DESCRIPTION
Title of the Invention: METHOD FOR PRODUCING POLypLOIDIZED
MEGAKARYOCYTE AND.PLATELETS
5
Technical Field
[00011 The present invention relates to a method for
efficiently polyploidizing megakaryocytes before
polyploidization, a method for producing platelets from
10 such megakaryocytes, and the like.
Background Art
[00021 Large numbers of blood cells are required for the
treatment of blood-related diseases or surgical treatments.
15 Among blood cells. a platelet which is a cell indispensable
for blood coagulation and hemostasis is one of particularly
important blood cells. The platelet is in high demand in
leukemia, bone marrow transplantation, anticancer treatment,
and the like so that necessity of stable supply of it is
20 high. The platelet has so far been supplied stably by, as
well as a method of collecting it from the blood donated by
donors. a method of administering a drug having a TPO-like
(mimetic) structure, a method of differentiating
megakaryocytes from the cord blood or bone marrow cells, or
25 the like method. Recently, there has been developed a
technology of inducing in vitro differentiation of
pluripotent stem cells such as ES cells or iPS cells to
prepare blood cells such as platelets.
[0003] The present inventors have established a
technology of inducing differentiation of megakaryocytes
and platelets from human ES cells and shown effectiveness
5 of ES cells as a source of platelets (Patent Document 1 and
Non-patent Document 1). In addition, the present inventors
have established a method for preparing megakaryocytes and
platelets from iPS cells and have enabled dissolution of
the problem of compatibility of a human leukocyte antigen
10 (HLA) which inevitably occurred in transfusion of platelets
derived from ES cells (Patent Document 2 ) .
Further, with a view to overcoming the problem of
the amount of platelets and the like prepared from stem
cells, the present inventors have found a method of
15 establishing and thereby preparing an immortalized
megakaryocyte progenitor el1 line from the stem cells and
thus, have developed an important technology for in vitro
preparation of a large amount of platelets and the like
(Patent Document 3).
20 [0004] In vivo, megakaryocytes form pseudopodial formation
called proplatelets (platelet progenitors), fractionate
their cytoplasm, and release platelets. Polyploidization
of megakaryocytes is thought to occur by endomitosis until
they release platelets. Endomitosis of megakaryocytes is
25 multipolar mitosis not accompanied with cleavage furrow
formation and spindle extension and caused by abnormal
karyokinesis and cytoplasm mitosis. As a result of
endomitosis, cells containing several segmented nuclei are
formed. polyploidization of megakaryocytes is induced by
repetition of such endomitosis.
Many study results have been reported to date on
5 polyploidization of megakaryocytes. Lodier, et al. have
elucidated (Non-patent Document 1) that in endomitosis of
megakaryocytes, localization of nonmuscle cell myosin I1 in
a contractile ring has not been recognized in spite of
formation of cleavage furrow and defects occur in
10 contractile ring formation and spindle extension. It has
been shown that such abnormalities in contractile ring or
spindle extension become more marked by inhibiting RhoA and
Rock activities (Non-patent Document 2). RhoA accumulates
at the cleavage furrow and promotes activation of some
15 effector factors including Rho kinase (Rock), citron kinase,
LIM kinase, and rnDia/formins. These results suggest that
by inhibiting the activities of factors such as RhoA and
Rock involved in formation of a contractile ring,
endomitosis of megakaryocytes is promoted. There is also a
20 report that when a Rho signal positioned downstream of
integrin alpha2/betal is reinforced, formation of
proplatelets of immature megakaryocytes before
polyploidization is inhibited.
[00051 It is reported that all trans retinoic acid (ATRA),
25 a transcription factor and valproic acid which is known as
a histone deacetylase inhibitor are involved in
differentiation of megakaryocytes. Schweinfurth, et al.
have found that treatment of immature megakaryocytes with
all trans retinoic acid or valproic acid promotes
polyploidization (Non-patent Document 3). Further, it is
reported that polyploidization of megakaryocytes is
5 promotedwhenp53, a cancer suppressor gene product is
knocked down (Non-patent Document 4).
It has also been shown that as an influence on a
differentiation procedure of megakaryocytes, culturing
immature megakaryocytes at 39OC, temperature higher than
10 conventional culturing temperature, promotes induction to
polyploidized mature megakaryocytes and formation of
proplatelets (Non-patent Document 5).
Citation List
15 Patent Documents
COO061 Patent Document 1: ~02~38/041370
Patent Document 2: W02009/122747
Patent Document 3: W02011/034073
Non-patent Documents
20 [00071 Non-patent Document 1: Takayama, et al., Blood,
111: 5298-5306 2008
Non-patent Document 2: Lordier, et al., Blood,
112: 3164-3174 2009
Non-patent Document 3: Schweinfurth, et al.,
25 Platelets. 21: 648-657 2010
Non-patent Document 4: Fuhrken, et al., J. Biol.
Chem., 283: 15589-15600 2008
Non-patent Document 5: Proulx et al., Biotechnol.
Bioeng., 88: 675-680 2004
Summary of the Invention
5 Problem to be Solved by the Invention
[0008] Finding that the amount of functional platelets
(platelets having in vivo activities such as hemostatic
action and characterized as CD42b+) available from
megakaryocytes whose "polyploidization" has not proceeded
10 sufficiently is too small to develop clinical application,
the present inventors thought that polyploidization of
megakaryocytes should be promoted in order to efficiently
produce functional platelets in vitro.
An object of the present invention is therefore to
15 provide a method of promoting polyploidization of
megakaryocytes and thereby preparing more polyploidized
rnegakaryocytes, a method of efficiently producing platelets
from polyploidized rnegakaryocytes, and the like.
20 Means for Solving the Problem
[0009] With the foregoing problem in view, the present
inventors tried to promote polyploidization of
megakaryocytes which have been preparsd from pluripotent
stem cells (ES cells, iPS cells, and the like) and whose
25 polyploidization has not proceeded sufficiently. First,
the present inventors made this trial with the immortalized
megakaryocytic progenitor cell line (refer to Patent
Document 3) prepared from pluripotent stem cells developed
by themselves. This immortalized megakaryocytic progenitor
cell line is the one which is imparted with enhanced
proliferative potential and is established (immortalized)
5 by inducing expression of an oncogene such as MYC or a gene
such as BMIl in the megakaryocyte progenitor cells derived
from pluripotent stem cells.
With a view to promoting polyploidization with this
immortalized megakaryocytic progenitor cell line, the
10 present inventors have succeeded in efficiently promoting
polyploidization by forcing expression of an apoptosis
suppressor gene when suppression of expression of an
oncogene and a polycomb gene is conducted.
The present inventors have also confirmed that as
15 well as forced expression of an apoptosis suppressor gene,
inhibiting expression or function of a p53 gene product
further increases efficiency of polyploidization. They
have further confirmed that subjecting the megakaryocytic
progenitor cell line to treatment with an ROCK (Rho-
20 associated coiled-coil forming kinase/~ho associated
kinase) inhibitor or an HADC inhibitor, to culture at 3g°C,
and the like is also effective for inducing
polyploidization. Moreover, they have found that treatment
with an actomyosin complex (complex of actin and myosin)
25 function inhibitor highly promotes polyploidization.
They have found that highly polyploidized
megakaryocytes produced by the present invention contain
megakaryocytes of 4N or 8N or greater at a ratio higher
than that of known ones and at the same time, contain such
cells at a ratio much higher than that of mature
megakaryocytes produced in vivo.
5 Further, the present inventors have found that in
sufficiently polyploidized mature rnegakaryocytes, the
number of platelets produced from one megakaryocyte shows a
drastic increase by suppressing the forced expression of an
apoptosis suppressor gene. In addition, they have
10 confirmed that the platelet production efficiency can be
further increased by culturing on a medium added with an
ROCK inhibitor. After studying the optimum conditions for
culturing period, culturing temperature, and the like, they
have completed the present invention.
15 [00101 The present invention relates to:
[ll a method of producing polypioidized
rnegakaryocytes, including a step of forcing expression of
an apoptosis suppressor gene in megakaryocytes before
polyploidization and culturing the cells;
20 [21 the method described above in [I], wherein the
apoptosis suppressor gene is a BCL-XL gene;
131 the method described above in [I] or [2],
wherein in the culturing step, expression Dr function of a
p53 gene product is inhibited;
25 [4] the method described above in any one of [I] to
[31, wherein in the culturing step, the megakaryocytes
before polyploidization are subjected to at least one of
the following (a) to (c) :
(a) treatment with an actomyosin complex function
inhibitor;
(b) treatment with an ROCK inhibitor; and
5 (c) treatment with an HDAC inhibitor;
[51 the method as described above in [41, wherein
the ROCK inhibitor is Y27632; the HDAC inhibitor is
valproic acid; and the actomyosin complex function
inhibitor is blebbistatin;
10 [61 the method described above in any one of 111 to
[SI, wherein the culturing step is conducted at a
temperature higher than 37°C;
[71 the method described above in any one of El] to
[61, wherein the megakaryocytes before polyploidization are
15 obtained by a step of forcing expression of an oncogene and
any of the following genes (i) to (iii) in the cells at any
differentiation stage from hematopoietic progenitor cells
to megakaryocytes before polyploidization:
(i) a gene suppressing expression of a p16 gene or
20 a p19 gene;
(ii) a gene suppressing expression of an Ink4a/Arf
gene; and
(iii) a polycomb gene; and culturing and
proliferating the cells;
25 [81 the method described above in [71 , wherein a c-
MYC gene is used as the oncogene and BMIl is used as the
any of the genes (i) to (iii) ;
[9] the method described above in [71 or [81 ,
wherein the hematopoietic progenitor cells are derived from
cells selected from the group consisting of iPS cells, ES
cells, hematopoietic stem cells derived from cord blood,
5 bone marrow blood, or peripheral blood, and hematopoietic
stem cells;
[lo] a blood cell composition containing the
polyploidized megakaryocytes produced by the method
described above in any one of [I] to [9] ;
10 [ll] a method of producing a platelet, including:
a step of obtaining polyploidized megakaryocytes by
using the method described above in any one of [I] to [91
and culturing the cells; and
a step of collecting a platelet from the culture of
15 the polyploidized megakaryocytes;
[I21 the method described above in [llj, wherein
the step of culturing the polyploidized megakaryocytes is
conducted while suppressing the expression of the apoptosis
suppressor gene that has been forcibly expressed or after
20 the apoptosis suppressor gene is removed from the cells;
[I31 the method described above in [Ill or [12],
wherein the step of culturing the polyploidized
megakaryocytes is conducted in the absence of a serum
and/or in the absence of a feeder cell;
25 [I41 the method described above in any one of [ll]
to [131, wherein the step of culturing the polyploidized
megakaryocytes is conducted from one day to 15 days;
[15] the method described in any one of [Ill to
[14], wherein the step of culturing the polyploidized
megakaryocytes is conducted at 37OC;
[16] the method described above in any one of [Ill
5 to [15], wherein in the step of culturing the polyploidized
megakaryocytes, an ROCK inhibitor and/or an actomyosin
complex function inhibitor is added to a medium;
[17] the method described above in [16], wherein
the ROCK inhibitor is Y27632 and the actomyosin complex
10 function inhibitor is blebbistatin;
1181 a platelet produced by the method described in
any of [Ill to [I71 ; and
[19] a blood product containing the platelet
described above in 1181 .
