U.S. patent application number 15/401832 was filed with the patent office on 2017-04-27 for cardiac cell culture material.
The applicant listed for this patent is METCELA INC.. Invention is credited to Takahiro IWAMIYA, Katsuhisa MATSUURA.
Application Number | 20170112880 15/401832 |
Document ID | / |
Family ID | 55063910 |
Filed Date | 2017-04-27 |
United States Patent
Application |
20170112880 |
Kind Code |
A1 |
IWAMIYA; Takahiro ; et
al. |
April 27, 2017 |
CARDIAC CELL CULTURE MATERIAL
Abstract
The purpose of the present invention is to provide a cardiac
cell culture material which specifically acts on cardiac cells. In
addition, another purpose of the present invention is to provide
artificial organ material obtained by culturing by using said
cardiac cell culture material, and a method for producing the same.
Thus, provided is a cardiac cell culture, wherein functional
cardiac tissue is favorably built by using a cardiac cell culture
material containing VCAM-1.
Inventors: |
IWAMIYA; Takahiro; (Tokyo,
JP) ; MATSUURA; Katsuhisa; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
METCELA INC. |
Yamagata |
|
JP |
|
|
Family ID: |
55063910 |
Appl. No.: |
15/401832 |
Filed: |
January 9, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/JP2015/050028 |
Jan 5, 2015 |
|
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15401832 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2502/1329 20130101;
C12N 1/00 20130101; C12N 2501/58 20130101; C12N 5/0657 20130101;
A61K 38/1774 20130101; A61P 9/00 20180101; A61K 35/33 20130101;
C12N 5/0656 20130101; C12N 5/10 20130101; A61F 2/24 20130101 |
International
Class: |
A61K 35/33 20060101
A61K035/33; A61K 38/17 20060101 A61K038/17; C12N 5/077 20060101
C12N005/077 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 2014 |
JP |
2014-142804 |
Claims
1. A cardiac cell culture material comprising a fibroblast
expressing VCAM-1 protein.
2. The cardiac culture material according to claim 1 used for
culturing to construct a cardiac tissue.
3. The cardiac cell culture material according to claim 1, wherein
the fibroblast is a cardiac-derived fibroblast.
4. The cardiac cell culture material according to claim 1, wherein
the fibroblast is an epicardial-derived cell-derived
fibroblast.
5. A cell culture substrate, wherein a wall surface and/or a bottom
surface of the culture substrate having the wall surface and/or the
bottom surface are coated with the cardiac cell culture material
according to claim 1.
6. An artificial organ material obtained by co-culturing a cardiac
cell with the cardiac cell culture material according to claim
1.
7. A method of producing an artificial organ material comprising a
step of co-culturing a cardiac cell with the cardiac cell culture
material according to claim 1.
8. A method for curing heart diseases by attaching a sheet to a
damaged part of the heart wherein, the sheet is constructed from
the cardiac cell culture material according to claim 1.
9. A method for constructing a functional cardiac tissue by
attaching a sheet to a damaged part of the heart wherein, the sheet
is constructed from the cardiac cell culture material according to
claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a continuation-in-part of International Application
PCT/JP2015/050028, filed on Jan. 5, 2015, and designated the U.S.,
and claims priority from Japanese Patent Application 2014-142804
which was filed on Jul. 11, 2014, the entire contents of each of
which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a cardiac cell culture
material and a cell culture substrate on which a wall surface
and/or a bottom surface of the culture substrate having the wall
surface and/or the bottom surface are coated with the cardiac cell
culture material. In addition, the present invention relates to an
artificial organ material obtained by culturing a cardiac cell by
using the cardiac cell culture material, and a method for producing
the same.
BACKGROUND ART
[0003] Fibroblasts exist in almost all of vertebrate, and when
tissue is injured by trauma and ischemia, the injured area is
replaced with fibrous tissue in accordance with fibroblasts
proliferation and the abundant extracellular matrix deposition.
Likewise, in a variety of heart disease such as myocardial
infarction and cardiomyopathies, a lot of cardiomyocytes were lost
and also fibrous tissue replaces that area, which leads to cardiac
remodeling and heart failure accompanied with excess hemodynamics
stress and neurohumoral stimulation. Although neurohumoral factors
such as angiotensin II and endothelin-1 are well known to
contribute to promote the cardiac remodeling via blood pressure
elevation, cardiomyocyte apoptosis and local inflammation, cardiac
fibroblasts have been reported to secrete those factors. Cardiac
fibroblasts are also known to play a critical role in heart
developments. Interconnected cellular processes in a cardiac
fibroblast form a network of collagen, fibroblasts and myocytes.
Although cardiomyocyte proliferation is indispensable process of
formation of thick ventricular wall and embryonic cardiac
fibroblasts have also been reported to promote myocardial mitotic
activity through .beta.-1 integrin signaling. The cardiac
fibroblasts dominant causative substance has been unclear. Herein
cardiac fibroblasts multifariously act on heart development and
pathogenesis and the importance of understanding of mutual
interaction and underlying mechanisms between cardiomyocytes and
cardiac fibroblasts have been widely recognized. However the
uncertain properties of cardiac fibroblasts were the bottle-neck
for it and it is required to reveal functional and molecular
biological characteristics of cardiac fibroblasts.
[0004] Heart tissue engineering is promising methods for not only
regenerative medicine, but also tissue models. Among cardiac tissue
engineering methods, cell sheet-based cardiac tissue using
temperature responsive culture dishes have been developed.
Previously, it was reported that layering of cardiac cell sheets
containing neonatal rats-derived cardiomyocytes, fibroblasts and
endothelial cells on the various types of vascular bed enabled to
fabricate three-dimensional vascularized viable cardiac tissue (Non
patent documents 1 to 3). Since cell sheet-based tissue engineering
does not need any scaffold, it requires some amounts of
extracellular matrices to construct cell sheets. Consistent with
the evidences that left ventricle is mainly composed of fibroblasts
and cardiomyocytes, some amounts of fibroblast are indispensable to
fabricate cardiac cell sheets when using purified embryonic stem
cell-derived cardiomyocytes (Non patent document 4). Since recent
reports have suggested that cell-cell interaction between
cardiomyocytes and non-myocytes is important for heart physiology
and pathogenesis (Non patent document 5), fibroblasts function
might also affect the function of the engineered cardiac tissue and
it might be prerequisite to select the suitable fibroblasts to
fabricate the cardiac tissue in vitro for tissue models. However it
remains unclear whether cardiac fibroblasts have the specific
function for cardiomyocytes compared with other types of
fibroblasts and the related molecular mechanisms.
[0005] As mentioned above, since the cardiac fibroblasts play an
important role in heart developments, and the onset or cure of
heart diseases, it is required to separate cardiac fibroblasts that
specifically act on cardiac cells such as cardiomyocytes from other
fibroblasts, and to sample the cardiac fibroblasts. According to
the recent studies, it has been revealed that fibroblasts, which
were previously considered as a uniform cell type, have a great
variety of phenotypes, and that the phenotypes differ depending on
a load state of existing organs, tissues or cells.
[0006] However, the function of fibroblasts is not clearly known,
and fibroblasts are only cells morphologically classified.
Therefore, among fibroblasts, it is difficult to select only one
type thereof having a specific function.
[0007] Meanwhile, with respect to vascular cell adhesion molecule-1
(VCAM-1, CD106) and .alpha.4 integrin, Kwee, et al. reported that
VCAM-1 was expressed on embryonic day 11.5 at epicardium,
cardiomyocytes, ventricular septum, and the like. It was also
reported that, although the expression of .alpha.4 integrin was
recognized at similar areas as those of VCAM-1, .alpha.4 integrin
was not expressed in ventricular septum (Non patent document 6).
Moreover, it was reported that, on embryonic day 11.5, there are
embryonic death resulting from inhibition of formation of the
placenta, and deformity due to decrease in dense layers of
ventricular myocardium and ventricular septum in an embryo that is
defective in VCAM-1. Yang, et al. also reported an epicardium
defect in .alpha.4 integrin null embryo of embryonic day 11.5 (Non
patent document 7). Accordingly, it is considered that VCAM-1 and
.alpha.4 integrin mainly contribute to formation of cardiac cells
and epicardium in the embryonic stage.
CITATION LIST
Non-Patent Document
[0008] Non-Patent Document 1: Shimizu T, et al., Fabrication of
pulsatile cardiac tissue grafts using a novel 3-dimensional cell
sheet manipulation technique and temperature-responsive cell
culture surfaces. Circulation research. 2002; 90:e40 [0009]
Non-Patent Document 2: Sekiya S, et al., Bioengineered cardiac cell
sheet grafts have intrinsic angiogenic potential. Biochemical and
biophysical research communications. 2006; 341:573-582 [0010]
Non-Patent Document 3: Shimizu T, et al., Cell sheet engineering
for myocardial tissue reconstruction. Biomaterials. 2003;
24:2309-2316 [0011] Non-Patent Document 4: Matsuura K, et al.,
Hagiwara N, Zandstra P W, Okano T. Creation of mouse embryonic stem
cell-derived cardiac cell sheets. Biomaterials. 2011; 32:7355-7362
[0012] Non-Patent Document 5: Deschamps A M, et al., Disruptions
and detours in the myocardial matrix highway and heart failure.
