U.S. patent application number 13/227113 was filed with the patent office on 2012-03-15 for method of mass producing growth factor using adipose derived adult stem cells.
This patent application is currently assigned to PROSTEMICS CO., LTD.. Invention is credited to BongGeun Choi, ByungSoon Park, ChulHong Park.
Application Number | 20120065129 13/227113 |
Document ID | / |
Family ID | 38309396 |
Filed Date | 2012-03-15 |
United States Patent
Application |
20120065129 |
Kind Code |
A1 |
Park; ByungSoon ; et
al. |
March 15, 2012 |
METHOD OF MASS PRODUCING GROWTH FACTOR USING ADIPOSE DERIVED ADULT
STEM CELLS
Abstract
The present invention relates to a method for producing large
amounts of human growth factors from human adipose-derived stem
cells. More specifically, the invention provides a method capable
of synthesizing human growth factors in significantly large amounts
by culturing adipose-derived stem cells extracted from human
adipose cells in suitable media and conditions. Also, stem cell
culture media produced according to the method of the invention,
and human growth factors isolated from the culture media, can be
advantageously used as raw materials for drugs and cosmetics.
Inventors: |
Park; ByungSoon; (Seoul,
KR) ; Choi; BongGeun; (Gwangjin-gu, KR) ;
Park; ChulHong; (Seoul, KR) |
Assignee: |
PROSTEMICS CO., LTD.
Seoul
KR
Park; ByungSoon
Seoul
KR
|
Family ID: |
38309396 |
Appl. No.: |
13/227113 |
Filed: |
September 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12161899 |
Jul 23, 2008 |
|
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PCT/KR06/04111 |
Oct 12, 2006 |
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13227113 |
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Current U.S.
Class: |
514/8.1 ;
514/7.6; 514/8.9; 514/9.1 |
Current CPC
Class: |
C07K 14/503 20130101;
C12N 5/0667 20130101; A61P 17/00 20180101; C07K 14/495 20130101;
C07K 14/52 20130101; C12N 2500/02 20130101; C12N 2500/99 20130101;
C12N 2510/02 20130101; C12N 2500/60 20130101; C12N 2500/38
20130101; C12N 2502/1382 20130101; C12N 2500/90 20130101 |
Class at
Publication: |
514/8.1 ;
514/7.6; 514/9.1; 514/8.9 |
International
Class: |
A61K 8/64 20060101
A61K008/64; A61Q 19/08 20060101 A61Q019/08; A61Q 19/00 20060101
A61Q019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 2006 |
KR |
10-2006-0008874 |
Claims
1. A cosmetic composition having a functionality and containing an
adipose-derived adult stem cell culture medium obtained by a method
comprising the steps of: (i) isolating adipose-derived adult stem
cells extracted from mammalian adipose cells; (ii) optionally
culturing the stem cells in a serum medium, and then subculturing
the stem cells in a serum-free medium; (iii) applying, to the
adipose-derived stem cells, at least one physical stimulation
selected from among low-oxygen culture, UV irradiation, nutrient
deficiency and mechanical friction; and (iv) optionally adding, to
the culture media, one or more vitamins selected from among vitamin
A, vitamin B, vitamin C and vitamin D, wherein step (iii) and step
(iv) are performed in conditions in which the highest production of
the human growth factors occurs.
2. The cosmetic composition of claim 1, wherein the adipose-derived
adult stem cell culture medium contains human growth factors.
3. The cosmetic composition of claim 1, wherein the adipose-derived
adult stem cell culture medium contains a human basic fibroblast
growth factor (bFGF).
4. The cosmetic composition of claim 1, wherein the adipose-derived
adult stem cell culture medium contains a human vascular
endothelial growth factor (VEGF).
5. The cosmetic composition of claim 1, wherein the adipose-derived
adult stem cell culture medium contains a human transforming growth
factor-beta (TGF-.beta.).
6. The cosmetic composition of claim 1, wherein the functionality
is an anti-wrinkle or anti-aging activity.