15
Effect of the Invention
[OOll]
The present invention makes it possible to
artificially promote polyploidization of megakaryocytes.
20 In particular, the present invention is effective also for
promotion of polyploidization of megakaryocytes prepared in
vitro as previously reported, and the invention makes it
possible to provide megakaryocytes (for example, a
megakaryocyte population having megakaryocytes of 4N or
25 oreater at a high ratio1 whose o- ol-w-l oidization level has
proceeded more than megakaryocytes available in vivo.
Further, the present invention makes it possible to
markedly increase the number of platelets produced per
polyploidized megakaryocyte.
It becomes possible to drastically decrease the
time necessary for producing platelets from stem cells and
5 then to conduct mass production of the platelets by
inducing megakaryocytes before polyploidization from stem
cells, proliferating the megakaryocytes before
polyploidization using, for example, the method described
in Patent Document 3, and polyploidizing the megakaryocytes
10 before polyploidization to produce platelets according to
the method of the present invention. The platelets
obtained as described above are CD42b positive and largely
contribute to clinical application.
15 Brief Description of the Drawings
[0012]
[FIG. lA] It shows the outline of a test conducted to
study cytokine dependence of iMKPC-type I proliferation.
[FIG. lB] It shows a change in the number of cells when
2 0 iMKPC-type I was cultured in media to which SCF and TPO
(S+T) , SCF and EPO (S+E) , SCF (S) , TPO (T) , and EPO (E) had
been added, respectively, according to the schedule shown
in FIG. 1A.
[FIG. lC] It shows flow cytometry histograms of the
25 expression of a surface marker of iMKPC-type I on Day 8 of
the schedule shown in FIG. 1A.
[FIG. ID] It shows the results of forcing expression of cMYC
and BMIl with CD34-positive cells derived from the cord
blood and promoting proliferation af megakaryocytes before
polyploidization.
[FIG. 2A] It shows the results of studying the influence
5 of cytokine on proliferation of iMKPC-type 11.
[FIG. 2B] It shows the results of studying the expression
of CD4la and CD42b in iMKPC-type 11.
[FIG. 31 It shows microscopic observations of
polyploidization of iMKPC-type I1 when forced expression of
10 BCL-XL and suppression of expression of a p53 gene were
conducted, and of polyploidization when blebbistatin was
added to the medium.
[FIG. 41 It shows microscopic observations in the studying
the influence of BCL-XL on proliferation of iMKPC-type 11.
15 [FIG. 51 Influence of an ROCK inhibitor on
polyploidization of megakaryocytes. After expression cf
MYC/BMI~ in megakaryocytes was suppressed (by culturing in
the presence of doxycycline and in the absence of
estradiol) , an ROCK inhibitor (Y27632) (10 pM) was added.
20 After culturing for 7 days, the degree of polyploidization
was studied. A shows flow cytometry histograms of cells
(vehicle) to which the ROCK inhibitor had not been added
and of cells (Rock i) to which the inhibitor had been added,
wherein these cells were stained with Hoechst, a nuclear
7 5 stain. then CD4l.a. a megakaryocyte marker was stained with
an anti-CD4la antibody. B is a graph showing the results
of A.
[FIG. 61 Studying results of the expression level of a
gene involved in maturation of megakaz-yocytes in culturing
at 3g°C. After expression of MYC/BMI~ in megakaryocytes
was suppressed, the resulting cells were cultured at 3g°C
5 for 5 days. The expression level of a gene group (GATA1
(A), PF4 (B), NFE2 (C), and P-tubulin (D)) indispensable
for maturation of megakaryocytes was studied through
quantitative PCR (q-PCR) . The expression level shown in
these graphs is a ratio to the expression level of GAPDH.
10 [FIG. 71 Studying results of the influence of BCL-XL, one
of apoptosis suppressor genes, on polyploidization of
megakaryocytes. The degree of polyploidization was studied
after suppressing expression of MYC/BMIl in megakaryocytes,
inducing expression of BCL-XL in the presence of an ROCK
15 inhibitor (10 pM), and culturing the resulting cells for 7
days. A shows flow cytometry histograms of each of
MYC/BMIl expressed cells (left graph), of cells treated
with an ROCK inhibitor after suppression of MYC/BMI~
expression (middle graph), and of cells subjected to
20 expression of BCL-XL in addition to suppression of
expression of MYC/BMI~ and treatment with an ROCK inhibitor
(right graph), wherein these cells were stained with a
nulcear stain, Hoechst, and then CD4la, a megakaryocyte
marker, was stained with an anti-CD4la antibody. B is a
25 graph showing the results of A. C includes micrographs of
cells having a nucleus of 2N, 4N, 8N, and 8N or greater.
[FIG. 81 Growth curve of BCL-XL expressed cells. It shows
the results of a change in the number of BCL-XL expressed
cells (CD4la+) (a) and the number of non-expressed cells
(CD4la+) (A) as a function of culturing days while
suppressing expression of MYC/BMI~ in megakaryocytes in the
5 presence of an ROCK inhibitor (10 pM) .
[FIG. 91 Influence of p53 knockdown on polyploidization.
The degree of polyploidization of CD4la+ cells was studied
by suppressing expression of MYc/BMI~ in megakaryocytes,
inducing expression of BCL-XL in the presence of an ROCK
10 inhibitor (10 pM) , knocking down a p53 gene, and then
culturing the resulting cells at 3g°C for 7 days. A shows
flow cytometry histograms of each of control cells
(control) in which p53 had not been knocked down and of
cells (SiP53) in which p53 had been knocked down, wherein
15 these cells were stained with a nuclear stain Hoechst, and
then CD4la1 a megakaryocyte marker was stained with an
anti-CD4la antibody. B is a graph showing the results of A.
[FIG. 101 Influence of valproic acid treatment on
polyploidization. The degree of polyploidization of CD4la+
20 cells was studied after suppressing the expression of
MYC/BMI~ in megakaryocytes, inducing expression of BCL-XL
in the presence of an ROCK inhibitor (10 pM), knocking down
a p53 gene, treating the resulting cells with valproic acid
(0.5 mM), and culturing at 3g°C for 7 days. A shows flow
2 5 cytometry histograms of each of cells (Si P531 not treated
with valproic acid and of cells (Sip53 VLP) treated with
valproic acid, wherein these cells were stained with a
nuclear stain Hoechst, and then CD4la, a megakaryocyte
marker was stained with an anti-CD4la antibody. B is a
graph showing the results of A.
[FIG. 111 Influence of a myosin heavy chain IIA/B ATPase
5 inhibitor (actomyosin complex function inhibitor) on
polyploidization of megakaryocytes. The degree of
polymerization was studied after suppressing the expression
of MYC/BMIl in megakaryocytes (by culturing in the presence
of doxycycline and in the absence of estradiol), adding
10 blebbistatin (10 pg/ml), a myosin heavy chain IIA/B ATPase
inhibit.or, and culturing for 7 days. A shows flow
cytometry histograms of cells ( - ) to which blebbistatin had
not been added and of cells (+) to which blebbistatin (10
pg/ml) had been added, wherein these cells were stained
15 with a nuclear stain Hoechst, and then CD4la, a
megakaryocyte marker was stained with an anti-CD4la
antibody. B is a graph showing the results of A.
[FIG. 121 Influence, on polyploidization of megakaryocytes,
of blebbistatin treatment used in combination with the
20 other treatments. The degree of polyploidization of CD4la+
cells were studied after suppressing the expression of
MYC/BMI~ in megakaryocytes, inducing expression of BCL-XL
in the presence of Y27632 (10 pM) and valproic acid (0.5
mM), knocking down a p53 gene, adding blebbistatin (10
25 pg/ml), and culturing at 3g°C for 7 days. A shows flow
cytometry histograms of each of cells ( - ) not treated with
blebbistatin and cells (+) treated with blebbistatin,
wherein these cells were stained with a nuclear stain,
Hoechst, and then a CD4la, a megakaryocyte marker was
stained with an anti-CD4la antibody. B is a graph showing
the results of A.
5 [FIG. 131 Growth curve of cells subjected to blebbistatin
treatment in combination with the other treatments. A
change in the number of the following cells was graphed (A)
as a function of culturing days: cells (CD4la+) treated
with blebbistatin (CD4la+) (A) and cells (CD4la+) not
10 treated with blebbistatin (a), each after expression of
MYC/BMI~ in megakaryocytes was suppressed, BCL-XL was
expressed in the presence of Y27632 (10 pM) and valproic
acid (0.5 mM), and a p53 gene was knocked down.
Micrographs of these cells are shown in B.
15 [FIG. 141 It shows the results of studying expression of
CD4la and CD42b in megakaryocytes and platelets in both
cases where expression of BL-XL was suppressed and where it
was not suppressed during a platelet release stage.
[FIG. 151 It shows cell counts measured at the time of
20 ON/OFF of BCL-XL expression based on the results of FIG. 14.
A shows the number of CD42b-positive platelets, B shows the
number of C~4la-positive/~~42b-positimveeg akaryocytes, and
C shows the number of CD4la-positive megakaryocytes.
[FIG. 161 It shows the results of studying the influence
25 af rultl-?rinn:t emperatl_?ress et at 35OT 37'C. 7g°C ~n the
number of platelets in both cases where expression of BCLXL
was suppressed and where it was not suppressed during a
platelet release stage.