Current heart failure reports. 2005; 2:10-17 [0013] Non-Patent
Document 6: Kwee L, et al., Defective development of the embryonic
and extraembryonic circulatory systems in vascular cell adhesion
molecule (vcam-1) deficient mice. Development (Cambridge, England).
1995; 121:489-503 [0014] Non-Patent Document 7: Yang J T, et al.,
Cell adhesion events mediated by alpha 4 integrins are essential in
placental and cardiac development. Development (Cambridge,
England). 1995; 121:549-560
SUMMARY OF INVENTION
Technical Problem
[0015] One purpose of the present invention is to provide a cardiac
cell culture material which specifically acts on cardiac cells, and
to provide a cell culture substrate on which a wall surface and/or
a bottom surface of the culture substrate having the wall surface
and/or the bottom surface are coated with the cardiac cell culture
material. In addition, another purpose of the present invention is
to provide an artificial organ material obtained by culturing a
cardiac cell by using the cardiac cell culture material, and a
method for producing the same.
Solution to Problem
[0016] It has been made clear that, in cardiac cell culturing, a
functional cardiac tissue is well constructed by using a cardiac
cell culture material containing VCAM-1 protein. Therefore, the
cardiac cell culture material is coated on a wall surface and/or a
bottom surface of a culture substrate having the wall surface
and/or the bottom surface, and can be used as a cell culture
substrate. A cardiac cell cultured by using the cardiac cell
culture material can be used as an artificial organ material.
[0017] Namely, the present invention includes followings.
[1] A cardiac cell culture material comprising VCAM-1 protein. [2]
The cardiac cell culture material according to [1], wherein the
VCAM-1 protein is a VCAM-1 separated and purified from an animal
material, a VCAM-1 recombinant protein, or a cell expressing VCAM-1
protein. [3] The cardiac culture material according to [1] or [2]
used for culturing to construct a cardiac tissue. [4] The cardiac
cell culture material according to [2] or [3], wherein the cell
expressing VCAM-1 protein is a fibroblast expressing VCAM-1
protein. [5] The cardiac cell culture material according to [4],
wherein the fibroblast is a cardiac-derived fibroblast. [6] The
cardiac cell culture material according to [4] or [5], wherein the
fibroblast is an epicardial-derived fibroblast. [7] A cell culture
substrate, wherein a wall surface and/or a bottom surface of the
culture substrate having the wall surface and/or the bottom surface
are coated with the cardiac cell culture material according to
[1]-[6]. [8] An artificial organ material obtained by co-culturing
a cardiac cell with the cardiac cell culture material according to
[1]-[6]. [9] A method of producing an artificial organ material
comprising a step of co-culturing a cardiac cell with the cardiac
cell culture material according to [1]-[6]. [10] A reagent for
screening a cardiac cell culture material containing an anti-VCAM-1
antibody. [11] A cardiac-derived fibroblast expressing VCAM-1
protein. [12] A method of producing the artificial organ material
according to [9] further including a step of separating and
collecting a cultured cell from a culture substrate. [13] A method
of producing the artificial organ material according to [12],
wherein the culture substrate is a temperature responsive culture
dish, and wherein the separation is performed by temperature
change. [14] A method of producing the artificial organ material
according to [9] further including the step of agglomerating the
co-cultured material by using scaffold having thickness in a
certain extent. [15] A method of producing the artificial organ
produced from the artificial organ material according to [9]
comprising step of using a 3D printer.
Advantageous Effects of Invention
[0018] A functional cardiac cell which can be used in a
regenerative medicine and an organizational model can be
constructed by culturing a cardiac cell by using the cardiac cell
culture material of the present invention. The cardiac cell culture
material can be coated on a wall surface and/or a bottom surface of
a culture substrate having the wall surface and/or the bottom
surface, which can be used as a cell culture substrate. Further, a
cardiac cell or a cardiac tissue obtained by culturing can be used
as an artificial organ material.
BRIEF DESCRIPTION OF DRAWINGS
[0019] FIG. 1 A microscopic observation of NCF, ACF and ADF
(photographs). (A) Bright field microscope images of each
fibroblast. (B-E) Representative Figures of DDR2, vimentin and
.alpha.SMA expression (Most of the fibroblasts were not expressing
calponin, cytokeratin 11 or NG 2).
[0020] FIG. 2 Differences in characteristics of mESC derived
cardiac cell sheets that were co-cultured with fibroblasts
(photographs). (A) Before separated, many cell masses that were
autonomously beating were observed on NCF and ACF co-culture sheet.
After decrease in temperature, cell sheet formation was not
observed in mESC derived cardiomyocytes and fibroblasts (-). (B)
Extracellular action potentials on each of the cell sheets. Action
potentials in ACF or NCF co-culture sheet were observed in each
channel. However, the action potentials occurred on a one-off basis
on the ADF co-culture sheet (encircling lines indicate the shapes
of the cell sheets). (C) Immunofluorescent stain in each of the
cell culture dishes which were observed by a confocal microscope.
YFP emitted green (yellow) fluorescence (YFP: excitation wavelength
514 nm, fluorescence wavelength 527 nm), and vimentin emitted red
fluorescence (cy3: excitation wavelength 512 nm, fluorescence
wavelength 552 nm), and the nucleus was stained in hoechst 33258
(blue) (hoechst 33258: excitation wavelength 352 nm, fluorescence
wavelength 461 nm). The confocal microscopy observation suggested
that, the cells co-cultured with NCF or ACF have a large number of
YFP (+) cells, compared with the cells co-cultured with fibroblasts
(-) or ADF. (D) Immunofluorescent stain in each of the cell culture
dishes observed by a confocal microscopy. cTnT was stained by Cy5
(Cy5: excitation wavelength 650 nm, fluorescence wavelength 530
nm), vimentin emitted red fluorescence (cy3: excitation wavelength
512 nm, fluorescence wavelength 552 nm), and the nucleus was
stained in hoechst 33258 (blue) (hoechst 33258: excitation
wavelength 352 nm, fluorescence wavelength 461 nm). The confocal
microscopy observation suggested that, the cells co-cultured with
NCF or ACF have a large number of cTnT (+) cells, compared with the
cells co-cultured with fibroblasts (-) or ADF. (E) The bar graphs
show increase in the numbers of YFP (+) cells or of cTnT (+) cells
in each of the cell culture dishes. The numbers of YFP (+) cells or
of cTnT (+) cells in fibroblasts (-) were set to 1. More numbers of
YFP (+) cells and cTnT (+) cells were observed in NCF or ACF
culture dish compared with those in the culture dish of ADF
co-culture or fibroblasts (-). In addition, there is no significant
relationship in the number of cardiomyocytes between NCF and ACF.
(N=3, **P<0.01)
[0021] FIG. 3 The number of cardiomyocytes at day 1 and day 5 from
the cell culture start in each of the cell culture dishes
(Photographs). (A) Immunofluorescent stain at day 1 from culture
start in each of the cell culture dishes which were used in a
confocal microscope. YFP emitted green (yellow) fluorescence (YFP:
excitation wavelength 514 nm, fluorescence wavelength 527 nm), and
cTnT emitted red fluorescence (cy3: excitation wavelength 512 nm,
fluorescence wavelength 552 nm), and the nucleus was stained in
hoechst 33258 (blue) (hoechst 33258: excitation wavelength 352 nm,
fluorescence wavelength 461 nm). (B) Immunofluorescent stain at day
5 from culture start in each of the cell culture dishes which were
observed by a confocal microscope. YFP emitted green (yellow)
fluorescence (YFP: excitation wavelength 514 nm, fluorescence
wavelength 527 nm), and cTnT emitted red fluorescence (cy3:
excitation wavelength 512 nm, fluorescence wavelength 552 nm), and
the nucleus was stained in hoechst 33258 (blue) (hoechst 33258:
excitation wavelength 352 nm, fluorescence wavelength 461 nm). (c)
The number of cardiomyocytes in each of the cell culture dishes.
The bar graphs show increase in the numbers of YFP (+) cells and of
cTnT (+) cells (The values at day 1 in fibroblasts (-) were set to
1). In the ACF and NCF culture dishes, more numbers of
cardiomyocytes were observed at day 5 from culture start compared
with those at day 1. However, in the other culture dishes, there
was no difference in the number of cardiomyocytes between day 1 and
day 5. No significant difference was observed between ACF and NCF.
(N=3, **P<0.01)
[0022] FIG. 4 Evaluation of proliferation in cardiomyocytes by
immunofluorescent stain (photographs). (A) Immunofluorescent stain
observation of Ki67 positive cardiomyocytes in each of co-culture
dishes by using the confocal microscope. cTnT was stained by Cy5
(Cy5: excitation wavelength 650 nm, fluorescence wavelength 530
nm), and Ki67 emitted red fluorescence (cy3: excitation wavelength
512 nm, fluorescence wavelength 552 nm), and the nucleus was
stained in hoechst 33258 (blue) (hoechst 33258: excitation
wavelength 352 nm, fluorescence wavelength 461 nm). (B) Percentage
of Ki67 (+) or phosphorylated histone 3 (phosphor S10; Phh3) (+)
cardiomyocytes in each of the culture dishes (N=4, **P<0.01).