7. A cosmetic composition for anti-wrinkle and/or anti-aging, which
comprises a culture medium of stem cells, in which the culture
medium is obtained by a method comprising the steps of: (i)
isolating adipose-derived adult stem cells extracted from mammalian
adipose cells; and (ii) culturing the stem cells in a serum medium,
and then subculturing the stem cells in a serum-free medium.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of application
Ser. No. 12/161,899 filed on Jul. 23, 2008, which is a U.S.
national phase application, pursuant to 35 U.S.C. .sctn.371, of
PCT/KR2006/004111 filed on Oct. 12, 2006, which claims priority to
Korean application 10-2006-0008874, filed Jan. 27, 2006. The entire
contents of the aforementioned patent applications are incorporated
herein by this reference.
TECHNICAL FIELD
[0002] The present invention relates to a method for producing
large amounts of growth factors from human adipose-derived stem
cells. More specifically, the present invention provides a method
which comprises culturing adipose-derived stem cells extracted from
human fat cells in suitable media and conditions, such that human
growth factors, for example, an acidic fibroblast growth factor
(acidic FGF), a basic fibroblast growth factor (basic FGF), an
insulin-like growth factor-1 (IGF-1), an insulin-like growth
factor-2 (IGF-2), a keratinocyte growth factor, a platelet-derived
growth factor (PDGF), a human transforming growth factor-alpha
(TGF-.alpha.), a human transforming growth factor-beta
(TGF-.beta.), a vascular endothelial growth factor (VEGF), an
epidermal growth factor (EGF) or a nerve growth factor, can be
synthesized in amounts significantly larger than using existing
methods involving stem cells.
BACKGROUND ART
[0003] As used herein, the term "stem cell" refers to a cell, which
remains undifferentiated into a particular cell type, and, if
necessary, has the potential to differentiate into all types of
cells constituting the body, including nerve, blood, cartilage,
etc. Methods capable of producing such stem cells are broadly
classified into two categories: (1) a method of producing stem
cells from embryos developed from fertilized eggs (embryonic stem
cells); and (2) a method of recovering stem cells stored in each
part of the adult human body (adult stem cells). Although embryonic
stem cells and adult stem cells are functionally different from
each other, they all have the ability to differentiate into various
cell types.
[0004] The embryonic stem cells have advantages of very good
differentiation potential and long telomeres, but have ethical
problems and in the disadvantage that they are difficult to obtain
in large amounts. In comparison, the adult stem cells can be
obtained in large amounts, but have the disadvantage that, when
these cells are transplanted into other persons, they carry the
risk of infection and have a relatively low differentiation
potential.
[0005] Despite the above-described disadvantages, adult stem cells
are considerably safe for use in medical applications.
Specifically, these cells do not cause cancer even when they are
transplanted into the body for organ regeneration, and they do not
cause immune rejection reactions because they have originated from
one's own body. Thus, these adult stem cells can be used for
autologous transplantation.
[0006] Also, the adult stem cells have site-specific
differentiation potential to differentiate according to the
characteristics of the peripheral tissues, and do not cause cancer
even when they are injected in an undifferentiated state. Thus,
these adult stem cells have the potential to produce cells
immediately after transplantation and also display the self-renewal
potential to create and store undifferentiated stem cells, if
necessary.
[0007] Due to the above-described advantages, the importance of
adult stem cells has recently been highlighted, and various studies
have been conducted to obtain adult stem cells in vivo.
[0008] Adipose tissue plays an important role in normal growth and
physiological action in vivo, but the importance thereof has been
previously unappreciated. The most general type of fat is white
adipose tissue, which is located below the skin (subcutaneous fat),
in the abdominal cavity (visceral fat) or around reproductive
organs (gonadal fat). Brown adipose tissue is a slightly less
general form of fat present in an adult, which plays an important
role in the production of heat during the infant stage (Gimble, New
Biol. 2(4): 304-12, (1990)).
[0009] However, in fact, reproductive capability and stage of
maturity are closely associated with adipose tissue storage in
individuals. Female and male adolescence are closely associated
with the production and secretion of adipose tissue-derived
hormones and with body fat composition. Also, adipose tissue plays
an important role in glucose metabolism and energy balance.