[FIG. 171 It shows the results of studying the -influence
of the presence or absence of a serum, feeder cells, and
blebbistatin on the number of platelets.
5 [FIG. 181 It shows the results of studying the influence
of a serum, feeder cells, and blebbistatin on the ratio of
CD42b platelets.
[FIG. 191 It shows one example of preferable culturing
conditions in the polyploidization step (MCB expansion) of
10 megakaryocytes and during a platelet release stage
(platelet production) .
[FIG. 201 It shows an increase in a ratio of CD42b
platelets by the suppression of expression of BCL-XL and a
further increase in a ratio of CD42b platelets by removing
15 the serum and feeder cells from the medium and adding
blebbistatin.
[FIG. 211 It shows the results of studying the influence
of a functional inhibitory antibody HIPl against CD42b on
the ristocetin agglutination effect of peripheral platelets.
2 0 [FIG. 221 It shows the results of studying the influence
of a functional inhibitory antibody HIPl against CD42b on
thrombus formation in vivo.
[FIG. 231 It shows the results of respectively
transplanting, to NOG mice, iPS cell-derived platelets
25 produced while adding KP-457 (S-45457), an ADAM17 inhibitor
and thereby increasing the expression level of GPIba
(CD42b) and iPS cell-derived platelets produced without
adding an ADAM17 inhibitor and measuring the number of
platelets which contributed to thrombus formation.
[FIG. 241 It shows the results of transplanting human
peripheral platelets spuriously deteriorated by adding 100
5 pm of CCCP, a platelet damaging agent in the presence of
KP-457, platelets to which CCCP was added in the absence of
KP-457, and fresh platelets, respectively and measuring the
number of platelets which contributed to thrombus formation.
10 Mode for Carrying out the Invention
[0013] (Method of producing polyploidized megakaryocytes)
The present invention provides a method of
promoting polyploidization of megakaryocytes and thereby
preparing polyploidized rnegakaryocytes.
15 One mode of the method of producing polyploidized
megakaryocytes according to the present invention includes
a step of forcing expression of an apoptosis suppressor
gene in megakaryocytes before polyploidization and
culturing the cells.
20 The term "megakaryocytes before polyploidization"
as used herein is not particularly limited and may refer to
megakaryocytes which are available from the cord blood or
bone marrow cells and whose polyploidization has not
proceeded sufficiently, or megakaryocytes which have been
3 5 i n d i ~ c t i ~ rd~ilf~f er r ~ n t i a t e df r o m RS cells. iPS c e l l s .
hematopoietic stem cells derived from cord blood, bone
marrow blood, or peripheral blood, progenitor cells, or the
like and whose polyploidization has not proceeded
sufficiently.
Further, the term "megakaryocytes before
polyploidization" as used herein embraces cells that are
5 characterized, for example, as CD4la positive/~~42a
positive/C~42b positive.
The term "polyploidized rnegakaryocytes" or
"megakaryocytes which have undergone polyploidization"
means cells or a cell population in which the number of
10 nuclei has increased relatively compared with
"megakaryocytes before polyploidization". For example,
when megakaryocytes to which the method of the present
invention is to be applied have a 2N nucleus, cells having
a 4N or greater nucleus correspond to "polyploidized
15 megakaryocytes" or "megakaryocytes which have undergone
polyploidization". Even in megakaryocytes before
polyploidization, the number of nuclei is not limited to
one. In a cell population, the number of nuclei in the
whole cell population shows a significant increase after a
20 predetermined term, the cell population before the
predetermined term may be called "megakaryocytes before
polyploidizationf' and the cell population after a
predetermined term may be called "megakaryocytes which have
undergone polyploidization".
25 LO0141 The present invention can also be applied to
megakaryocytes before polyploidization which have been
inductively differentiated from pluripotent stem cells
(such as ES cells and iPS cells), hematopoietic stem cells
derived from cord blood, bone marrow blood, or peripheral
blood, and progenitor cells. For example, megakaryocytes
available from a net-like structure (which may also be
5 called ES-sac or iPS-sac) prepared from ES cells or iPS
cells are preferred. Here, the "net-like structure"
prepared from ES cells or iPS cells means a steric sac
(having an internal space) like structure derived from ES
cells or iPS cells. It is made of an endothelial cell
10 population and the like and contains therein hematopoietic
progenitor cells (refer to Patent Document 1, Patent
Document 2, and Non-patent Document 2).
No particular limitation is imposed on ES cells to
be used in the present invention and it is possible to use
15 those established finally as an ES cell line by culturing
fertilized eggs in the blastocyst stage together with
feeder cells, isolating proliferating cells derived from
the inner clump of cells into individual cells, and
repeating subculture.
20 When iPS cells are used, cells of any origin can be
used insofar as they have acquired pluripotent
differentiation similar to ES cells by introducing several
kinds of transcription factor (which will hereinafter be
called "pluripotent differentiation factor") genes capable
34 of nrn~rirji-nu snrnatir r ~ 1 1 - s (for exarn~jp f j h m h l a g t s nr
blood cells) with pluripotent differentiation. As the
pluripotent differentiation factors, many factors have
I
already been reported. Examples include, but not limited
to, Oct family (ex. 0ct3/4), SOX family (ex. SOX2, SOX1.
SOX3, SOX15, and SOX17). Klf family (ex. Klf4, and Klf2),
MYC family (ex. c-MYC. N-MYC, and L-MYC), NANOG, and LIN28.
5 [0015] The present inventors have reported that by forcing
expression of an oncogene such as MYC and a gene such as
BMIl in megakaryocytes before polyploidization (including
those called "megakaryocytic progenitor cells" in Patent
Document 3) derived from pluripotent stem cells, the
10 resulting megakaryocytes have enhanced proliferative
capacity (Patent Document 3, JEM, 207: 2817-2830 2010).
The megakaryocytes before polyploidization obtained
using the above-mentioned method are suited for use in the
method of the present invention.
15 In the method of producing polyploidized
megakaryocytes according to the present invention, as the
megakaryocytes before polyploidization, those obtained by a
step of, in any differentiation stage from hematopoietic
progenitor cells to megakaryocytes before proliferation.
20 forcing expression of an oncogene and any of the following
genes (i) to (iii) :
(i) a gene suppressing the expression of a p16 gene
or a p19 gene;
(ii) a gene suppressing the expression of an
25 Ink4a/~rf gene ; and-
(iii) a polycomb gene;
and culturing and proliferating the resulting cells.
Examples of the oncogene include MYC family gene,
Src family gene, Ras family gene, Raf family gene, and
protein kinase family genes such as c-Kit, PDGFR, and Abl.
Examples of the genes (i) to (iii) include BMI1, Me118,
5 ~ingla/b, ~hc1/2/3, ~bx2/4/6/7/8, Ezh2, Eed, Suzl2, HADC,
and Dnmtl/3a/3b1 with a BMIl gene being particularly
preferred. Control of the expression of the oncogene and
the polycomb gene can be conducted by those skilled in the
art in a conventional manner. For example, the method
10 described in detail in Patent Document 3 and the like can
be used. The oncogene and any one of the genes (i) to
(iii) may be introduced into the cells at any stage of
hematopoietic progenitor cells to megakaryocytes before
polyploidization. However, this is not limited as
15 expression of these genes is induced in the megakaryocytes
before polyploidization to be used in the present invention.
[00161 The oncogene and the genes (i) to (iii) (for
example, a BMIl gene) to be used in the present invention
include not only genes having an already known cDNA
20 sequence but also homologs identified using prior art based
on homology to the known cDNA sequence.
For example, among MYC family genes, the c-MYC gene
is a gene having a nucleic acid sequence of SEQ ID NO: 1.
Homologs of the c-MYC gene are genes having a cDNA sequence
35 s ~ l b s t a n t i a l l vt h e same a s t h e n i ~ c l e i ca cid sequence of SRQ
ID NO: 1. The cDNA having a sequence substantially the
same as the nucleic acid sequence of SEQ ID NO: 1 is a DNA
having about 60% or greater sequence identity, preferably
about 70% or greater sequence identity, more preferably
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity,
5 most preferably about 99% sequence identity to a DNA having
a sequence of SEQ ID NO: 1, or a DNA capable of being
hybridized with a DNA having a sequence complementary to
the nucleic acid sequence of SEQ ID NO: 1 under stringent
conditions, where a protein encoded by such a DNA
10 contributes to amplification of cells at a differentiation
stage such as megakaryocytes before polyploidization.
[0017] The EM11 gene is a gene having a nucleic acid
sequence of, for example, SEQ ID NO: 2. A homolog of the
BMIl gene is a gene having a cDNA sequence substantially
15 the same as the nucleic acid sequence of, for example, SEQ
ID NO: 2; The cDNA having a sequence substantially the
same as the nucleic acid sequence of SEQ ID NO: 2 is a DNA
having about 60% or greater sequence identity, preferably
about 70% or greater sequence identity, more preferably
20 about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity,
most preferably about 99% sequence identity to a DNA having
a sequence of SEQ ID NO: 2, or a DNA capable of being
hybridized with a DNA having a sequence complementary to
25 the nucleic acid sequence of SEQ ID NO: 2 under stringent
conditions, where a protein encoded by such a DNA
suppresses oncogene-induced senescence in the cells in
which the oncogene such as the MYC family gene has been
expressed, thereby promoting amplification of the cells.
[0018] The above-mentioned oncogene and the genes (i) to
(iii) are necessary for cell proliferation, but they may
5 inhibit promotion of polyploidization or release of
platelets so that expression of these genes may be
suppressed prior to a polyploidization step. Suppression
of the expression of these genes in the cells facilitates
release of functional platelets (Patent Document 3).
10 [0019] The term "apoptosis suppressor gene" as used herein
is not particularly limited insofar as it is a gene
suppressing apoptosis. Examples of it include a BCL2 gene,
a BCL-XL gene, Survivin, and MCL1.
The present inventors have found that when forced
15 expression of the oncogene and any of the genes (i) to
(iii) is suppressed, death of the proliferated
megakaryocytes before polyploidization can be induced. As
shown later in Examples, suppression of expression of the
oncogene and any of the genes (i) to (iii) in the
20 megakaryocytes before polyploidization and forced
expression of an apoptosis suppressor gene in the cells
promote the polyploidization of the megakaryocytes,
resulting in efficient production of platelets from the
megakaryocytes before polyploidization.