(C) Immunofluorescence stain observation of phosphorylated histone
3 (phosphor S10; Phh3) positive cardiomyocytes in each of the
culture dishes by using the confocal microscope. cTnT was stained
by Cy5 (Cy5: excitation wavelength 650 nm, fluorescence wavelength
530 nm), and phosphorylated histone 3 (phosphor S10; Phh3) emitted
red fluorescence (cy3: excitation wavelength 512 nm, fluorescence
wavelength 552 nm), and the nucleus was stained in hoechst 33258
(blue) (hoechst 33258: excitation wavelength 352 nm, fluorescence
wavelength 461 nm). (D) Percentage of phosphorylated histone 3
(phosphor S10; Phh3) (+) cardiomyocytes in each of the culture
dishes (N=4, **P<0.01). (E) (F) BrdU FACS assay of
cardiomyocytes in each of the culture dishes (N=3, **P<0.01).
(G) Immunofluorescence stain observation of YFP (+) and of cTnT (+)
at day 1 and day 5 from culture start in the insert culture dishes
by using the confocal microscope. YFP emitted green (yellow)
fluorescence (YFP: excitation wavelength 514 nm, fluorescence
wavelength 527 nm), and cTnT emitted red fluorescence (cy3:
excitation wavelength 512 nm, fluorescence wavelength 552 nm), and
the nucleus was stained in hoechst 33258 (blue) (hoechst 33258:
excitation wavelength 352 nm, fluorescence wavelength 461 nm). (H)
The bar graphs show increase in the numbers of YFP (+) cells and of
cTnT (+) cells at day 1 and at day 5. The numbers of YFP (+) cells
and of cTnT (+) cells at day 1 were set to 1. The proliferation of
cardiomyocytes was observed at day 5 (N=4, **P<0.01).
[0023] FIG. 5 (A) Comprehensive gene cluster analysis of ADF and
NCF (photograph). This gene heat map shows a remarkable difference
between ADF and NCF. This map was divided into two groups. The
first group consisted of only ADF, and the second group consisted
of only NCF. (B) The VCAM-1 gene expression level was examined by
real time PCR. The VCAM-1 expression level was significantly high
in NCF. The number of VCAM-1 genes in NCF was 16 times higher than
that in ADF (N=3, *P<0.05). (C-D) The expression level of VCAM-1
protein in NCF and ADF in western blot analysis. The following
transient overexpression cell lysate was used as a positive
control: Sol8 (SantaCruz, Calif., USA). The label peak of
.beta.-actin of each cell was set to 1 (N=3, **P<0.01). (E)
Immunofluorescence stain of the VCAM-1 receptor (.alpha.4.beta.1)
on mESC derived cardiomyocytes. (F) Western blot analysis of the
VCAM-1 receptor on mESC derived cardiomyocytes. The following
transient overexpression cell lysate was used as a positive
control: Jurkat whole cell lysate.
[0024] FIG. 6 Identification of cardiac growth factor by
immunofluorescence stain analysis (photographs). (A-B)
Immunofluorescence stain observation of the effect of neutralizing
antibodies on cardiomyocytes at day 5. YFP emitted green (yellow)
fluorescence (YFP: excitation wavelength 514 nm, fluorescence
wavelength 527 nm), and cTnT emitted red fluorescence (cy3:
excitation wavelength 512 nm, fluorescence wavelength 552 nm), and
the nucleus was stained in hoechst 33258 (blue) (hoechst 33258:
excitation wavelength 352 nm, fluorescence wavelength 461 nm). When
NCF and cardiomyocytes were co-cultured by using a VCAM-1
neutralizing antibody, the number of cardiomyocytes was decreased
at day 5. Meanwhile, when an isotype control was used, there was no
effect on the number of cardiomyocytes at day 5. (N=3,
**P<0.01). (C-D) Immunofluorescence stain observation of the
effect of VCAM-1 soluble protein on cardiomyocytes at day 5. YFP
emitted green (yellow) fluorescence (YFP: excitation wavelength 514
nm, fluorescence wavelength 527 nm), and cTnT emitted red
fluorescence (cy3: excitation wavelength 512 nm, fluorescence
wavelength 552 nm), and the nucleus was stained in hoechst 33258
(blue) (hoechst 33258: excitation wavelength 352 nm, fluorescence
wavelength 461 nm). Cardiomyocyte growth effect was obtained by
culturing with VCAM-1 soluble protein (10 .mu.g/mL). Moreover, the
number of cardiomyocytes on Day 5 was comparable to that in
co-culture with NCFs. (N=3, *P<0.05, **P<0.01).
[0025] FIG. 7 The results of FACS analysis of cardiac fibroblasts
derived from neonatal mice. (A-C) The results of staining with an
anti-VCAM-1 antibody are shown. (D) The result of a negative
control by staining dermal fibroblasts only with a secondary
antibody is shown.
[0026] FIG. 8 The optimum compounding concentration of VCAM-1 (+)
cardiac fibroblasts. (A) Phase difference images of VCFs (VCAM-1
(+)) and VNCFs (VCAM-1 (-)) isolated by a magnetic cell separator
(MACS). (B) Fluorescence images (photographs) of VCFs (VCAM-1 (+))
and VNCFs (VCAM-1 (-)) isolated by a magnetic cell separator
(MACS). VCAM-1 emits red fluorescence, and Hoechst 33258 emits blue
fluorescence. Scale bar=200 .mu.m.
[0027] FIG. 9 Evaluation of optimum compounding ratio of VCFs. (A)
Fluorescence images (photographs) showing the results of
co-culturing cardiomyocytes expressing GFP with ADFs, NCFs, or VCFs
and/or VNCFs. ADFs=mouse adult-derived dermal fibroblasts,
VCFs=VCAM-1 positive mouse neonatal cardiac fibroblasts,
VNCFs=VCAM-1 negative mouse neonatal cardiac fibroblasts, and
NCFs=neonatal mouse cardiac fibroblasts. GFP cardiomyocytes emit
green fluorescence, and Ki 67 emits red fluorescence, and cTnT was
stained by Cy5 (Cy5: excitation wavelength 650 nm, fluorescence
wavelength 530 nm). Magnification is .times.20. (B) A bar graph
showing the results of co-culturing cardiomyocytes expressing GFP
with ADFs, NCFs, or VCFs and/or VNCFs. The numbers of GFP (+) cells
and cTnT (+) cells were set to 1 when cardiomyocytes and ADFs were
co-cultured at the concentrations of 80% and 20%, respectively.
N=4, **P<0.01.
[0028] FIG. 10 Evaluation of localization (N=4) of cardiomyocytes
and fibroblasts at day 5 in a tissue created at the concentration
of 80% cardiomyocytes and 20% VCFs.
[0029] FIG. 11 Evaluation of division (proliferation) effect,
migration effect and ability of constructing a network of
cardiomyocytes for 5 days of culturing under the condition of
co-culturing with NCFs and ADFs (photographs). YFP expressing
cardiomyocytes emit green fluorescence, and DsRed fibroblasts emit
red fluorescence. Magnification is .times.200.
[0030] FIG. 12 A shows fluorescence images indicating division
(proliferation) of cardiomyocytes for three days of culturing at
the time when co-culturing ES-derived cardiomyocytes with VCFs
(VCAM-1 (+)) or VNCFs (VCAM-1 (-)) (photographs). GFP expressing
cardiomyocytes emit green fluorescence. Magnification is
.times.100. B shows the results evaluating the migratory ability by
calculating the total migratory distance for 3 days of culturing at
the time when co-culturing ES-derived cardiomyocytes, and VCFs or
VNCFs. N=5, *P<0.05.
[0031] FIG. 13 The results of analyzing the localization of
Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) and
VCAM-1 positive cardiac fibroblasts with flow cytometry.
[0032] FIG. 14 (A) Localization of VCFs in a biological heart
(N=3). (B) Localization of VCFs in cardiac fibroblasts (N=3). (C)
Localization of CD31-positive fibroblasts in a biological heart
(N=3). (D) Localization of CD31 positive fibroblasts in cardiac
fibroblasts (N=3).
DESCRIPTION OF EMBODIMENTS
[0033] The present embodiment relates to a cardiac cell culture
material containing VCAM-1. In the present embodiments, the
"cardiac cell culture material" may be any material that is used
when culturing a cardiac cell. For example, the material includes
but is not limited to a reagent such as a protein, and a peptide,
etc. to be added to a culture medium, and a material, etc. for
coating a bottom surface or a wall surface of a culture substrate
of a culture vessel, etc. such as a petri dish and a flask, and the
like. Examples of these cell culture substrate, in which a wall
surface and/or a bottom surface of the culture substrate having the
wall surface and/or the bottom surface are coated with the cardiac
cell culture material include microcarrier, and cell culture bag,
etc.
[0034] VCAM-1 (vascular cell adhesion molecule-1) is a known
protein as a cell adhesion molecule that expresses in a vascular
endothelial cell, and the like. For example, in the case of humans,
VCAM-1 includes but not limited to a protein encoded by a gene
described in accession number NM_001078, etc. of NCBI (National
Center for Biotechnology Information), and also includes an isoform
obtained by alternative splicing. The VCAM-1 protein in the present
embodiment includes VCAM-1 which is expressing on a cell surface, a
soluble VCAM-1, various mutants one or a plurality of, for example,
1-20, 1-15, 1-10 or 1-5 of amino acids of which have been deleted
from, substituted from, or added to an amino acid of VCAM-1 protein
and having the same activity as VCAM-1 protein. A VCAM-1 protein in
an animal material which has been separated and purified by a
well-known method and a recombinant protein may be used as the
VCAM-1 in the present embodiment. For example, the animal material
includes but is not limited to humans; experimental animals such as
mice, rats, guinea pigs, hamsters, pigs, monkeys and rabbits; and
bacteria such as E. coli, etc. A commercially available recombinant
protein may be also used.