[0010] For a few years, there has been a significant advancement in
the biomaterial field. On the basis of this, many materials are
currently developed and used. Despite this progress, many studies
have, in fact, not been conducted on the use of human adipose
tissue. However, since it was recently reported that adult stem
cells are present in adipose tissue (Zuk P A, et al., Molecular
Biology of Cell, 13: 4279-4295 (2002); Rodriguez A M, et al.,
Biochimie, 87: 125-128 (2005)), various studies on the use of
adipose-derived cells have been conducted.
[0011] Also, with the development of the fields of biochemistry and
molecular biology, small amounts of signaling substances (growth
factors) have recently been found in the human body, and on the
basis of these findings, theories on in vivo aging have been
re-established (Stanley Cohen, Nobel Lecture 1986, Dec. 8).
Moreover, it has been found that the signaling substances (growth
factors) decrease as age increases, and this decrease in the growth
factors is closely associated with the aging of the human body
(Sporn M and Roberts A, Handbook of Experimental Pharmacology, Vol.
1, Vol. 95/1, 1990, Springer-Verlag, DE, Berlin., pp. 667-698).
[0012] Thus, it has been reported that, when external growth
factors are administered to the body, the aging of the body can be
inhibited, and studies on special effects of these substances have
been conducted (G E Pierce and T A Mustoe, Annu Rev Med, 46.
467-481 (1995)). Specifically, studies on the structure and
synthesis of the signaling substances (growth factors) have been
conducted, but the most of the growth factors have protein
structures, which are three-dimensionally complex, thus posing
various problems in chemical synthesis and significantly increasing
costs for the synthesis.
[0013] Accordingly, the present inventors have conducted studies on
a method of obtaining active human growth factors at low cost while
maintaining the activity thereof in the human body and, as a
result, paid attention to the fact that adipose-derived adult stem
cells secrete growth factors (Rehman, J. et al., Circulation, 109:
1292-1298 (2004)).
[0014] However, studies on adipose-derived adult stem cells mainly
relate to the use or differentiation of the cells themselves, and
methods of synthesizing growth factors from these cells have not
been studied.
[0015] Korean Patent Laid-Open Publication No. 2004-94910, entitled
"Improved fat cell-differentiated, adipose-derived adult stem cells
and the use thereof" discloses a method capable of increasing the
in vivo survival rate of adipose-derived adult stem cells, but does
not disclose the meaningful synthesis of growth factors. Korean
Patent Laid-Open Publication No. 2005-6408, entitled "PBR ligand
having function of regulating fat cell differentiation, derivative
compounds thereof, and composition for regulation of fat cell
differentiation, containing the same", merely discloses a method of
differentiating fat cells into other particular cells.
[0016] Also, Korean Patent Laid-Open Publication No. 2005-99274,
entitled "Animal serum-free medium composition for culture of human
stem cells, and method for induction of differentiation into liver
cells", discloses a method of differentiating human stem cells
using animal serum-free medium, and Korean Patent Registration No.
484550, entitled "Method for production of cells for cell
transplantation", discloses the use of stem cells for cell
transplantation.
[0017] The reason that studies on the synthesis of growth factors
using adipose-derived adult stem cells are insufficient is that the
methods of using adult stem cells are focused on their use through
differentiation into other cells and that studies on the
cytological differences between stem cells are insignificant.
[0018] Also, as described above, methods for synthesizing growth
factors using recombinant genetic techniques have only been
studied, as disclosed in Korean Patent Registration No. 101436,
entitled "Method for producing recombined human endothelial cell
growth factors", Korean Patent Registration No. 62551, entitled
"Method for producing human epithelial cell growth factors by
genetic recombination technology", Korean Patent Laid-Open
Publication No. 2003-45032, entitled "Method for producing
biologically active human acidic fibroblast growth factors and use
thereof for stimulation of angiogenesis."
[0019] Accordingly, the present inventors have discovered a method
for producing large amounts of growth factors using adipose-derived
adult stem cells and, as a result, found that adipose-derived adult
stem cells obtained through the establishment of suitable culture
conditions and physical and/or chemical stimulation synthesized and
secreted growth factors in significantly effective amounts compared
to adipose-derived adult stem cells to which specific stimulation
were not applied.
[0020] The above information disclosed in this Background section
is only for enhancement of understanding of the background of the
invention and therefore it may contain information that does not
form the prior art that is already known in this country to a
person of ordinary skill in the art.