2 5 .Ac-: -g h n ~ ~lnat er i-n E.*arnnl_~~m ~q3kar\rgr\rtps cnnt in il?
long-term proliferation by forcing expression of an
apoptosis suppressor gene.
[0020] Apoptosis suppressor genes such as BCL-XL gene and
BCL2 gene to be used in the present invention include not
only genes whose cDNA sequence has already been published
but also homologs identified by prior art based on homology
5 to the known cDNA sequence. For example, a BCL-XL gene,
one of apoptosis suppressor genes, is a gene having a
nucleic acid sequence of SEQ ID NO: 3. A homolog of the
BCL-XL gene is a gene having a cDNA sequence substantially
equal to the nucleic acid sequence of SEQ ID NO: 3. The
10 cDNA having a sequence substantially equal to the nucleic
acid sequence of SEQ ID NO: 3 is a DNA having about 60% or
greater sequence identity, preferably about 70% or greater
sequence identity, more preferably about 80%, 81%, 82%, 839,
84%, 85%, 86%, 87%, 88%, 89%, go%, 91%, 92%, 93%, 94%, 95%,
15 96%, 97%, or 98% sequence identity, most preferably 99%
sequence identity to a DNA having a sequence of SEQ ID NO:
3, or a DNA capable of being hybridized with a DNA having a
sequence complementary to the nucleic acid sequence of SEQ
ID NO: 3 under stringent conditions, where a protein coded
20 by such DNA is effective for suppressing apoptosis.
[00211 The term "stringent conditions" as used herein
means hybridization conditions easily determined by those
skilled in the art, and are empirical experimental
conditions that typically depend on a probe length, a
25 washing temperature, and a salt concentration. Usually, a
temperature for proper annealing becomes higher when a
longer probe is used, and becomes lower when a shorter
probe is used. Hybrid formation usually depends on
reannealing ability of a complementary strand placed in an
environment where a temperature is slightly lower than its
melting point.
5 Under low stringent conditions, for example, in a
filter washing stage after hybridization, a filter is
washed in a 0-lxSSC, 0.1% SDS solution under temperature
conditions of from 37OC to 42OC. Under high stringent
conditions, for example, in the washing stage, a filter is
10 washed in a 5xSSC, 0.1% SDS solution at 65OC.
Polynucleotide with higher homology can be obtained by
making the stringent conditions higher.
[0022] In order to force expression of genes such as
oncogene, genes (i) to (iii), and apoptosis suppressor gene
15 in cells, any method well known to those skilled in the art
may be employed. For example, the gene may be introduced
into cells by using a gene introduction system with a
lentivirus or a retrovirus, and then expressed. When gene
expression is conducted using a viral gene introduction
20 vector, a target gene may be expressed by operably linking
the gene to the downstream of an appropriate promoter,
inserting the resulting gene into the gene introduction
vector, and then introducing it into the cells. Here, the
term "operably linking" means that a target gene is linked
target gene. In embodiments of the present invention, for
example, the target gene may be constantly expressed using
a CMV promoter, an EF1 promoter, or the like.
Alternatively, an appropriate promoter (inducible promoter)
may be placed under control of an element having activity
controlled by a trans factor, e.g., a drug response element
5 such as tetracycline response element and a target gene may
be inductively expressed by conducting such a control as
drug addition. Since such a gene expression system using a
drug can realize desired expression control of the oncogene
or the genes (i) to (iii), an appropriate system can be
10 easily selected by those skilled in the art. A
commercially available kit may be used in order to conduct
such expression. The oncogene and the genes (i) to (iii) ,
which are the target genes in expression control, may be
inserted into respective vectors or in one vector.
15 [0023] Suppression of expression of the oncogene or any of
the genes (i) to (ii.i.j in megakaryocytes may be achieved,
for example, by removing the drug or the like and thereby
releasing the induction of expression using the abovementioned
inductive expression system. Alternatively, the
20 oncogene or any of the genes (i) to (iii) which has been
introduced may be removed using a Cre/lox system or the
like to suppressively control the expression of these genes.
A commercially available kit or the like may be used as
needed in order to suppressively regulate the expression of
25 the oncogene or the gene (i) to (iii).
[00241 One mode of the met-hod of producing polyploidized
rnegakaryocytes according to the present invention includes
a step of forcing expression of an apoptosis suppressor
gene in cells, and in parallel, inhibiting expression or
function of a p53 gene product in the cells. The term
"expression" is used as a concept encompassing
5 transcription and translation. For example, the term
"inhibiting expression" may include the meaning "inhibiting
in a transcription level" or "inhibiting in a translation
level".
The p53 gene product is widely known as a tumor
10 suppressor gene and its sequence and the like in various
animal species are known.
A method of inhibiting the function of the p53 gene
product in megakaryocytes can be achieved by a conventional
technology in this technical field. Examples of the method
15 include a method of introducing mutation (substitution,
insertion, or deletion, or alteration or modification) into
a P53 gene and thereby inhibiting production of the gene
product and a method of directly inhibiting the function of
the gene product. Examples of the method of directly
20 introducing mutation (substitution, insertion, or deletion,
or alteration or modification) into a gene include a method
of destructing the whole p53 gene through homologous
recombination while making use of an appropriate gene
targeting vector and a method of introducing mutation in a
7 5 r e q i n n i m n o r t a n t f o r a c t i v i tv of the gene prodi~ct by makinq
use of a Cre/lox system or the like.
LO0251 As a method of inhibiting the function of the p53
gene product, a dominant negative method may be used. The
dominant negative method is a method of inducing in cells
abundant expression of a p53 protein having mutation
introduced therein to reduce or deprive its activity,
5 making a ratio of the p53 protein inert to the normal p53
protein in cells overwhelmingly high, and thereby obtaining
cells showing a behavior of cells which have lost the
function of the p53 protein.
LO0261 As the method suppressing the expression of the p53
10 gene product, an antisense method, a ribozyme method, an
RNAi method, or the like may be used.
LO0271 The antisense method is a method of suppressing
expression of a gene by using a single stranded nucleic
acid having a base sequence complementary to a target gene
15 (basically, an mRNA as a transcription product) and having
usually a length of from 16 bases to 100 bases, preferably
from 15 bases to 30 bases. The gene expression is
inhibited by introducing an antisense nucleic acid in cells
and hybridizing it with the target gene. The antisense
20 nucleic acid is not completely complementary to the target
gene insofar as an expression inhibiting effect of the
target gene can be produced. The antisense nucleic acid
can be designed as needed by those skilled in the art by
using known software or the like. The antisense nucleic
25 acid may be any of DNA, RNA, and DNA-RNA chimera, or may be
modified.
LO0281 A ribozyme is a nucleic acid molecule catalytically
hydrolyzes a target RNA and is composed of an antisense
region having a sequence complementary to the target RNA
and a catalyst center region involved in cleavage reaction.
A ribozyme can be designed as needed in a known manner by
5 those skilled in the art. A ribozyme is usually an RNA
molecule, but a DNA-RNA chimera molecule may be used
instead.
[0029] The RNAi method is a sequence-specific gene
expression suppressing mechanism induced by a double
10 stranded nucleic acid. The method has high target
specificity and in addition, is highly safe because it
utilizes a gene expression suppressing mechanism originally
present in vivo.
Examples of the double stranded nucleic acid having
15 an RNAi effect include siRNA. When siRNA is used for
mammalian cells, it is a doublz stranded RNA having usually
from about 19 to 30 bases, preferably from about 21 to 25
bases. The double stranded nucleic acid having an RNAi
effect usually has, as one of the strands, a sequence
20 complementary to a portion of a target nucleic acid and, as
the other strand, a sequence complementary thereto.
The double stranded nucleic acid having an RNAi
effect can be designed in a known manner based on the base
sequence of a target gene. The double stranded nucleic
7 5 a c i d h a ~ r j n g an RNAi effect may be any of a double stranded
RNA, a DNA-RNA chimera type double stranded nucleic acid,
an artificial nucleic acid, and a nucleic acid subjected to
various modifications.
The siRNA, antisense nucleic acid, and ribozyme can
be expressed in cells by introducing, into the cells,
vectors (for example, lentivirus vectors) containing the
5 nucleic acids encoding them, respectively. As the siRNA,
DNAs encoding two strands, respectively, may be used or a
DNA encoding a single stranded nucleic acid obtained by
linking the two strands of a double stranded nucleic acid
via a loop may be used. In the latter case, the single
10 stranded RNA obtained by intracellular transcription has a
hairpin type structure because the complementary portion of
it is hybridized in the molecules. This RNA is called
shRNA (short hairpin RNA). When the ShRNA is exported to
the cytoplasm, the loop portion is cleaved by an enzyme
15 (Dicer) to be a double stranded RNA and it produces an RNAi
effect .
[0030] As another method of inhibiting the function of the
p53 gene product in megakaryocytes, a method of directly or
indirectly inhibiting the function of the p53 gene product,
20 a method of inhibiting phosphorylation of p53 and thereby
indirectly inhibiting the activation of p53, or the like
method can be employed.
[00311 As described later in Examples, megakaryocytes
before forced expression of an apoptosis suppressor gene
25 and inhibition of expression or function of the p53 gene
product, continue proliferation, wherein the megakaryocytes
whose cytokine dependence is SCF and platelets released
therefrom are not CD42b positive are included. When forced
expression of an apoptosis suppressor gene and inhibition
of expression or function of the p53 gene product are
conducted, megakaryocytes are partially polyploidized while
5 continuing proliferation and release many CD42-positive
platelets. At this stage, the cytokine dependence of
megakaryocytes changes from SCF to TOP and proliferation
and maturation proceed in parallel to each other.
[00321 One mode of the method of producing polyploidized
10 megakaryocytes according to the present invention includes
at least one of a step of treating the cells in which an
apoptosis suppressor gene has been forcibly expressed
while culturing them, with (a) an actomyosin complex
function inhibitor, (b) an ROCK inhibitor, and (c) an HDAC
15 inhibitor. By the above-mentioned treatment, more stable
proliferation and polyploidization.~roceed.