[0035] Moreover, a cell that is expressing VCAM-1 may be used as
VCAM-1 of the present embodiment. In order to screen a cell that is
expressing VCAM-1, a publicly known cell sorting method may be
used. For example, the cell sorting method includes but not limited
to flow cytometry using an anti-VCAM-1 antibody, magnetic bead
method, affinity column method, and panning method.
[0036] Anti-VCAM-1 antibodies are not particularly limited.
Commercially available anti-VCAM-1 antibodies may be used, and a
product produced by a known method by using VCAM-1 as an antigen
may be also used. Moreover, as far as the cells that are expressing
VCAM-1 may be screened, either monoclonal antibody or polyclonal
antibody may be used; however, it is preferred to use monoclonal
antibody from the viewpoint of specificity.
[0037] Namely, the methods of screening the cardiac cell culture
materials of the present embodiment include, a step of preparing
cells, a step of performing cell sorting to the cells by using a
VCAM-1 antibody, and a step of collecting only cells that have been
judged to be expressing VCAM-1 as a result of the cell sorting.
[0038] As the cell that is expressing VCAM-1, the types are not
limited as far as VCAM-1 is expressed. However, it is preferred to
use fibroblasts. The fibroblasts include all the cells that will
ultimately become fibroblasts or myofibroblasts. Namely, the scope
of fibroblasts of the present embodiment includes the cells that
are in the middle of differentiation or a maturation stage and
cannot be identified as fibroblasts or myofibroblasts at that time
as far as the cells will ultimately become fibroblasts or
myofibroblasts. Moreover, the scope of fibroblasts of the present
embodiment includes the cells that are not called as fibroblast,
such as stromal cells, interstitial cells, progenitor cells,
precursor cells, stem cells, or the like, as far as the cells have
the same functions and activities as fibroblasts and express
VCAM-1.
[0039] Derivation of fibroblasts is not limited. Pluripotent stem
cells such as ES cells, iPS cells and muse cells, and adult stem
cells such as mesenchymal stem cells may be differentiated and
used, and primary cells taken from animals may be used, and
established cells may be used. However, cardiac-derived fibroblasts
are preferably used, and among them, epicardium-derived fibroblasts
are in particular preferred to be used. In a case where established
cells are used, processing of cell sorting may be omitted by
selecting the cells that are known to express VCAM-1. The animals
from which fibroblasts are derived may be appropriately selected in
accordance with the animals from which the cells to be co-cultured
are derived. The animals, for example, include humans; experimental
animals such as mice, rats, guinea pigs, hamsters, pigs, monkeys
and rabbits; pet animals such as dogs, cats and birds; and
livestock such as cattle, horses, sheep and goats. In a case where
fibroblasts are taken from animals, the fibroblasts may be of at
any time of the animals such as fetus, neonate, infant, adult, and
there is no limit.
[0040] The cardiac cell culture material of the present embodiment
may be a composition containing physiological saline, cell culture
solution, or cell preservation solution, etc. for maintenance or
preservation of VCAM-1 protein or cells that are expressing VCAM-1
protein. There is no limit on the contents contained in the
composition as far as the contents do not impair the function of
VCAM-1. Moreover, the state of the cardiac cell culture material of
the present embodiment may be liquid, gel-like, freezed, or
freeze-dried, and the state thereof is not limited.
[0041] Further, the cardiac cell culture material may include
fibroblasts regardless of presence or absence of VCAM-1 protein.
The fibroblasts include all the cells that will ultimately become
fibroblasts or myofibroblasts. Namely, even if the cells are in the
middle of differentiation or a maturation stage and cannot be
identified as fibroblasts or myofibroblasts at that time, if the
cells are those that will ultimately become fibroblasts or
myofibroblasts, the cells may be used without limit. Among them,
fibroblasts that are expressing CD31 (vascular endothelial cell
marker) are preferred. When fibroblasts that are expressing VCAM-1
protein are used as VCAM-1 protein, the ratio of VCAM-1 protein
expressing cells (cell number):CD31 expressing cells (cell number)
are preferably 5:5-9:1, more preferably 5:5-8.2, even more
preferably 6:4-8.2, and may be 7:3-8:2.
[0042] The present embodiment relates to an artificial organ
material in which the ratio of cardiac fibroblasts expressing
VCAM-1 to all the cells is 50% or higher. The ratio of the cardiac
fibroblasts is 50% or higher, and preferably 60% or higher, and
more preferably 70% or higher, and even more preferably 80% or
higher, and most preferably 90% or higher. The artificial organ
material may be obtained by co-culturing cardiac fibroblasts that
express VCAM-1 with cardiomyocytes, and the ratio of the cardiac
fibroblasts at the start of co-culturing is normally 3% or higher,
preferably 4% or higher, more preferably 6% or higher, and even
more preferably 8% or higher, and most preferably 9% or higher.
Meanwhile, the ratio of the cardiac fibroblasts at the start of
co-culturing is preferably 30% or lower, preferably 20% or lower,
and most preferably 20%. The present invention also relates to an
artificial organ material obtained by culturing a cardiac cell with
the above-mentioned cardiac cell culture material, or a method for
producing the same. Namely, the cardiac cell culture material of
the present embodiment can construct a functional cardiac tissue
that can be used in a regenerative medicine and organizational
model by culturing with a cardiac cell. The cardiac tissue can be
used as an artificial organ material. The artificial organ material
can be of any form. For example, it can be adhered to a damaged
part of organs such as heart in the form of sheet. Therefore, one
embodiment is a method for curing heart diseases by attaching the
sheet to a damaged part of the heart. The artificial organ material
can be also transplanted to a defect site of an organ after it is
laminated or it is agglomerated by using scaffold, which has
thickness in a certain extent. The material of the scaffold
includes but not limited to hydroxyapatite, atelocollagen, and gel.
Further, the artificial organ material can be used for cell
transplantation, academic research, etc. as it is in the state of
the culture cell, without making it to be a particular form.
Furthermore, an artificial organ can be produced from the
artificial organ material by using a 3D printer. The produced
artificial organ not only can be used for transplantation but also
can be widely used for safety pharmacology test and preclinical
research, etc.
[0043] In the present embodiment, "constructing a cardiac tissue"
means constructing a tissue having at least one of the cardiac
functions such as promoting division of cardiac cells, and
providing uniform beating throughout a whole tissue, which can be
used for regenerative medicine and a tissue model. The cardiac
functions include all the known cardiac functions such as
autonomous pulsating ability, contraction and relaxation ability,
impulse conduction ability, and hormone secretion ability, etc. The
cardiac functions are not limited to functions which only the heart
has. For example, a muscle cell also has the contraction and
relaxation ability. However, even if other cells have an equivalent
function, it does not affect the definition of the cardiac
functions of the present embodiment. Further, with respect to the
cardiac functions, there is no limit on highness and lowness of the
functions as long as they are suitable for use purpose of a cardiac
tissue. For example, for the purpose of producing an artificial
heart, it is required to have a contraction and relaxation ability
to the extent that it can pump out blood thought out the body;
however, for the purpose of academic research, etc. of contraction
and relaxation ability in vitro, it is satisfied if contraction and
relaxation ability is detected by some means.
[0044] In the artificial organ material, or the method to produce
the same of the present embodiment, cardiac cells to be used
include all the cells that constitute the heart such as
cardiomyocytes, smooth muscle cells, pacemaker cells and vascular
endothelial cells. The derivation of the cardiac cells can be
appropriately set in accordance with the purpose of use as an
artificial organ material. For example, for the purpose of
transplantation to humans, human-derived cardiac cells may be used,
and for the purpose of constructing a tissue model in a mouse
experiment, mouse-derived cardiac cells may be used. Furthermore, a
cardiac cell of any period from fetus, newborn, pediatric and adult
can be used, and there is no limit on the period. The cardiac cell
of the present embodiment is preferred to be produced from
pluripotent stem cells such as ES cells, iPS cells, and muse cells,
and adult stem cells such as mesenchymal stem cells.
[0045] The "culturing" of the present embodiment can be carried out
by a publicly known cell culturing method, and there is no limit on
the condition of the culturing as long as a cardiac cell culture
material and a cardiac cell are present in a culture vessel, or are
immersed in the same culture medium. In a case where the cardiac
cell culture material are cells which are expressing VCAM-1
protein, the mixing percentage of the cells (cell number) that are
expressing VCAM-1 to cardiac cells are preferably 3-20%, more
preferably 6-18% and most preferably 9-16%.
[0046] In the present embodiment, a culture liquid used for the
culturing can be appropriately set in accordance with a kind of
cell to be cultured. For example, DMEM, .alpha.-MEM, RPMI-1640, and
the like may be used. Nutritional substances such as FCS and FBS
and antibiotics may be added to the culture liquid. Growth factor
and cytokines such as fibroblast growth factors (FGF) may also be
added to the culture liquid.