SUMMARY OF THE DISCLOSURE
[0021] It is an object of the present invention to produce large
amounts of human growth factors from adipose-derived adult stem
cells, the produced human growth factors having excellent in vivo
activity compared to growth factors synthesized by recombinant or
chemical methods.
[0022] Another object of the present invention is to provide safe
and effective drugs or cosmetics containing either human growth
factors produced in large amounts from adipose-derived stem cells,
or culture media of the growth factors.
[0023] To achieve the above objects, in one aspect, the present
invention provides a method for producing large amounts of human
growth factors from adipose-derived stem cells, the method
comprising the steps of: (i) isolating adipose-derived adult stem
cells extracted from mammalian adipose cells; (ii) optionally
culturing the stem cells in a serum medium, and then subculturing
the stem cells in a serum-free medium; (iii) applying, to the
adipose-derived stem cells, at least one physical stimulation
selected from among low-oxygen culture, UV irradiation, nutrient
deficiency and mechanical friction; and (iv) optionally adding, to
the culture media, one or more vitamins selected from among vitamin
A, vitamin B, vitamin C and vitamin D, wherein step (iii) and step
(iv) are performed in conditions where the highest production of
the human growth factors occurs.
[0024] In another aspect, the present invention provides a
functional cosmetic composition containing human growth factors
produced by said method.
[0025] In still another aspect, the present invention provides a
functional cosmetic composition containing an adipose-derived adult
stem cell culture medium obtained by said method.
[0026] According to the present invention, it is possible to
produce human growth factors in large amounts from adipose-derived
adult stem cells. It was found that the growth factors produced
using the inventive production method had excellent safety and
activity compared to those of growth factors produced according to
existing production methods. Furthermore, the growth factors
produced using the inventive production method could act in the
same fashion as the existing growth factors of the human body.
Also, it is expected that a culture medium of adipose-derived stem
cells and growth factors isolated from the culture medium can be
advantageously applied in drugs, quasi drugs and cosmetics for
anti-wrinkle, wound healing, and scar removing.
BRIEF DESCRIPTION OF DRAWINGS
[0027] FIG. 1 is an optical microscope image of stem cells isolated
from adipose tissue;
[0028] FIG. 2 shows the results of flow cytometry of originally
isolated PLA cells;
[0029] FIG. 3 is a graphic diagram showing the results of flow
cytometry after subculture;
[0030] FIG. 4 is an electrophoresis image of TGF.beta.-1, bFGF and
VEGF RT-PCR amplification products from adipose-derived stem cells,
confirming the expression of TGF.beta.-1, bFGF and VEGF RT-PCR;
[0031] FIG. 5 is a graphic diagram showing the concentrations of
bFGF and VEGF secreted in culture media during the culture of
adipose-derived stem cells;
[0032] FIG. 6 is a graphic diagram showing the measured collagen
synthesis of fibroblasts in mixed culture of fibroblasts and
adipose-derived stem cells;
[0033] FIG. 7 is a graphic diagram showing the number of fibroblast
cells in varying concentrations of adipose-derived stem cell
culture media;
[0034] FIG. 8 is a graphic diagram showing the comparison of cell
proliferation potential between adipose-derived stem cell culture
media and recombinant growth factor-containing media;
[0035] FIG. 9 is a graphic diagram showing the comparison of
secretion of a basic fibroblast growth factor (bFGF) of
adipose-derived stem cells cultured in physical and chemical
conditions according to the present invention;
[0036] FIG. 10 is a graph showing the comparison of the secretion
of a vascular epithelial growth factor (VEGF) of adipose-derived
stem cells cultured in physical and chemical conditions according
to the present invention;
[0037] FIG. 11 is a graph showing the comparison of the secretion
of transforming growth factor-beta (TGF-.beta.) of adipose-derived
stem cells cultured in physical and chemical conditions according
to the present invention;
[0038] FIGS. 12, 13, 14 and 15 are photographs showing the
wrinkle-reducing activity of a composition containing growth
factors isolated from autologous adipose-derived adult stem cells;
and
[0039] FIG. 16 is a photograph showing the results of Western blot
analysis for the collagen synthesis of fibroblasts, in
adipose-derived stem cell culture media.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0040] The first aspect of the present invention relates to a
method for producing large amounts of growth factors using
adipose-derived stem cells.