The term "actomyosin complex" as used herein means
a complex between actin and myosin I1 and it constitutes,
for example, a contractile ring which will appear at the
20 time of cytokinesis. In the actomyosin complex, myosin I1
functions as a motor protein while interacting with actin
and is involved in contraction of the contractile ring and
the like. The "actomyosin complex function inhibitor" in
the present invention may inhibit the function by any
25 mechanism. It includes. for example. those inhibiting the
formation of an actomyosin complex and thereby inhibiting
the function of the actomyosin complex; those inhibiting
myosin heavy chain (MHC) IIA/IIB ATPase and thereby
inhibiting the function of the actomyosin complex; and
those inhibiting myosin light chain kinase (MlCK) and
thereby inhibiting the function of the actomyosin complex.
5 The myosin heavy chain IIA/B ATPase is a molecule having an
important role in contraction of a contractile ring, while
the myosin light chain kinase phosphorylates L2, among
myosin light chains, and induces a sliding movement between
actin and myosin.
10 It has been reported to date that an ROCK inhibitor
suppresses endomitosis of megakaryocytes and promotes
polyploidization. The myosin heavy chain IIA/B ATPase or
myosin light chain kinase that controls formation or
function of an actomyosin complex functions downstream of
15 an ROCK signal and more directly controls contraction of a
contractile ring via formation or function regulation of an
actomyosin complex. The actomyosin complex function
inhibitor is therefore presumed to suppress endomitosis of
megakaryocytes more effectively and promote
20 polyploidization more, compared with the ROCK inhibitor.
Examples of the actomyosin complex function
inhibitor usable in the present invention include
blebbistatin (Science, 299: 1743-1747 2003), a myosin heavy
chain IIA/B ATPase inhibitor and ML7, a myosin light chain
25 kinase inhibitor. As the myosin heavy chain I1A/B ATPase
inhibitor or myosin light chain kinase inhibitor, nucleic
acids (for example, shRNA) or antibodies inhibiting the
activity of myosin heavy chain IIA/B ATPase or myosin light
chain kinase can also be used.
It is to be noted that the term "treatment" as used
herein means an operation conducted to produce the effect
5 of an inhibitor or the like in target cells, for example,
addition of an adequate amount of an inhibitor or the like
to a culture medium of cells to incorporate it in the cells.
In some cases, an operation that promotes incorporation of
it in cells may be used in combination.
10 [0033] One mode of the method of producing megakaryocytes
according to the present invention includes a step of
treating the cells in which an apoptosis suppressor gene
has been forcibly expressed with an ROCK inhibitor while
culturing them.
15 Examples of the ROCK (Rho-associated coiled-coil
forming kinase/~ho associated kinase) inhibitor include
[ (R) - (+) -trans-N-( 4-pyridyl)- 4-( 1-aminoethyl)- cyclohexane
carboxamide.2HC1~HzO] (Y27632 ) . In some cases, antibodies
or nucleic acids (for example, shRNA) that inhibit Rho
20 kinase activity can also be used as the ROCK inhibitor.
LO0341 One mode of the method of producing megakaryocytes
according to the present invention includes a step of
treating the cells in which an apoptosis suppressor gene
has been forcibly expressed with an HDAC inhibitor while
25 culturing the cells.
The HDAC inhibitor has an action of inhibiting
histone deacetylase (HDAC) activity. Many HDAC inhibitors
have been known to date. Examples of it include valproic
acid, trichostatin A, SAHA (suberoylanilide hydroxamic
acid), and APHA (aroyl-pyrrolyl-hydroxyamide). In
particular, valproic acid and trichostatin A are preferably
5 used. When a drug to be used is provided in the form of
salt, the inhibitor may be used in the form of salt.
[00351 The optimum concentration when the cells are
treated with an actomyosin complex function inhibitor, ROCK
inhibitor, HDAC inhibitor, or the like can be determined in
10 advance by those skilled in the art based on the results of
a preliminary test. The treatment time, method or the like
can also be selected as needed by those skilled in the art.
For example, when the cells are treated with blebbistatin,
a myosin heavy chain I1 ATPase inhibitor, it may be added
15 in an amount of from about 2 to 15 pg/ml or from about 5 to
10 pg/ml to a culture medium and cultured, for example,
preferably for from about 5 to 10 days, particularly
preferably for from about 6 to 7 days. Y27632, a ROCK
inhibitor may be added in an amount of from 5 to 15 pM or
20 from 8 to 12 pM, preferably from about 10 pM, while
valproic acid, an HDAC inhibitor may be added in an amount
of from about 0.1 to 1 mM or from about 0.2 to 0.7 mM,
preferably about 0.5 mM. Treatment time with Y27632 or
valproic acid may be for from 10 to 21 days, preferably for
25 about 14 days.
100361 One mode of the method of producing polyploidized
megakaryocytes according to the present invention includes
placing megakaryotic cells at a temperature of 37OC or
greater, in which an apoptosis suppressor gene in the cells
has been forcibly expressed while culturing the cells.
It has been confirmed that culturing megakaryocytes
5 at a usual temperatures of 37°C or greater promotes
differentiation of megakaryocytes which have undergone
polyploidization. The "temperatures of 37OC or greater" is,
for example, from about 37OC to about 42OC. preferably from
about 37OC to about 39OC, because temperatures not giving
10 damage to cells are adequate. Although a culturing term at
temperatures of 37' or greater can be determined as needed
while monitoring the number of megakaryocytes which have
undergone polyploidization, it is, for example, from 10
days to 28 days, preferably from 14 days to about 21 days.
15 100371 No particular limitation is imposed on the other
culturing conditions in the step of conducting forced
expression of an apoptosis suppressor gene in
megakaryocytes which have not undergone polyploidization
and then culturing the resulting cells insofar as the
I 20 effect of the present invention can be produced preferably
under the conditions. Known culturing conditions or
I conditions equivalent thereto can be used. For example, ~
TPC, IL-1, IL-3, IL-4, I L 5 , IL-6, IL-9, IL-11, EPO, GM-CSF,
SCF, G-CSF, Flt3 ligand, and Heparin may be used either
7 5 s i n g l y or i-n combination of two or more and added to a
medium.
Alternatively, a feeder cell may be used as needed
for culturing.
[0038]
The megakaryocytes which have undergone
polyploidization obtained using the above-mentioned method
5 efficiently produces CD42b-positive functional platelets.
As shown later in Examples, the CD42b-positive platelets
have high thrombus forming ability both in vivo and in
vitro. In addition, the megakaryocytes which have
undergone polyploidization can produce functional platelets
10 even when thawed after cryopreservation.
[0039]
The present invention also provides a blood cell
composition having a high content of polyploidized
megakaryocytes. The term "blood cell compositionrr may
15 comprise, as well as "polyploidized megakaryocytes" whose
polyploidization has been promoted by the method of the
present invention, megakaryocytes prepared using another
method, and the other blood cells.
Treatment of the megakaryocytes before
20 polyploidization by the method of the present invention can
promote differentiation of them into polyploidized
megakaryocytes of 4N or greater. Therefore, application of
the method of the invention to a population of
megakaryocytes differentiated, for example, from
25 pluripotent stem cells or the like makes it possible to
obtain a blood cell composition having a high content of
polyploidized megakaryocytes of 4N or greater. When the
population of megakaryocytes is treated using the method of
the present invention, it is possible to increase the
content of polyploidized megakaryocytes with 4N or greater
to at least 20% or greater, 30% or greater, preferably 40%
or greater, 50% or greater, more preferably 80% or greater
(refer to, for example, FIG. 11B). Accordingly, the
present invention makes it possible to prepare a population
of megakaryocytes or a population of blood cells having a
high existence ratio of poly-ploidized megakaryocytes.
Such a blood cell composition can also be
cryopreserved. Therefore, such a blood cell composition is
distributed in a frozen state and the method of producing
platelets which will be described later may be conducted on
the user side.
[00401 Megakaryocytes and the like which have been treated
to promote poly-ploidization by the method of the present
invention are effective also for transplanting them in vivo
and producing functional platelets in vivo by a proper
method.
At present, hematopoietic stem cells have been
transplanted through bone marrow transplantation, cord
blood transplantation, or the like. In particular, cord
blood transplantation enables reduction of the problems of
bone marrow transplantation such as shortage of number of
donors and a great burden for donors so that there has
recently been more opportunities of cord blood
transplantation. The megakaryocytes produced in vivo by
cord blood transplantation have however not undergone
polyploidization sufficiently and it takes time to produce
a sufficient number of platelets in vivo. When a
production capacity of platelets should be increased
5 rapidly, cord blood transplantation cannot satisfy this
demand sufficiently at present.
Transplantation of polyploidized megakaryocytes
obtained by using the method of the present invention can
overcome the problems of bone marrow transplantation such
10 as shortage of the number of donors and a heavy burden of
donors and the problems of cord blood transplantation such
as platelet production capacity in vivo. The method of the
present invention is therefore much superior to the
conventional transplantation methods.
15 [00411 (Production method of platelets)
The method of producing platelets according to the
present invention, on the other hand, produces platelets in
vitro from the polyploidized megakaryocytes and the like
obtained using the method of the present invention.
20 The method of producing platelets according to the
present invention includes a step of culturing the
polyploidized megakaryocytes obtained by the abovementioned
method and collecting platelets from the cultured
product.
25 Although no limit-ation is imposed on the culturing
conditions, the polyploidized megakaryocytes may be
cultured for from about 7 to 15 days, for example, in the
presence of TPO (from about 10 to 200 ng/mL, preferably
from about 50 to 100 ng/mL) or in the presence of TPO (from
about 10 to 200 ng/mL, preferably from about 50 to 100
ng/mL), SCF (from 10 to 200 ng/m~,p referably about 50
5 ng/m~),a nd Heparin (from about 10 to 100 u/~L,p referably
about 25 U/ml) .
[0042] In one mode of the method of producing platelets
according to the present invention, in the culturing step
of polyploidized megakaryocytes, the above-mentioned forced
10 expression of an apoptosis gene is suppressed or the abovementioned
apoptosis suppressor gene is removed from the
polyploidized megakaryocytes.
The suppression of expression of an apoptosis
suppressor gene may also be achieved, for example, by
15 removing the chemical or the like to release the induction
of expression by the above-mentioned inductive expression
system. Alternatively, the apoptosis suppressor gene
introduced may be removed by using a Cre/lox system and
thereby expression of this gene may be suppressively
20 controlled. A commercially available kit or the like can
also be used as needed in order to suppressively regulate
the expression of an apoptosis suppressor gene.