[0047] With respect to the cultivation period, the number of days
until the desired cell number and/or function are obtained may be
appropriately set. For example, the periods include 1 day, 2 days,
3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11
days, 12 days, 13 days, 14 days, 1 month, 2 months, 3 months, 4
months, 5 months, and 6 months. The cultivation temperature may be
appropriately set in accordance with the kinds of cells to be
cultured. For example, the temperature may be 10-60.degree. C.,
preferably 20-50.degree. C., and more preferably 30-40.degree.
C.
[0048] The production method of the present embodiment may further
include the step of collecting cultured cells. The "cultured cells"
may include both fibroblasts and cardiac cells, and may only
include the cardiac cells. With respect to the step to collect a
cell, the cell may be separated and collected by using proteases
such as trypsin. However, it is preferred that cell is separated
and collected by the change in temperature by using a temperature
responsive culture dish capable of separating a cell while
retaining an extracellular matrix, etc.
EXAMPLES
[0049] The present invention is further described below in detail
with reference to the following examples; however, it should be
construed that the invention is no way limited to those
examples.
Example 1
Materials and Methods
<Animals and Reagents>
[0050] Wild-type C57BL/6 mice were purchased from Japan SLC
(Shizuoka, Japan). B6 Cg-Tg (CAG-DsRed*MST) 1Nagy/J mice were
purchased from The Jackson Laboratory (Bar Harbor, Me.). All the
experimental protocols were approved by the Institutional Animal
Care and Use Committee of Tokyo Women's Medical University. The
following antibodies were used for immune cytochemistry, western
blot and flow cytometric analysis (FACS): rabbit polyclonal
anti-discoidindomein receptor tyrosine kinase 2 (DDR2) (GeneTex,
Irvine, Calif.); guinea pig monoclonal anti-vimentin (Progen,
Heidelberg, Germany); mouse monoclonal anti-NG2 (Millipore,
Temecula, Calif.); Rabbit polyclonal anti-alpha smooth muscle actin
(Abcam, Cambridge, UK); mouse monoclonal anti-cardiac troponin T
(cTnT) (Thermo Scientific, Rockford, Ill.); mouse monoclonal
anti-cytokeratin11 (EXBIO, NadSafinou, CZ); rabbit polyclonal
anti-Ki67 (Abcam, Cambridge, UK); rabbit polyclonal anti-Histon H3
(phosphor S10) (Abcam, Cambridge, UK); rat monoclonal anti-integrin
.alpha.4/.beta.1 (Abcam, Cambridge, UK); recombinant mouse
VCAM-1/CD106 Fc chimera (R&D systems, Minneapolis, Minn.).
Unless otherwise specified, all reagents were purchased from
Sigma-Aldrich. Secondary antibodies were purchased from Jackson
ImmunoResearch Laboratories (West Grove, Pa.).
[0051] <Mouse ES Cell Cultures>
[0052] The maintenance of mESC expressing the neomycin
phosphotransferase gene under the control of the .alpha.-myosin
heavy chain promoter and cardiomyocyte differentiation and
purification were described previous report (Matsuura K, et al.
Biomaterials. 2011; 32:7355-7362). Briefly, for cardiac induction
and cardiomyocyte purification, trypsinized ES cells were seeded at
5.times.10.sup.4 cells/mL (total, 125 mL/flask) into spinner flasks
(Integra Biosciences, Zizers, Switzerland) and cultured with DMEM
supplemented with 10% FBS for 10 days, then these differentiated
cells were treated with neomycin for further 8 days.
[0053] <Fibroblast Isolations>
[0054] Fibroblasts were obtained from Wild-type C57BL/6 mice
(Neonatal, 1 day; Adult, 10-12 weeks).
[0055] Neonatal cardiac fibroblasts (NCFs) were obtained from
hearts of neonatal mice (1 day of age) as described previous report
(Matsuura K, et al, Biomaterials. 2011; 32: 7355-7362). NCFs from
passage 3 were used for the experiments.
[0056] Adult cardiac fibroblasts (ACFs) were obtained from hearts
of adult mice (10-12 weeks) using the explant culture method as
follow. First hearts were washed with PBS (-) and cut into circa 5
mm.sup.2 species. These species were covered with sterilized cover
glasses and cultured with DMEM supplemented with 10% FBS on 10 cm
culture dishes. 2 weeks after starting culture, cells were
dissociated with 0.25% Trypsin/EDTA and subcultured to other 10 cm
dishes. ACFs from passage 3 were used for the experiments.
[0057] Adult dermal fibroblasts (ADFs) were obtained from dorsal
dermal tissue of adult mice (10-12 weeks). First harvested dermal
tissues were treated with Dispase I [1000 U/mL] (Eidea inc.) over
night at 4.degree. C. Next, the tissues were cut into circa 1
mm.sup.2 species. These species were covered with sterilized cover
glasses and cultured with DMEM supplemented with 10% FBS on 10 cm
culture dishes. 2 weeks after starting culture, cells were
dissociated with 0.25% Trypsin/EDTA and subcultured to another 10
cm dishes. ADFs from passage 3 were used for the experiments.
[0058] In some experiments, NCFs and ADFs were isolated from
B6.Cg-Tg (CAG-DsRed*MST) 1Nagy/J mice (Neonatal: 1 day, Adult: 10
weeks) with the same methods as described above.
[0059] <Cell Sheet Preparation>
[0060] Before seeding cells, the surface of temperature-responsive
culture dishes (UpCell; CellSeed inc.) was coated with FBS for 2 h.
mESC-derived cardiomyocytes were co-cultured with each type of
fibroblasts at the ratio of 8:2 with DMEM supplemented with 10% FBS
(3.2.times.10.sup.5 cells/cm.sup.2). After 5 days of culture, the
cells were incubated at 20.degree. C. for detaching cell sheets.
Bright field images of samples were obtained by a Nikon ECLIPSE Ti
(Nikon, Tokyo, Japan).
[0061] <Electrophysiological Analysis>
[0062] The electrical activities of the cardiomyocyte sheets were
obtained from the extracellular potentials measured by a
multi-electrode array (MED) system (Alpha MED Sciences inc.) as
described previous report (Matsuura K, et al, Biomaterials. 2011;
32:7355-7362).
[0063] <Immunocytochemistry>
[0064] Cells were fixed with 4% paraformaldehyde and subjected to
immunostaining as described previous report (Matsuura K, et al,
Biomaterials. 2011; 32:7355-7362). Images of the stained samples
were obtained by an ImageXpress Ultra Confocal High Content
Screening System (Molecular Devices, CA, USA). Image analysis data
was obtained by a MetaExpress software (Molecular Devices
inc.).
[0065] <FACS Analysis>
[0066] Incubating cells (5.times.10.sup.5 cells) were stained with
BrdU at a final concentration of 10 .mu.M in cell culture medium.
BrdU staining for a FACS analysis was performed as described in a
BrdU Flow Kits Instruction Manual (BD Pharmingen, Franklin Lakes,
N.J.). Briefly, cells were fixed and permeabilized with BD
Cytofix/Cytoperm Buffer, then exposed incorporated BrdU with DNase.
BrdU staining was performed with APC-anti-BrdU antibody (BD
Pharmingen, Franklin Lakes, N.J.). Samples were analysed with a
Gallios (Beckman Coulter, Brea, Calif.). The following reagents
were used for the analysis: BD Cytofix/Cytoperm Buffer (BD
Pharmingen, Franklin Lakes, N.J.); BD Perm/Wash Buffer (10.times.)
(BD Pharmingen, Franklin Lakes, N.J.); BD Cytoperm Plus Buffer
(10.times.) (BD Pharmingen, Franklin Lakes, N.J.); BrdU (10 mg/mL)
(BD Pharmingen, Franklin Lakes, N.J.); DNase (BD Pharmingen,
Franklin Lakes, N.J.).
[0067] <Time-Laps Photography>
[0068] Samples were observed five days in 5% CO.sub.2 at 37.degree.
C. with a BZ-9000 Fluorescence Microscope (Keyence, Osaka,
Japan).
[0069] <RNA Extraction and Comprehensive Genetic
Analysis>
[0070] Total RNA was extracted using TRIzol reagent (Invitrogen,
Carlsbad, Calif.) according to the manufacturer's instructions.
Total RNA was further purified using the Qiagen RNeasy Mini Kit
(QIAGEN, Valencia, Calif.) according to the manufacturer's
instructions.
[0071] RNA quantity and quality were determined using a Nanodrop
ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham,
Mass.) and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto,
Calif.), as recommended.
[0072] For cRNA amplification and labeling, total RNA was amplified
and labeled with Cyanine 3 (Cy3) using Agilent Low Input Quick Amp
Labeling Kit, one-color (Agilent Technologies, Palo Alto, Calif.)
following the manufacturer's instructions. Briefly, 100 ng of total
RNA was reversed transcribed to double-strand cDNA using a poly
dT-T7 promoter primer. Primer, template RNA and quality-control
transcripts of known concentration and quality were first denatured
at 65.degree. C. for 10 min and incubated for 2 hours at 40.degree.
