared with buffer A (lOmM
Tris-XCl pH 8.0, 0.1 mM EDTA) and eluted with a linear
gradient of buffer A and 2 M NaCl in buffer A. The flow
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Wo92/o6l78 ~ ~ ~ 3 (3 ~ 9 -26- PCT/US91/0715
rate was 2ml/min and 5ml fractions were collected. The
bioactivity was found to be in the higher salt
concentration or downside of the major peak during
elution with the salt gradient, the major portion of the
activity being in the fractions from about l.25 to l.6 M
NaCl.
The fractions may be assayed in a 96 titer well
plate, with 4 replicates. The fractions are dialyzed
against PBS. The medium comprising 5-10% of the
fractions to be assayed in X-Vivo (Whittaker), lmg/ml
heparin, are seeded with 8 x 104 cells/well and fed every
three days. After two weeks the cells are inspected for
megakaryocyte morphology and staining with Megacolor.
It is evident from the above results, that an
efficient economic procedure has been provided for
producing human me~akaryocytes in culture. Thus, a
continuous source of platelets is provided for treatment
and study of the function of platelets, as well as their
formation. Also, megakaryopoiesis may be studied. In
addition, a factor associated with megakaryopoiesis is
provided and other factors may be similarly isolated.
All publications and patent applications mentioned
in this specification are indicative of the level of
skill of thc ~ skilled in the art to which this
invention pertains. All publications and patent
applications are herein incorporated by reference to the
same extent as if each individual publication or patent
. : ' , ' ~ ' . ' :: ' . .
. . ;,' ~ , , ,, -
WO92~0617X ~ PCT/US91/~7159
-27-
application was specifically and individually indicated
to be incorporated by reference.
The invention now being fully described, it will be
apparent tO one of ordinary skill in the art that many
changes and modifications can be made thereto without
departing from the spirit or scope of the appended
claims.
, . ,, , ~ .