As shc~m later in Examples, when the expression of
an apoptosis suppressor gene which has been forcibly
25 expressed to promote polvploidization leads to an increase
in production efficiency of CD4la-positive/CD42b-positive
functional platelets.
Suppression of expression of or removal of an
apoptosis suppressor gene is started 15 days, preferably 10
days, more preferably from 3 to 7 days, still more
preferably about 3 days before collection of platelets.
In this step, expression of not only an exogenous
apoptosis suppressor gene but also an endogenous apoptosis
suppressor gene may be suppressed. Inhibition of the
expression or function of a p53 gene product may be
conducted successively after the present step.
10 LO0431 The culturing temperature is not particularly
limited insofar as the effect of the present invention can
be produced. Culturing may be conducted at from 35OC to
40°C, with from 37'C to 39' being suited as shown later in
Examples.
15 LO0441 In the production method according to the present
invention, the step of culturing polyploidized
megakaryocytes may be conducted under serum-free and/or
feeder cell-free conditions. As shown later in Examples,
no large difference was found in the production amount of
20 platelets between culturing in a medium containing a fetal
bovine serum and culturing in a serum-free medium. However,
a ratio of CD42b-positive platelets was greater when the
cells was cultured in a serum-free medium or a feeder cellfree
medium. If the platelet production step can be
25 conducted in a serum-free and feeder cell-free medium, the
platelets thus obtained can be used clinically without
ca-using the problem of immunogenicity.
Production of platelets without using a feeder cell
can suppress a production cost and is suited for mass
production because adhesion of the feeder cell is not
required and therefore suspension culture can be conducted
5 in a flask or the like. When the feeder cell is not used,
a conditioned medium may be used. The conditioned medium
is not particularly limited and can be prepared by those
skilled in the art in a known manner. For example, it can
be obtained, for example, by culturing a feeder cell as
10 needed and then removing the feeder cell from the cultured
product by using a filter or the like.
100451 In one mode of the method of producing platelets
according to the present invention, a ROCK inhibitor and/or
actomyosin complex function inhibitor is added to a medium.
15 The ROCK inhibitor and actomyosin complex function
inhibitor similar to those used in the above-mentioned
method of producing poly-ploidized megakaryocytes can be
used. Examples of the ROCK inhibitor include Y27632.
Examples of the actomyosin complex function inhibitor
20 include blebbistatin, a myosin heavy chain I1 ATPase
inhibitor. The ROCK inhibitor may be added singly, the
ROCK inhibitor and the actomyosin complex function
inhibitor may be added individually; or they may be added
in combination.
2.5 The ROCK inhibitor and/or actomyosin complex
function inhibitor is added preferably in an amount of from
0.1 pM to 30 pM, for example, from 0.5 pM to 25 pM, from 5
pM to 20 pM, or the like.
The culturing term after addition of the ROCK
inhibitor and/or actomyosin complex function inhibitor may
be for one day to 15 days. It may be for 3 days, 5 days, 7
5 days, or the like. By adding the ROCK inhibitor and/or
actomyosin complex function inhibitor, a ratio of CD42bpositive
platelets can be increased further.
[0046] The embodiment of the present invention includes a
kit for promoting polyploidization of megakaryocytes and
10 producing mature megakaryocytes and/or platelets. The kit
includes, as well as an expression vector and the like
necessary for inducing intracellular expression of the
oncogene, any of the above-mentioned genes (i) to (iii) , a
BCL-XL gene, or the like, and a reagent, a medium for
15 culturing cells, a serum, a supplement such as growth
factor (for example, TPO, EPO, SCF, Heparin, IL-6, IL-11,
or the like), an antibiotic, and the like. In addition,
the kit includes, when, for example, cells derived from ES
cells or iPS cells are used, an antibody (for example,
20 antibody against Flkl, CD31, CD34, UEA-I lectin, or the
like) to confirm a marker for identifying a net-like
structure prepared from these cells. The reagent, antibody,
and the like included in the kit are supplied in any kind
of vessels that enable a constituting ingredient to
25 effectively keep its activity and cause neither adsorption
of it to the material of the vessel nor deterioration of it.
The kit of the present invention may further
include megakaryocytes before polyploidization in which the
oncogene and any of the above-mentioned genes (i) to (iii)
have been forcibly expressed.
[0047] The "cells" described herein are derived from
5 humans and non-human animals (for example, mice, rats,
cattle, horses, pigs, sheep, monkeys, dogs, cats, and
birds). Although no particular limitation is imposed,
human-derived cells are particularly preferred.
lo0481 The present invention will hereinafter be described
10 in further detail by showing Examples. It should however
be borne in mind that the present invention is not limited
to or by Examples.
EXAMPLES
15 [0049] 1. Preparation of megakaryocytes before
polyploidization
1-1. Preparation of megakaryocytes before polyploidization
from ES cells
In order to study polyploidization of
20 megakaryocytes, megakaryocytes before polyploidization were
prepared from ES cells (refer to Patent Document 3 for
details).
A human ES cell line [KhES-3) was cultured for 14
days in the presence of 20 ng/ml of VEGF to prepare a net-
2 5 like str11ctllre Hematonoi~tic nroqenitor cells removed
from the resulting net-like structure were recovered and
seeded on 10T1/2 cells to give a cell count of lxl~~/well.
The hematopoietic progenitor cells thus prepared
were infected with a c-MYC-2A-BMI1-containingp Mx tet off
c-MYC 2A BMIl retrovirus vector three times every 12 hours
at MOI=10 (confirmed using Jurkat cells) to induce
5 expression of c-MYC and BMIl (Patent Document 3). The pMx
tet off c-MYC 2A BMIl vector allows the expression of a c-
MYC gene and a BMIl gene in the presence of estradiol,
while it suppresses the expression of the c-MYC gene and
the BMIl gene in the presence of doxycycline (Dox) and
10 absence of estradiol.
Simultaneously with the first infection, 2 mM of
estradiol was added and 12 hours after the final infection,
the virus was removed. In that stage, the released amount
of CD42b-positive platelets was small even if the
15 expression of the c-MYC gene or BMIl gene was turned off,
suggesting that the cells were immature megakaryocytes.
These immature cells may hereinafter be called "iMKPC-type
The cytokine dependence of iMKPC-type I was studied
20 by seeding 2x10~i MKPC-type I cells on lOT1/2 feeder cells
and culturing them for 14 days at 37OC under the conditions
shown in FIG. 1A in the presence of 2 uM of P-estradiol
while using the following cytokines: SCF (50 ng/ml), TPO
(50 ng/ml) , and EPO (6 U/ml) . A population in which
25 proliferation was confirmed on Day 4 and Day 11 was counted
for cell number, and 2 x 1 0 ~ cells were replated, while the
medium of the other populations was changed.. The number of
cells was counted on Day 8 and Day 14 and 2x10~ cells were
replated. At the same time, on Day 8, some of the cells
were analyzed with a flow cytometer after staining with a
CD41 antibody, a CD42b antibody, and a GPA antibody.
5 The results are shown in FIGS. 1B and C. As shown
in FIG. lB, proliferation of iMKPC-type I cells strongly
depended on SCF. As shown in FIG. lC, the cells of any
population were almost CD41 positive, but in the
populations without SCF, the CD41+/CD42b- population showed
10 a marked reduction in proliferation. The ~ ~ 4 1 + / ~ ~ 4 2 b -
population is a population showing good proliferation among
the iMKPC-type I cells.
These findings including SCF dependent
proliferation suggest that iMKPC-type I cells are immature
15 megakaryocytes.
[0050] 1-2. Preparation of megakaryocytes before
polyploidization from CD34-positive cells derived from cord
blood
It was confirmed that megakaryocytes before
20 polyploidization can be prepared from CD34-positive cells
derived from the cord blood in a similar manner to that
using ES cells or iPS cells.
More specifically, CD34-positive cells derived from
the cord blood were infected three times with pMx-c-MYC and
75 DN~arn RMT1 ~ r i r l l s e q (each r e t r o ~ r i r l ~vesc tor) at MOI=10 and
the number of CD4la (megakaryocyte marker) positive cells
on Day 14 and Day 21 was counted using FACS. Mock (Empty
vector) was used as a control.
The results are shown in FIG. ID. Compared with
Mock, proliferation of CD41 positive megakaryocytes was
observed from a population in which c-MYC and BMIl had been
5 forcibly expressed. It was therefore confirmed that
megakaryocytes before polyploidization can be obtained from
cells derived from the cord blood in a similar manner to
that described in 1-1.
[0051] 2. Preparation of polyploidized megakaryocyte from
10 megakaryocytes before polyploidization
2-1. Influence of BCL-XL expression on polyploidization
On Day 23 after infection with a pMx tet off c-MYC
2A BMIl retrovirus vector containing c-MYC-2A-BMI1 in
Section 1-1, the cells were infected once with doxycycline-
15 inducible Lv-BCL-XL-GFP (lentivirus vector) at MOI=10. The
vector was prepared by introducing PCR-amplified cDNA of
BCL-XL into an Ai-Lv tet on vector (clontech) treated with
EcoRI and BamHI by using an In-Fusion advance PCR cloning
kit (clontech). Twenty four hours after infection, the
20 virus was removed. By removing estradiol and adding
Doxycyline, expression of c-MYC and BMIl was suppressed and
at the same time, expression of BCL-XL was started.
[00521 2-2. Influence of knock drown of p53 gene on
polyploidization
25 A p53 gene was knocked down by infecting it with a
FG12-sh p53 lentivirus vector in addition to doxycyclineinducible
Lv-BCL-XL-GFP lentivirus vector. The cells thus
obtained may hereinafter be called "iMKPC-type 11" cells.
100531 2-3. CyLokine dependence of iMKPC-type I1
On 10T1/2 feeder cells, 2x10' iMKPC-type I1 cells
were seeded, followed by culturing in the presence of 0.5
pg/ml of Dox at 3g°C for 21 days under the conditions of
cytokines, that is, SCF (50 ng/ml) , TPO (50 ng/ml) , and EPO
(6 U/ml). The results are shown in FIG. 2A. It was
confirmed that the iMKPC-type I1 cell line shows
proliferation in the absence of cytokine, but proliferation
is promoted further by the addition of TPO. Although the
iMKPC-type I cells were SCF-dependent and TPO had no
influence on their proliferation, the type I1 cells showed
enhanced proliferation in the presence of TPO and SCF had
no influence on proliferation.