[0041] A growth factor contained in adipose-derived stem cells is
selected from the group consisting of an acidic fibroblast growth
factor (acidic FGF), a basic fibroblast growth factor (basic FGF),
an insulin-like growth factor-1 (IGF-1), an insulin-like growth
factor-2 (IGF-2), a keratinocyte growth factor (KGF), a
platelet-derived growth factor (PDGF), a human transforming growth
factor-alpha (TGF-.alpha.), a human transforming growth factor-beta
(TGF-.beta.), a vascular endothelial growth factor (VEGF), an
epithelial growth factor (EGF), a nerve growth factor (NGF), and
mixtures thereof.
[0042] In the present invention, adipose-derived stem cells were
used to specifically stimulate the synthesis of the following
growth factors.
[0043] 1) Basic fibroblast growth factor (hereinafter, referred to
as "bFGF")
[0044] bFGF or heparin-binding growth factor 2 (HBGF-2) includes
seven kinds of factors having a homology of about 30-50% at the
amino acid sequence level (Burgess, W. H and Maciag, T., Annu Rev.
Biochem., 58: 575-606 (1989); Baird, A. and Klagsbrun, M., Cancer
Cells, 3(6): 239-43 (1991)). bFGF is isolated from nerve tissue,
the pituitary body, the adrenal cortex, corpora leutea, and the
placenta.
[0045] bFGF isolated from the body has a size of about 18 kDa.
Several studies have reported the existence of larger species of
bFGF having a size of about 24 kDa, which result from the extension
of the amino terminal end of the protein due to translation
initiation at a region containing no AUG start codon (Burgess, W. H
and Maciag, T., Annu Rev. Biochem., 58: 575-606 (1989); Baird, A.
and Klagsbrun, M., Cancer Cells, 3(6): 239-43 (1991); Prats, H. et
al., PNAS, 86: 1836-1840 (1989); Quarto, N. et al., J. Cell.
Physiol., 147(2): 311-8 (1991); Bugler, B. et al., Mol. Cell.
Biol., 11(1): 573-7 (1991)). Such phenomena result in the
localization of bFGF in cell nuclei rather than cytoplasm (Quarto,
N. et al., J. Cell. Physiol., 147(2): 311-8 (1991); Bugler, B. et
al., Mol. Cell. Biol., 11(1): 573-7 (1991)). bFGF proteins produced
by general recombinant or chemical methods are based on a 18-kDa
region. Production of recombinant or synthetic bFGF differs from
naturally-produced bFGF, because recombinant or synthetic bFGF
morphologically lacks a hydrophobic signal peptide base sequence
(Mignatti, P. et al., Proc. Natl. Acad. Sci. USA 88: 11007
(1991)).
[0046] 2) Vascular endothelial growth factor (hereinafter, referred
to as "VEGF")
[0047] VEGF (Ferrara, N. and Henzel, W. J. Biochem Biophys Res
Commun, 161(2): 851-8 (1989)) is known as a vascular permeable
factor (Senger, D. R. et al., Science, 219(4587): 983-5 (1983)), is
a homodimer, has a molecular weight of 34-42 kDa, and is a
heparin-bound glycoprotein. This protein has activity as an
angiogenesis factor, and thus can promote the mitosis of epithelial
cells and improve vascular permeability.
[0048] VEGF is expressed as a primary structure known as a limited
base sequence and has homology with the A and B chains of a
platelet-derived growth factor (PDGF). Such growth factors have
eight conserved cysteine residues, which are involved in forming
interior and solvent exposed disulfide bonds. cDNA encoded by the
VEGF protein has 53% homology to the platelet-derived growth factor
(PDGF) at the amino acid sequence level. VEGF was isolated from a
cDNA library of the human placenta (Maglione, D. et al., PNAS, 88:
9267 (1991)). This protein is called "placenta growth factor"
(PGF), and is currently recognized as a member of the VEGF family.
Based on homology with VEGF, the placenta growth factor (PGF) is
suggested to be an angiogenesis factor.
[0049] Genes for the human VEGFs are combined in eight exons.