C. with 5.times. first strand Buffer, 0.1 M DTT, 10 mM dNTP mix,
and Affinity Script RNase Block Mix. The AffinityScript enzyme was
inactivated at 70.degree. C. for 15 min.
[0073] cDNA products were then used as templates for in vitro
transcription to generate fluorescent cRNA. cDNA products were
mixed with a transcription master mix in the presence of T7 RNA
polymerase and Cy3 labeled-CTP and incubated at 40.degree. C. for 2
hours. Labeled cRNAs were purified using QIAGEN's RNeasy mini spin
columns and eluted in 30 .mu.l of nuclease-free water. After
amplification and labeling, cRNA quantity and cyanine incorporation
were determined using a Nanodrop ND-1000 spectrophotometer and an
Agilent Bioanalyzer.
[0074] For Sample hybridization, 1.65 .mu.g of Cy3 labeled cRNA
were fragmented, and hybridized at 65.degree. C. for 17 hours to an
Agilent Mouse GE 4x44Kv2 Microarray (Design ID: 026655). After
washing, microarrays were scanned using an Agilent DNA microarray
scanner.
[0075] For data analysis of microarray, intensity values of each
scanned feature were quantified using Agilent feature extraction
software version 10.7.3.1, which performs background
subtractions.
[0076] We only used features that were flagged as no errors
(present flags) and excluded features that were not positive, not
significant, not uniform, not above background, saturated, and
population outliers (marginal and absent flags). Normalization was
performed using Agilent GeneSpring GX version 11.0.2. (per chip:
normalization to 75 percentile shift; per gene: normalization to
median of all samples). There are total of 39,429 probes on Agilent
Mouse GE 4x44Kv2 Microarray (Design ID: 026655) without control
probes.
[0077] The altered transcripts were quantified using the
comparative method. We applied 2-fold change in signal intensity to
identify the significant differences of gene expression in this
study.
[0078] <Quantitative Real-Time PCR Analysis>
[0079] Complementary DNA was generated from total RNA with High
Capacity cDNA Reverse Transcription Kit (Applied biosystems). As
the PCR-related primers, VCAM-1 Gene Express Assays (life
Technology) was used.
[0080] Each RT-PCR included 10 minutes at 25.degree. C., 120
minutes at 37.degree. C., and 5 seconds at 85.degree. C. with
iCycler (BIO-RAD). cDNA template (1 .mu.g) was used from each
sample. TaqMan probe real-time PCR studies were performed with
TaqMan Gene Expression Assays (Applied biosystems). All experiments
were conducted in triplicate. Samples were cycled 40 times with an
7300 Real Time PCR System (Applied Biosystems) as follows: 2
minutes at 50.degree. C. and 10 minutes at 95.degree. C., followed
by 40 cycles of 15 seconds at 95.degree. C. and 1 minute at
60.degree. C. Relative quantification was calculated according to
the .DELTA..DELTA.CT method for quantitative real-time PCR using a
Gap DH gene as endogenous control.
[0081] <Western Blotting>
[0082] NCFs or ADFs were lysed in Laemmli sample buffer (BIO-RAD,
CA, USA), protease inhibitor (Boehringer Mannheim, Indianapolis,
Ind.) and 2-mercaptoethanol (Wako Pure Chemical Industries, Japan).
The samples were separated on a 4% to 12% Bis-Tris Gels (Life
Technologies, MD, United States), electrotransferred to a iBlot
Transfer Stack, nitrocellulose, regular-size (Life technologies,
MD, United States) with iBlot 7-Minute Blotting System (Life
technologies, MD, United States), and processed for
chemiluminescence analysis with Amersham ECL Prime Western Blotting
Detection Reagent (GE Healthcare, PA, United States). Band
intensity was analyzed using LAS4000 (Fujifilm, Tokyo, Japan) and
NIH image software (version 1.46r). The following cell transient
overexpression lysates were used for positive controls: K562 (Human
erythromyeloblastoid leukemia cell line) for Col11a1 (Abcam, CB,
UK); Sol8 (SantaCruz, Calif., USA) for Vcam-1; ITGB1 293T for
.beta.1/CD29 (Abnova, Taipei, Taiwan); Jurkat Whole Cell Lysate for
integrin .alpha.4.beta.1 (SantaCruz, Calif., USA).
[0083] <Neutralizing Antibodies Assay>
[0084] The following antibodies and culture dishes were used for
neutralizing antibody assay: anti-VCAM-1 (LifeSpan Biosciences,
Seattle, Wash.); goat IgG isotype control (LifeSpan Biosciences,
Seattle, Wash.). Cell Culture Inserts for 24-well plates. 0.4 .mu.m
pores, Translucent, High Density PET Membrane (BD Pharmingen,
Franklin Lakes, N.J.).
[0085] After the pretreatment with the antibodies at 10 .mu.g/mL
for 30 min, fibroblasts were seeded onto the upper layer of insert
culture dishes (2.4.times.10.sup.5 cells). mESC-derived
cardiomyocytes were seeded onto the below layer (4.8.times.10.sup.5
cells). The culture medium with the antibody at 10 .mu.g/mL was
changed every day until 5 days.
[0086] <Statistical Analysis>
[0087] All data were presented as the mean.+-.SD. The significance
of the variation among different groups was determined by One-Way
ANOVA Analysis. And then, the difference between two groups was
determined by Tukey-Kramer Multiple Comparison Test using Statcel
Software. p value<0.05 was considered to be significantly
different.
[0088] 2. Results of Experiments
<Cell Sheet Creation Using mESC-Derived Cardiomyocytes and
Fibroblast>
[0089] At first we evaluated the characterization of cells that we
were going to use for the co-culture experiments. The phase
contrast images revealed that cells isolated from neonatal hearts,
adult hearts and adult dermal tissue showed the fibroblast-like
morphology (See FIG. 1A). Since there are not specific antibodies
for fibroblasts, we tried to examine the expression of the proteins
that are known to be expressed in fibroblasts such as DDR2
(CD167b), vimentin and .alpha.SMA. As shown in FIGS. 1B to 1E,
almost all of each type of cells expressed DDR2, vimentin and
.alpha.SMA, but not Calponin (smooth muscle cell marker),
Cytokeratin (epithelial cell marker) and NG2 (pericyte marker). On
the basis of these findings, we used these cells as fibroblasts
following experiments.
[0090] According to our previous findings that certain extent
amounts of fibroblasts were necessary for fabricating cell sheet
using mESC-derived cardiomyocytes and the optimal ratio of
cardiomyocytes/fibroblasts was 8:2 (Biomaterials.
2011:32:7355-7362), we tried to create cardiac cell sheets using
mESC-derived cardiomyocytes and 3 types of fibroblasts (ACFs, ADFs
and NCFs) on UpCell temperature-responsive culture dishes. When
cardiomyocytes were co-cultured with ACFs or NCFs, beating
cardiomyocytes were equally distributed all over the area.
Conversely, when cardiomyocytes were co-cultured with ADFs, nearby
beating cells were aggregated. After 5 days cultivation, when the
cultivation condition were changed from 37.degree. C. to 20.degree.
C., monolayered cell sheets were created in every condition with
fibroblasts, but not in the condition without fibroblasts (FIG.
2A).
[0091] Next we examined the electrophysiological evaluation of the
cell sheets using a MED system (Biomaterials. 2011; 32:7355-7362,
Biomaterials. 2006; 27:4765-4774). Consistent with the
microscopical observation (FIG. 2A), the extracellular action
potential was observed at each channel in cell sheets with ACFs and
NCFs (FIG. 2B). Since it was recognized that, in these cell sheets,
the entire sheets were uniformly beating, it was suggested that an
electronic network was fabricated in the sheets and these cell
sheets can carry out electric propagation. Meanwhile, in the cell
sheets co-cultured with ADFs, the extracellular action potential
was only observed in limited areas.
[0092] To confirm the difference of cardiomyocytes distribution
among cell sheets, cofocal microscopic analysis was performed. As
shown in FIGS. 2C to 2E, the number of YFP (+) cells and cardiac
troponin T (cTnT) (+) cells, indicatives of mESC-derived cells, in
cell sheets with ACFs and NCFs were more than those in cell sheets
with ADFs. The number of cardiomyocytes in cell sheets with ADFs
was comparable to that in condition without fibroblasts. In
addition, there is no significant correlation on the number of
cardiomyocytes between cell sheets co-cultured with ACFs and NCFs.
These findings suggest that every kind of fibroblasts was useful
for fabricating cell sheet, but fibroblasts derived from hearts
might be better for fabricating more functional cardiac cell
sheets.
[0093] <Cardiomyocyte Proliferation in Cellsheets>
[0094] To investigate the cause of the different number of
cardiomyocytes between cell sheets co-cultured with heart-derived
fibroblasts and dermal tissue-derived fibroblasts, the number of
cardiomyocytes was examined at day 1 and day 5 in co-culture (FIGS.