[0054] 2-4. Analysis of surface marker of iMKPC-type I1
On Day 21 after addition of cytokine, the cells
were analyzed with a flow cytometer after staining with a
CD41 antibody, a CD42b body, and a GPA antibody. The
results are shown in FIG. 2B. The TPO-added population
showed higher expression in CD4la and CD42b compared with
the other populations, which suggests that it is a
population further committed to megakaryocytes.
[00551 2-5. Morphological change and influence of
blebbistatin
Microscopic observation results of iMKPC-type I and
type I1 are shown in FIG. 3. It was observed that by
turning off the expression of c-MYC and BMI1, forcing
expression of BCL-XL, and knocking down the p53 gene, the
cells become increasingly polyploidized. It was also
confirmed that the cells were polyploidized further by the
addition of blebbistatin (5 pg/ml).
5 As shown in Sections 2-3 to 2-5, in the stage of
iMKPC-type 11, megakaryocytes proliferate, a ratio of CD42b
positive platelets in all the released platelets became
higher, some of the cells were polyploidized, and cytokine
dependence changed to TPO, suggesting that proliferation
10 and maturation occured in parallel to each other.
[0056] 2-6. Influence of suppression of BCL-XL expression
On 10T1/2 feeder cells, 2x10' iMKPC-type I1 cells
were seeded and long-term culture was conducted at 39OC in
the presence of 0.5 pg/ml of Dox (BCL-XL ON) or absence of
15 it (BCL-XL OFF). Every two to five days, the number of
cells was counted and 2x10~ cells were replated.
The results are shown in FIG. 4. Suppression of
BCL-LX by Dox OFF decreased a proliferation rate of iMKPCtype
I1 cells and after a long term (after 100 days), the
20 cells lost their proliferation capacity, suggesting that
BCL-XL is indispensable for long-term proliferation of
iMKPC-type I1 cells.
LO0571 2-7. Study of the influence of the other treatments
on polyploidization
25 2-7-1. Influence of ROCK inhibitor
After introduction of MYC and MBIl genes, the
resulting megakaryocytes (about 10') were cultured for
about 30 days in the absence of doxycycline and presence of
estradiol under the culturing conditions of 37OC and
presence of 5%C02 to proliferate them to about 10".
Culturing was continued further while changing the
5 condition to the presence of doxycycline and absence of
estradiol in order to suppress the expression of the MYC
gene and the.BMI1 gene in the thus proliferated
megakaryocytic cell line and adding an ROCK inhibitor
(Y27632; product of Wako Pure Chemicals) to the culture
10 medium to give a concentration of 10 pM in order to
determine the influence of Y27632 on polyploidization. On
Day 7 after culturing was started while adding Y27632 to
the culture medium, the degree of polyploidization was
studied using FACS (FIG. 5). The cells added with the ROCK
15 inhibitor showed an increase in the number of cells with 4N
(upper graph of FIG. 5 (Rock i), open sqnare in the lower
graph) compared with that in the cells added without the
inhibitor (upper graph of FIG. 5 (vehicle), filled square).
This revealed that the ROCK inhibitor promotes
20 polyploidization of megakaryocytes before polyploidization
which were derived from ES cells and acquired proliferation
capacity as a result of expression of a C-MYC gene and a
BMIl gene.
[00581 2-7-2. Influence of ROCK inhibitor + culturing
25 under high-temperature condition
It has been reported to date that as a result of
culturing immature megakaryocytes at a temperature, for
example 3g°C, higher than the normal culturing temperature,
maturation of megakaryocytes is promoted, including
polyploidization and proplatelet formation (Non-patent
Document 5). In order to confirm this in megakaryocytes
5 before polyploidization which have been derived from ES
cells, an expression level of genes (GATA1, PF4, NFE2, and
P-tubulin) known to show enhanced expression with
maturation of megakaryocytes was studied using quantitative
PCR.
10 Proliferation of megakaryocytes before
polyploidization was promoted. In order to suppress the
expression of the MYC gene and BMIl gene in the resulting
megakaryocytes before polyploidization, culturing was
conducted for 5 days under changed conditions, that is, in
15 the presence of doxycycline and the absence of estradiol
and at a culturing temperature of 39OC. Then, quantitative
PCR was conducted to measure the expression level of the
respective genes (FIG. 6). As a result, it was found that
the expression levels of the genes serving as an indicator
20 of maturation of megakaryocytes were greater in culturing
at 3g°C than in culturing at 37OC.
[0059] 2-7-3. Influence of ROCK inhibitor + forced
expression of BCL-XL gene
Expression of MYC/BMI~ in megakaryocytes before
25 polyploidization was suppressed and at the same time, a
lentivirus vector similar to that used in 2-1 for inducing
expression of BCL-XL was introduced into the cells in the
presence of doxycycline.
It was studied if polyploidization of
megakaryocytic progenitor cells derived from an ROCK
inhibitor by the presence or absence of expression of a
5 BCL-XL gene (FIG. 7) .
The degree of polyploidization was studied (ploidy
assay) by suppressing expression of MYC/BMI~ in the
presence of 10 pM of Y27632 while inducing suppression of
BCL-XL, and culturing the resulting cells for 7 days. It
10 was confirmed that the number of polyploidized cells with
8N or greater significantly increased in the BCL-XL
expressed cell line (a shaded bar in FIG. 7B) compared with
a cell line (a blank bar in FIG. 7B) in which BCL-XL was
not expressed. In addition, it was observed that the
15 number of the cells in which BCL-XL was expressed showed a
gradual increase (. in FIG. 8) while the number of the
cells in which BCL-XL was expressed decreases (A in FIG. 8) .
This suggests that in order to avoid oncogene
dependence in megakaryocytes before polyploidization which
20 acquired high proliferation capacity as a result of forced
expression of the oncogene, genes suppressing apoptosis
such as BCL-XL gene were effective.
[00601 2-7-4. Influence of ROCK inhibitor + forced
expression of BCL-XL gene + suppression of p53 gene
25 It was studied whether suppression of expression of
p53 promoted polyploidization of megakaryocytes before
polyploidization or not.
Expression of the p53 gene was suppressed as in 2-2
by lentivirus infection at ~v101=10 by using a lentivirus
FG12 vector in which one promoter, shp53 had been
introduced.
5 After suppression of expression of MYC/BMI~, forced
expression of BCL-XL, and suppression of expression of p53
in the presence of Y27632, culturing was conducted for 7
days at 39OC. After culturing, the degree of
polyploidization was studied. As a result, it was found
10 that compared with control cells (black bar in FIG. 9B) in
which p53 was not knocked down, cells (blank bar in FIG.
9B) in which p53 was knocked down showed an increase in the
number of the cells with 8N and polyploidization was
promoted.
15 [0061] 2-7-5. Influence of ROCK inhibitor + forced
expression of BCL-XL gene + suppression of p53 gene +
valproic acid
Influence on polyploidization was studied by
further treating the cells, which was subjected to the
20 treatment of 2-7-4, with valproic acid. After suppression
of expression of MYC/BM11, forced expression of BCL-XL,
treatment with the ROCK inhibitor (10 pM), and suppression
of expression of p53, valproic acid (final concentrstion:
0.5 rnM) was added to the medium and culturing was conducted
25 at 3g°C for 7 days. As a result, it was found that cells
(blank bar in FIG. 10B) treated with valproic acid showed
promoted polyploidization compared with control cells
(black bar in FIG. 10B) which was not subjected to valproic
acid treatment.
[0062] 2-7-6. Influence of forced expression of BCL-XL
gene + myosin heavy chain IIA/B ATPase inhibitor and
5 influence of ROCK inhibitor + forced expression of BCL-XL
gene + suppression of p53 gene + valproic acid + myosin
heavy chain IIA/B ATPase inhibitor
It was studied whether treatment of megakaryocytes
the polyploidization of which have not proceeded
10 sufficiently with blebbistatin, that is, a myosin heavy
chain IIA/B ATPase inhibitor had an influence on the degree
of polyploidization. After suppression of expression of
MYC/BMIl, forced expression of BCL-XL, and treatment with
blebbistatin (10 pg/ml), the resulting cells were cultured
15 at 3g°C for 7 days. It was found that the number of cells
with 8N or greater was greater in the cells subject,= d to
blebbistatin treatment (blank bar in FIG. 11) than in the
cells not subjected to blebbistatin treatment (black bar in
FIG. llb), showing promoted polyploidization.
20 Next, the degree of polyploidization when the
blebbistatin treatment was used in combination with the
other treatments was studied. The cells were subjected to
blebbistatin treatment in addition to the above-mentioned
treatments in Section 2-7-5 and influence of them on
75 pnl TI oi d i x a t i on was studied. After suppression of
expression of MYC/BMIl, forced expression of BCL-XL,
treatment with a ROCK inhibitor (10 pM), suppression of
expression of p53, and treatment with valproic acid (0.5
mM), treatment with blebbistatin (10 pg/ml) was conducted
and then, the resulting cells were cultured at 3g°C for 7
days. It was found that the number of cells with 8N or
5 greater was greater in the cells subjected to blebbistatin
treatment (blank bar in FIG. 12) than in the control cells
not subjected to blebbistatin treatment (black bar in FIG.
12b), showing that the treatments promoted polyploidization.
In addition, after culturing for 7 days, the cells treated
10 with blebbistatin showed a slight deterioration in
proliferation capacity (upper graph in FIG. 13) but
hypertrophy of cytoplasm was observed. Induction to mature
megakaryocytes was therefore confirmed (lower graph in FIG.
13).
15 [0063] 3. Production of platelets from polyploidized
megakaryocytes
3-1. Influence of suppression of expression of BCL-XL on
platelet production (1)
On 10T1/2 feeder cells, 2x10~ iMKPC-type I1 cells
20 obtained in Section 2-2 were seeded, followed by culturing
at 3g°C in the presence of 0.5 pg/ml of Dox (BCL-XL ON) or
after removal of Dox from the culture medium (BCL-XL OFF).
On Day 3-4, the megakaryocytes and the platelets in the
culture medium were analyzed with a flow cytometer after a
25 staining with a CD41 antibody and a CD42 antibody.