Alternative splicing yields base sequences of 121, 165, 189 and 206
amino acids, which encode four monomeric VEGFs. Each of the base
sequences has 26 signal peptide amino acid residues, and thus can
be detected. VEGF121 and VEGF165 are diffusible proteins exposed to
extracellular matrixes, and VEGF189 and VEGF206 have high affinity
for heparin, and thus form proteoglycan bonds with heparin in
extracellular lipids. VEGF, which was originally described as a
glycoprotein, contains an N-linked glycosylation site.
[0050] The VEGF proteins produced by general recombinant or
chemical methods are based on the sequences of the diffusible
proteins VEGF121 and VEGF165. It was reported that the expression
of recombinant human VEGF in E. coli was not different from
original VEGF in vitro with respect to biological functions
(Connolly, D. T. J. Cell. Biochem, 47(3): 219-23 (1991); Schott, R.
J. and Morrow, L. A. Cardiovasc, Res., 27(7): 1155-61 (1993);
Neufeld, G. et al., Prog. Growth Factor Res., 5(1): 89-97 (1994);
Senger, D. R. et al., Cancer and Metastasis Reviews, 12(3-4):
303-24 (1993)).
[0051] 3) Transforming growth factor-.beta. (hereinafter, referred
to as "TGF-.beta.")
[0052] A human transforming growth factor (TGF), which is a factor
that stimulates the transformation of fibroblasts differentiating
into a tumor-like phenotype, consists of a mixture of two proteins
TGF-.alpha. and TGF-.beta. and is associated with tumor-inhibitory
factors rather than a tumor-stimulating factors (Lawrence, D. A.
Eur. Cytokine Netw, 7(3): 363-74 (1996); Cox, D. A. and Maurer, T.,
Clin. Immunol. Immunopathol, 83(1): 25-30 (1997); Alevizopoulos, A.
and Mermod, N., Bioassays. 19(7): 581-91 (1997)).
[0053] The two molecules are members of the TGF super-family,
including TGF-.beta.1 having five kinds of bone morphogenic
proteins as active and inactive substances (Kingsley, D. M., Genes
Dev, 8: 133 (1994)). It is known that human TGF-.beta.1 has a
molecular weight of 25 kDa, consists of disulfide bonds and
bi-glycosyl homodimers, and has a 100% conserved gene sequence
among almost all mammalian species. TGF-.beta.1 initiates
intracellular signaling through the transfer of a disulfide bond
from a precursor by a protease similar to subtilisin (Dubois, C. M.
et al., J. Biol. Chem., 270: 10618 (1995)), and it is generally
secreted as an inactive material or a composite of two (Gleizes,
P-E. et al., Stem Cells, 15: 190-197 (1997)). A TGF-.beta.1
signaling process includes two receptors (Ten Dijke, P. Curr. Opin.
Cell Biol, 8(2): 139-45 (1996); Derynck, R. and Feng X. H.,
Biochim. Biophys. Acta 1333(2): F105-50 (1997); Padgett, R. W. et
al., Bioessays, 20(5): 382-90 (1998)), and a TGF-.beta. RII dimer
as a 75-kDa ligand-binding protein has an intracellular
serine-threonine kinase enzyme, which is continually activated.
TGF-.beta. RII phosphorylates the 53-kDa signaling dimer TGF-.beta.
RI through binding to TGF-.beta.1. The phosphorylated TGF-.beta. RI
activates protein kinase and induces initiating a downstream signal
via intracellular protein SMADS.
[0054] The TGF receptor involved in the signaling process is found
in all cells and affects almost all physiological actions. The
systematic and cell-specific activation thereof is a very complex
mechanism, but shows three basic activities. TGF-.beta.1 generally
regulates the proliferation of cells such as inhibitory factors,
promotes the precipitation of protein hydrolysates in addition to
cell membranes by repeating the inhibition of protein degradation
and the synthesis of proteins, and stimulates immune inhibitory
reactions through various mechanisms.
[0055] A TGF-.beta.1 protein produced by prior recombinant or
chemical synthesis is an active protein having a size of 25 kDa,
and thus shows biological activity similar to the original
TGF-.beta.1 protein in vitro, but it contains only some of the
inherent properties of the TGF-.beta.1 protein.