3A to C). At day 1, the number of cardiomyocytes was identical
among conditions, suggesting that each type of fibroblasts did not
affect the initial adherence of cardiomyocytes after seeding. In
co-culture with ACFs and NCFs, the number of YFP (+) and cTnT (+)
cardiomyocytes at day 5 was significantly higher than that at day
1. On the other hand, in co-culture with ADFs or in cardiomyocytes
monoculture condition, the number of cardiomyocytes at day 5 was
similar to that at day 1. The time-lapse image analysis using YFP
(+) cardiomyocytes and fibroblasts isolated from DsRed mice showed
that cardiomyocytes migrated and proliferated and constructed the
mutual network formation in co-culture with NCFs. Conversely in
co-culture with ADFs, cardiomyocytes showed less proliferation and
did not construct the network formation. These findings suggest
that fibroblasts from hearts, but not fibroblasts from dermal
tissue might induce proliferation of mESC-derived cardiomyocytes in
co-culture condition.
[0095] The proliferation of cardiomyocytes among conditions was
confirmed by the immune cytochemical analysis. As shown in FIG. 4A
to D, the percentage of Ki67 (+) cells and phospho histone H3
(PHH3) (+) cardiomyocytes in co-culture with NCFs were significant
higher than those in co-culture with ADFs and in cardiomyocytes
monoculture condition. Furthermore BrdU incorporation assay also
showed the significant increase of the percentage of proliferative
cardiomyocytes in co-culture with NCFs compared with that in
co-culture with ADFs and in cardiomyocytes monoculture condition
(FIGS. 4E and F). These findings strongly suggest that
heart-derived fibroblasts induce proliferation of
cardiomyocytes.
[0096] To investigate the underlying mechanisms on the
proliferation of cardiomyocytes in co-culture with NCFs,
mESC-derived cardiomyocytes and NCFs were cultured using cell
culture inserts. In this experiment, NCFs were cultured on the
upper layer and cardiomyocytes were cultured on the lower layer.
The number of cardiomyocytes at day 5 was remarkably higher than
that at day 1 in the presence of NCFs (FIG. 4G). However, the
degree of the increase on cardiomyocyte number in the cell culture
insert experiments between day 1 and day 5 (.about.1.8 times) (FIG.
4H) was lower than that in co-culture condition (.about.2.5 times).
These findings indicate it might promote the cardiomyocyte
proliferation that the soluble factors secreted from NCFs and the
cell-cell interaction between cardiomyocytes with cardiac
fibroblasts.
[0097] <Comprehensive Genetic Analysis of NCFs and ADFs>
[0098] To identify the factors that are responsible for involved in
these effects, we performed comprehensive genetic analysis between
NCFs and ADFs using a microarray analysis. As shown in FIG. 5A,
many differences in gene expression were observed between NCFs and
ADFs. Over 500 genes showed more than 10 times enhanced expression
in NCFs compared with ADFs. After choosing the
cardiovascular-related genes from the lists, 20 genes were
remained. Furthermore when we selected genes that were reported the
embryonic lethal phenotype causing a disorder to generate heart in
knock out mouse model and also act as a soluble factor and an
sdhesive factor, Vcam-1 was remained. The enhanced expression of
Vcam-1 in NCFs compared with ADFs was confirmed by quantitative
RT-PCR and western blot analysis (FIGS. 5B to D).
[0099] <VCAM-1-Dependent Cardiomyocyte Proliferation in
Co-Culture with Cardiac Fibroblasts>
[0100] Since integrin .alpha.4.beta.1 is known to be the principal
co-receptor of VCAM-1, we examined the integrin .alpha.4.beta.1
expression in mESC-derived cardiomyocytes. As shown in FIGS. 5E and
F, almost all of mESC-derived cardiomyocytes showed generation of
integrin .alpha.4.beta.1.
[0101] Next we elucidated whether VCAM-1 contributed to cardiac
fibroblasts-mediated cardiomyocyte proliferation using neutralizing
antibodies. After the pretreatment of NCFs with anti-VCAM-1
antibodies, NCFs and mESC-derived cardiomyocytes were cultured
using cell culture inserts. Anti-VCAM-1 antibody treatment
significantly inhibited cardiac fibroblast-mediated increase of
cardiomyocyte number (FIGS. 6A and B).
[0102] Finally we evaluated the direct effects of VCAM-1 on the
proliferation of cardiomyocytes. One day after starting culture,
cardiomyocytes were treated with VCAM-1 recombinant protein until
day 5. As shown in FIGS. 6C and D, VCAM-1 treatment increased the
number of cardiomyocytes compared with control. These findings
suggest heart-derived fibroblasts might induce cardiomyocyte
proliferation through fibroblasts-mediated VCAM-1 and integrin
.alpha.4.beta.1 in cardiomyocytes.
[0103] To confirm importance of VCAM-1 positive cells in
constructing functional cardiac cell sheets, we measured the
percentage of VCAM-1 positive cells in organism-derived cardiac
fibroblasts.
[0104] Cardiac fibroblasts were dissected and collected from
neonatal mice (1 day) of C57/BL6 mice, and skin fibroblasts were
dissected and collected from adult mice (10-12 weeks). Each of the
fibroblasts were adhesion-cultured up to passage 3, and the cell
volume of 1.times.10.sup.7 cells per condition was obtained.
Passage 3 is the same condition with the culture condition of the
above-mentioned cardiomyocytes produced by cell sheets.
[0105] Both fibroblasts were subjected to primary immunofluorescent
stain with Goat polyclonal anti-VCAM-1 antibodies (R&D systems,
Minneapolis, Minn.), and were subjected to secondary
immunofluorescent stain with Alexa Fluor 488 Donkey anti-goat IgG
(Life Technologies, MD, United States). Subsequently, FACS analysis
was conducted at Gallios (Beckman Coulter, Brea, Calif.), and
VCAM-1 positive cell rate was measured (N=3). Calculation of
significant difference was carried out by Student's t-test.
[0106] The results of cardiac fibroblasts (NCFs) were shown in
FIGS. 7A-C. It was found that the percentage of VCAM-1 positive
cells in NCFs was approximately 60% (FIG. 7A: 66.57%, FIG. 7B:
58.95%, FIG. 7C: 54.73%). Conversely, the percentage of VCAM-1
positive cells in skin fibroblasts (ADFs) was approximately 5%, and
it turned out that the percentage of VCAM-1 positive cells in NCFs
is significantly more than that of ADFs (P<0.001).
[0107] It was suggested that cardiac fibroblasts containing many
VCAM-1 positive cells contribute to construction of functional
myocardial tissues by proliferating cardiomyocytes derived from
mice ES through the expressing VCAM-1. Further, it was considered
that VCAM-1 positive cardiac fibroblasts originate from an outer
membrane-derived cell from the view point of embryology, and we
obtained the suggestion that it is effective to classify
fibroblasts from the view point of embryology, and not to conduct
morphological classification but to conduct functional
classification as a cell source for constructing a functional
tissue.
[0108] It is considered that, in NCFs, the majority of the cells
that are not expressing VCAM-1 express CD31 (vascular endothelial
cell marker). The reason for this is as follows: it is known that
tissue-resident cardiac fibroblasts are produced from
epicardium-derived cells through epithelial mesenchymal transition
(EMT), and also are differentiated from vascular endothelial cells
through endothelial mesenchymal transition (EndMT). Furthermore, as
is the case with cardiac fibroblasts, kidney fibroblasts that
differentiate from vascular endothelial cells through EndMT are
expressing CD31 (J Am Soc Nephrol 19:2282-2287, 2008). This may
also become one of the bases for supporting that NCFs are
expressing CD31.
[0109] From the above, it was clarified that, not skin fibroblasts
but cardiac fibroblasts enhance proliferation of mouse embryonic
stem cell (mESC) derived cardiomyocytes, and contribute to
construction of more functional cardiac cell sheets. Moreover, it
was indicated that cardiac fibroblasts are more abundantly
expressing VCAM-1 compared with skin fibroblasts, and that the
VCAM-1 of cardiac fibroblasts play an important role in
proliferation of cardiac cells and construction of cardiac tissues
that are functionally biologically-designed.
Example 2
[0110] Example 1 reveals that when a myocardial tissue derived from
a pluripotent stem cell is constructed, cardiac fibroblasts cause
cell proliferation of cardiomyocytes through VCAM-1 which is
protein highly expressed by the cardiac fibroblasts, prompt beating
in the created whole myocardial tissue, and significantly improve
the functionality. However, it is also revealed that cardiac
fibroblasts have a heterozygous character even in a local area
named heart, and that all the cardiac fibroblasts do not
necessarily express VCAM-1. Therefore, we carry out a study to
elucidate that, when fibroblasts are not classified by the
morphological features in a conventional manner but are classified
by protein expressed with molecular biology, to what extent cardiac
fibroblasts expressing VCAM-1 (VCFs) should be compounded so as to
create a highly functional myocardial tissue.
[0111] 1. Experimental Method
(1) Animals and Reagents
[0112] Wild-type C57BL/6 mice were purchased from Japan SLC
(Shizuoka, Japan). B6 Cg-Tg (CAG-DsRed*MST) 1Nagy/J mice were
purchased from The Jackson Laboratory (Bar Harbor, Me.). All the
experimental protocols were approved by The Keio University
Institutional Animal Care and Use Committee. The following
antibodies were used for immunofluorescent staining and flow
cytometry.
[0113] guinea pig monoclonal anti-vimentin (Progen, Heidelberg,
Germany),
[0114] mouse monoclonal anti-cardiac troponin T (cTnT) (Thermo
Scientific, Rockford, Ill.),
[0115] rabbit polyclonal anti-Ki67 (Abcam),
[0116] Rat monoclonal anti-VCAM-1 (Biotin) (Abcam),
[0117] Rat monoclonal anti-CD31 (Abcam),
[0118] Rabbit monoclonal anti-VCAM-1 (Abcam).