The results are shown in FIG. 14. It was found
that the population (B) in which expression of BclxL was
suppressed by Dox OFF becomes composed mainly of mature
megakaryocytes in which CD42b was expressed (B) compared
with the megakaryocytes (A) in which BCL-XL was expressed
by Dox ON. It was also found that with regard to the
platelets released from them, a ratio of the platelets in
which CD42b necessary for function expression was expressed
became greater (D) than that of the platelets (C) released
from the megakaryocytes of BCL-XL ON.
[00641 3-2. Influence of suppression of expression of BCLXL
on platelet production (2)
F I G . 15 shows the measurement results of the number
of cells when BCL-XL expression was ON or OFF, based on the
results similar to those shown in Section 3-1. The number
of platelets became markedly greater in the population in
which expression of BCL-XL has been suppressed.
A shows the number of CD42b-positive platelets; B
shows the number of CD4la-positive/C~42b-positive
megakaryocytes, and C shows the number of CD4la-positive
megakaryocytes.
No influence of BclxL expression on the number of
megakaryocytes in which CD41 was expressed could be found
( C ) , but it was presumed that an expression ratio of CD42b
in CD41+ increased due to suppression of expression of
BclxL and the number of mature megakaryocytes increased.
[ O f 1 6 5 1 3 - 3 . T n f l ~ ~ e n coef cul.turing temperature on platelet
production
On 10T1/2 feeder cells, from 2 to 3x10~ iMKPC-type
I1 cells were seeded and cultured for 3 days at culturing
temperatures of 35, 37, and 3g°C while adding or not adding
0.5 pg/m~ of Dox. The platelets contained in the
supernatant were analyzed with a flow cytometer after
staining with a CD41 antibody and a CD42b antibody. With
37OC Dox+ as 1, the mean and standard deviation of the
number of CD41+ CD42b+ platelets of each population are
shown in FIG. 16.
It was found from the results that the culturing
temperature of 37OC or 3g°C is adequate. In the tests
conducted later, the culturing temperature was set at 37OC.
[0066] 3-4. Influence of feeder cells, use of serum for
culture, and presence or absence of blebbistatin on
platelet production
A platelet production efficiency was measured by
using the following conditions in combination as shown in
FIG. 17: use/non-use of feeder cells, use/non-use of
Conditioned Medium, use of serum medium/use of serum-free
medium, and administration/non-administration of
blebbistatin.
Conditioned Medium was prepared by seeding 8x10~
MMC-treated 10~1/2 cells on a 10-cm dish subjected to
gelatin coating, changing the medium with 10 ml of a
differentiation medium (EBM) containing SCF (50 ng/ml) and
TPO (50 ng/ml) and further containing 15% serum or not
containing a serum on the next day (after adhesion of
cells), recovering the medium and adding 10 ml of a new
medium (containing SCF and TPO) 24 hours later, pooling
three-day's Conditioned Medium, and filtering the medium
through a 0.22-pm filter to remove the 10T1/2 cells. When
the resulting medium was used for tests, SCF and TPO were
5 added newly.
In a feeder cell using population, from 2 to 3x10'
iMKPC-type I1 cells were seeded on 10T1/2 feeder cells,
while in the non-use population, the cells were seeded on a
gelatin-coated dish similarly. In the serum medium using
10 population, a 15% serum-containing differentiation medium
or Conditioned Medium was used, while in the blebbistatin
administration population, 5 pM of blebbistatin was added
to the medium. Culturing was conducted at 37OC for 3 days.
The platelets contained in the supernatant of the
15 medium were analyzed with a flow cytometer after staining
with an antibody CD41 and an antibody CD42b. With the
cells obtained by culturing on feeder cells while adding
15% serum without adding blebbistatin as normal condition
cells, the mean and standard deviation of a ratio of the
20 number of CD41+ CD42b+ platelets in each population with
respect to those of the normal condition are shown in bar
graph in FIG. 17. No significant difference in the
platelet production amount was found among the populations.
Comparison in the expression level of CD42b in
-7 c-; C n 4 l --n o s l t i ~ r ep l a t ~ 1 _ e t ci, n earh nnm~alt i o n fa r a t i o nf
average fluorescence intensity of each population to
average fluorescence intensity of normal condition) is
shown in FIG. 18. Under serum-free and feeder cell-free
conditions, the platelets showing the highest CD42b
expression were produced. Influence of blebbistatin was
not found.
5 100671 One example of the optimized conditions is shown in
FIG. 19. A ratio of CD41-positive and CD42b (which may be
called "GP1ba")-positive platelets when cultured under the
optimized conditions is shown in FIG. 20.
As shown in FIG. 20, in the platelets produced from
10 the polyploidized megakaryocytes obtained by the present
invention, about 20% were CD41-positive and CD42b positive.
When expression of BCL-XL was suppressed, the ratio
increased to 55% and when culturing was conducted while
removing the feeder cells and the serum in addition, the
15 ratio increased even to 81%.
[0068] 4. Importance of CD42b expression for platelet
function (reference)
An inhibitory effect of an HIPl antibody on a
ristocetin agglutination reaction (agglutination reaction
20 via vWF and a receptor [hetero pentamer composed of GPIba,
GPIX, and the like] on platelets) was measured using human
peripheral blood platelets. The HIPl antibody was a
function inhibiting antibody of GPIba.
After the effect of GPIba was inhibited by
25 suspending 1x10~ platelets in 50% blood plasma, adding an
HIPl antibody thereto, and pre-culturing at 37OC for 3
seconds, ristocetin (final concentration: 2 mg/ml) was
added to induce an agglutination reaction and light
transmission was monitored for 7 minutes. The maximum
light transmission (showing agglutination intensity) of
each population is shown in bar graph in FIG. 21. The HPIl
5 antibody completely inhibited agglutination due to
GPIb/alpha-von Willebrand factor (vWF) association at a
concentration of 10 pg/ml or greater.
[00691 Next, 100 ug of an HIPl antibody or control IgG was
administered into NOG mice and platelets stained with TAMRA
(red dye) were transplanted. The vascular endothelium was
exposed to laser to damage it and induce thrombus formation.
The number of human platelets (red) which had contributed
to the thrombus formation was counted.
The mean and standard deviation of the number of
15 human platelets in the thrombi corrected with a unit blood
vessel length are shown in bar graph (FIG. 22). In the
HIPl antibody administered group, contribution of human
platelets to thrombus was inhibited.
It was therefore found that GPIba (CD42b) is a
20 molecule playing an important role in thrombus formation
both in vivo and in vitro.
Industrial Applicability
[00701 The present invention provides a method of
7 5 promoting po1yploi.d i zation of megakaryocyt ic proqenitor
cells and moreover, efficiently inducing release of
platelets. In particular, the method of the present
invention is very effective for the preparation of
megakaryocytes or platelets in vitro from various stem
cells and it greatly contributes to the development of
remedies in medical fields or blood products.

L 3 ' CLAIMS
1. A method of producing polyploidized
megakaryocytes, comprising forcing expression of an
5 apoptosis suppressor gene in megakaryocytes before
polyploidization, and culturing the cells.
2. The method according to claim 1, wherein the
apoptosis suppressor gene is a BCL-XL gene.
3. The method according to claim 1 or 2, wherein
10 in the culturing step, expression or function of a p53 gene
product is inhibited.
4. The method according to any one of claims 1 to
3, wherein in the culturing step, the megakaryocytes before
polyploidization are subjected to at least one of the
15 following (a) to (c) :
(a) treatment with an actomyosin complex function
inhibitor;
(b) treatment with an ROCK inhibitor; and
(c) treatment with an HDAC inhibitor.
20 5. The method according to claim 4, wherein the
ROCK inhibitor is Y27632; the HDAC inhibitor is valproic
acid; and the actomyosin complex function inhibitor is
blebbistatin.
6 . The method according to any one of claims 1 to
25 5, wherein the culturing step is conducted at a temperature
higher than 37OC.
7. The method according to any one of claims 1 to
6, wherein the megakaryocytes before polyploidization are
obtained by forcing expression of an oncogene and any of
the following genes (i) to (iii) in the cells at any
differentiation stage from hematopoietic progenitor cells
5 to megakaryocytes before polyploidization:
(i) a gene suppressing expression of a p16 gene or
a p19 gene;
(ii) a gene suppressing expression of an Inkla/Arf
gene; and
10 (iii) a polycomb gene, and
culturing and proliferating the resulting cells.
8. The method according to claim 7, wherein a c-
MYC gene is used as the oncogene and BMIl is used as the
any of the genes (i) to (iii) .
15 9. The method according to claim 7 or 8, wherein
the hematopoietic progenitor cells are derived from cells
selected from the group consisting of iPS cells, ES cells,
hematopoietic stem cells derived from cord blood, bone
marrow blood, or peripheral blood, and hematopoietic stem
20 cells.
10. A blood cell composition comprising the
polyploidized megakaryocytes produced by the method
according to any one of claims 1 to 9.
11. A method of producing a platelet, comprising:
25 obtaining polyploidized megakaryocytes by using the
method according to any one of claims 1 to 9 and culturing
the cells; and
collecting a platelet from the culture of the
polyploidized rnegakaryocytes.
12. The method according to claim 11, wherein the
step of culturing the polyploidized megakaryocytes is
5 conducted while suppressing the expression of the apoptosis
suppressor gene that has been forcibly expressed, or after
the apoptosis suppressor gene is removed from the cells.
13. The method according to claim 11 or 12,
wherein the step of culturing the polyploidized
10 megakaryocytes is conducted in the absence of a serum
and/or in the absence of a feeder cell.
14. The method according to any one of claims 11
to 13, wherein the step of culturing the polyploidized
megakaryocytes is conducted from 1 day to 15 days.
15 15. The method according to any one of claims 11
to 14, wherein the step of culturing the polyploidized
megakaryocytes is conducted at 37OC.
16. The method according to any one of claims 11
to 15, wherein in the step of culturing the polyploidized
20 megakaryocytes, an ROCK inhibitor and/or an actomyosin
complex function inhibitor is added to a medium.
17. The method according to claim 16, wherein the
ROCK inhibitor is Y27632 and the actomyosin complex
function inhibitor is blebbistatin.
25 18. A platelet produced by the method according to
any one of claims 11 to 17.
19. A blood product comprising the platelet
according to claim 18.
Dated this 29.1 1.2013 wp WHA ASTAVA]
OF REMFRY & SAGAR
ATTORNEY FOR THE APPLICANT[S]