[0119] Secondary antibodies were purchased from ImmunoResearch
Laboratories (West Grove, Pa.).
(2) Culture of Mouse ES-Derived Cardiomyocytes
[0120] Maintenance, cardiomyocyte differentiation, and purification
of mouse ES cells (mESC) that express neomycin phosphotransferase
genes under the control of .alpha.-myosin heavy chain promoter, and
that express yellow fluorescent protein (YFP) are conducted in
accordance with the method as described previous report (Matsuura
K, et al., Biomaterials. 2011; 32:7355-7362).
[0121] Briefly described, for the purposes of induction to
cardiomyocytes and cardiomyocyte purification, 5.times.10.sup.4
cells/mL of trypsin-treated ES cells (total 125 mL/flask) were
seeded to a spinner flask (Integra Biosciences, Zizers,
Switzerland), and cultured with DMEM supplemented with 10% FBS for
10 days, and subsequently the differentiated cells were treated
with neomycin for 8 days.
[0122] Mouse ES-derived cardiomyocytes (Cor.At) to which puromycin
resistance gene and green fluorescent protein (GFP) were introduced
under .alpha.-myosin heavy chain promoter were purchased from
Axiogenesis AG (Cologne, Germany). Mouse ES-derived cardiomyocytes
were treated with puromycin for 2 days, and were cultured in a
medium not containing puromycin for 2 weeks.
(3) Isolation of VCAM-1 Positive Cardiac Fibroblasts
[0123] Cardiac fibroblasts isolated from a one day old wild-type
C57BL/6 neonatal mouse were cultured, and VCFs were isolated with a
magnetic cell sorter (Magnetic-activated cell sorting, MACS). The
isolated VCFs were re-cultured, and an experiment was carried out
after removing dead cells.
(4) Immunofluorescent Staining
[0124] Cells were fixed with 4% paraformaldehyde, and
immunofluorescent staining was carried out. The
immunofluorescent-stained cells were analyzed with a confocal
quantification image cytometer CQ1 (Yokogawa Electric Corporation,
Tokyo, Japan).
(5) Flow Cytometry
[0125] Tissues of the heart collected from a wild-type C57BL/6
mouse were dissociated with gentleMACS Octo Dissociator (Miltenyi
Biotec, Gladbach, Germany), and homogenized to the cellular level.
The obtained cells were immunofluorescent-stained, and subsequently
analyzed with an S3 cell sorter (BIO-RAD, CA).
(6) Time-Lapse Photography and Analysis
[0126] As evaluation of the ability of forming a network, YFP
positive ES cell-derived cardiomyocytes and cardiac fibroblasts
isolated from a DsRed mouse were co-cultured, and the inside of a
BZ-9000 fluorescent microscope was kept at the concentration of 5%
CO2 at 37.degree. C., and time-lapse observation was carried out
for 5 days (Keyence, Osaka, Japan). With respect of the evaluation
of the migratory ability of a single cardiomyocyte, GFP expressing
type cardiomyocytes were seeded at the concentration of
5.45.times.10.sup.4 cells/cm.sup.2' and fibroblasts isolated with
MACS were seeded at the concentration of 1.36.times.10.sup.4
cells/cm.sup.2, which were weaker than the concentration at the
time when a myocardial tissue was constructed, and time-lapse
photography was carried out with a BZ-X700 fluorescent microscope
for 3 days. The photographed time-lapse images were analyzed with a
Motion Analyzer so as to calculate the total migratory distance
(mm) of cardiomyocytes for three days of culturing, and the
evaluation of the migratory ability was carried out (Keyence).
[0127] 2. Results
Optimum Compounding Concentration Rates of VFCs and
Cardiomyocytes
[0128] It was found out that both of VCFs and VCAM-1 negative
cardiac fibroblasts (VNCFs) that were isolated and cultured with
MACS show a fusiform fibroblast-like morphology in phase difference
images (FIG. 8A). However, by using immunofluorescent staining,
while expression of VCAM-1 protein emitting red fluorescence was
observed in almost all the cells in VCFs, expression of VCAM-1
protein was not observed in VNCFs (FIG. 8B).
[0129] Subsequently, VCFs and VNCFs collected from cardiac
fibroblasts and dermal fibroblasts were co-cultured with
cardiomyocytes at each compounding ratio (Table 1), the highest
mitogenic effect of cardiomyocytes was observed at Day 5 under the
condition where 20% of VCFs and 80% of cardiomyocytes were seeded
(FIGS. 9A and 9B). In addition, when the number of fibroblast were
calculated by deducting the number of triple-positive cells of
cTnT/GFP/Hoechst from the total number of Hoechst 33258 positive
nuclei in a myocardial tissue at day 5 to which VCFs was compounded
at 20%, it was revealed that cardiomyocytes existed at 9.5%, and
fibroblasts existed at 90.5% (FIG. 10).
TABLE-US-00001 TABLE 1 Compounding ratio of each type of cell
VCAM1(+) VCAM1(-) Sample Cardiomyocytes NCFs NCFs NCFs ADFs A 80%
-- -- -- 20% B 80% 0% 20% -- -- C 80% 4% 16% -- -- D 80% 8% 12% --
-- E 80% 12% 8% -- -- F 80% 16% 4% -- -- G 80% 20% 0% -- -- H 80%
-- -- 20% --
[0130] Evaluation of Migratory Ability of Cardiomyocytes
[0131] It was revealed by time-lapse photographing that, when YFP
positive ES cell-derived cardiomyocytes and cardiac fibroblasts
isolated from a DsRed mouse were co-cultured, cardiomyocytes were
divided at day 5, and a strong network was constructed (FIG. 11).
As mentioned before, cardiac fibroblasts prompt cell division of
cardiomyocytes through the expressing VCAM-1 protein. VCFs and
VNCFs isolated with MACS were co-cultured with GFP expressing type
cardiomyocytes, and the total migratory distance (mm) of the
cardiomyocytes for 3 days of culturing was calculated by time-lapse
photographing, and the evaluation of migratory ability was carried
out. It was revealed that, when VCFs were compounded, the number of
GFP positive cardiomyocytes grows, and it was suggested by video
analysis that the migratory ability of cardiomyocytes was
significantly high, and took part in forming of a high level
network (FIG. 12).
[0132] Evaluation of Localization of VCFs in a Biological Heart
[0133] It was revealed that, when a myocardial tissue was
constructed by co-culturing at the concentration of 20% VCFs and
80% cardiomyocytes, cardiomyocytes grew at the highest level in the
myocardial tissue, and obtained high migratory ability (FIGS. 9, 11
and 12). Further, it was revealed that 9.5% cardiomyocytes and
90.5% fibroblasts existed in a myocardial tissue created under the
above-mentioned condition (FIG. 10). In order to evaluate whether
the localization of cardiomyocytes and fibroblasts was different
from a biological heart, a heart of a one day old mouse was
collected, and crushed into the cellular level by enzyme treatment,
and the evaluation of localization of cardiomyocytes and VCFs were
carried out with flow cytometry. It was revealed that VCFs existed
at 14.8% in the heart, and that 55.6% of fibroblasts expressed
VCAM-1 protein (FIGS. 13, 14A and 14B). Moreover, it was revealed
that fibroblasts that express CD31 existed at 3.7% in the heart,
and that 16.1% of cardiac fibroblasts expressed CD31 protein (FIGS.
13, 14C and 14D).
[0134] 3. Conclusion
[0135] The present study revealed that the optimum seeding
concentration of VCFs required to create a high functional
myocardial tissue was 20% by evaluating the proliferation level of
cardiomyocytes by compounding VCFs sorted by a magnetic cell
separator (Magnetic-activated cell sorting, MACS) and ES
cell-derived cardiomyocytes at each concentration.
[0136] Furthermore, it was revealed that the localization of
cardiomyocytes and fibroblasts in a myocardial tissue with 20% VCFs
at day 5 after culturing was 9.5% and 90.5%, respectively. When
VCFs were seeded at the concentration of 20% and cardiomyocytes
were seeded at the concentration of 80%, and the evaluation of the
migratory ability of cardiomyocytes was carried out for 3 days with
time-lapse photographing, it was revealed that VCFs provides
cardiomyocytes with high migratory ability, and it was suggested
that the provision of high migratory ability takes part in
formation of a high level myocardial network in a myocardial
tissue.
[0137] As a result of flow cytometry, it was revealed that
localization of cardiomyocytes and fibroblasts in a created
VCFs-compounded myocardial sheet was greatly different from a
biological heart, and it was revealed that fibroblasts which
express Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1,
CD31) other than VCFs are localized in a heart.
INDUSTRIAL APPLICABILITY
[0138] By culturing using the cardiac cell culture materials of the
present invention, functional cardiac tissues are preferably
constructed. The cardiac cells obtained by the culture can be used
as regenerative medicines such as transplantation, or as artificial
organ materials such as cardiac tissue models.
[0139] While the invention has been described in detail and with
reference to specific embodiments thereof, it will be apparent to
one skilled in the art that various changes and modifications can
be made therein without departing from the spirit and scope
thereof.
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