[0056] The method according to the first aspect of the present
invention comprises the steps of: (i) isolating adipose-derived
adult stem cells extracted from mammalian adipose cells; (ii)
selectively culturing the stem cells in a serum medium and then
subculturing the stem cells in a serum-free medium; (iii) applying,
to the adipose-derived stem cells, at least one physical
stimulation selected from among low-oxygen culture, UV irradiation,
nutrient deficiency and mechanical friction; and (iv) selectively
adding, to the culture media, at one or more vitamins selected from
among vitamin A, vitamin B, vitamin C and vitamin D, wherein the
step (iii) and the step (iv) are performed in conditions where the
highest production of the human growth factors occurs.
[0057] Collection of Stem Cells
[0058] Adipose-derived adult stem cells according to the present
invention can be collected through a purification process from
cells present in adipose tissue. Preferably, human adipose-derived
adult stem cells are collected, and for this purpose,
adipose-derived stem cells are separated from human adipose
tissue.
[0059] Adipose tissue is brown or white adipose tissue derived from
subcutaneous, network membrane, intestinal, breast genital or other
adipose tissue sites and subcutaneous white adipose tissue can be
conveniently obtained using liposuction.
[0060] Regarding the source of adipose tissue, it is possible to
use adipose tissue discarded in the liposuction process, which is
commonly performed. That is, the utility of the adipose tissue from
liposuction is increased, because the need to perform invasive
surgery for adipose tissue is eliminated. The separated liposuction
material is washed, and only the adipose tissue is separated from
the washed material. The extracellular matrix of the adipose tissue
is treated with collagenase, and then centrifuged to collect a
stromal vascular fraction containing a high density of stem cells.
The pellets thus obtained are washed, and then passed through a
cell filter to separate other tissues. Then, monocytes and cell
fragments containing red blood cells are isolated from the
remaining tissue using a monocyte isolation solution. The isolated
monocyte cells are cultured in non-inducing media, and nonadhesive
cells are removed.
[0061] Culture of Stem Cells
[0062] In the present invention, special culture media were
established for the in vitro culture of the adipose-derived stem
cells obtained in the above-described process.
[0063] Before the start of the cell culture, biological samples
extracted from a supply source can be repeatedly washed with a wash
medium containing general antibiotics to minimize the possibility
of contamination in subsequent culture.
[0064] In the present invention, culture media are optimized so as
to maximize the synthesis and secretion of growth factors in the
adipose-derived stem cells.
[0065] Specifically, the in vitro culture of the adipose-derived
stem cells is performed by culturing the cells in a serum medium
and then subculturing the cells in a serum-free medium, such that
the synthesis of growth factors is maximized.
[0066] The serum-containing medium for the initial cell culture is
preferably a medium suitable for maintaining and storing cell types
such as adipose-derived stem cells.
[0067] In the present invention, the medium is based on Dulbecco's
Modified Eagle's Medium (DMEM), which is commonly used for cell
culture in the art, and contains serum, which is commonly used in
cell culture.
[0068] Herein, the initial medium may also be a frozen medium
obtained by adding 7-10% of dimethyl sulfoxide (DMSO) thereto, and
thus the stem cells can be frozen, and then, if necessary, can be
thawed before use.
[0069] Regarding the serum, 0.1-20% fetal bovine serum (FBS) is
preferably added to the medium. More preferably, the medium also
contains antibiotic agents, antifungal agents and reagents for
preventing contamination caused by the growth of mycoplasma.
[0070] Regarding the antibiotic agents, it is possible to use any
antibiotic agent used in general cell culture, including
penicillin-streptomycin. Regarding the antifungal agent, it is
preferable to use amphotericin B, and as the mycoplasma inhibitory
agent, it is preferable to use tylosin. In addition, mycoplasma
contamination can be prevented with gentamicin, ciprofloxacin,
azithromycin, etc. If necessary, oxidation nutrients such as
glutamine, and energy metabolites such as sodium pyruvate, can
further be added to the medium.
[0071] A more preferred medium contains 1-2 mM glutamine, 0.5-1 mM
sodium pyruvate, 0.1-10% FBS, 1% antibiotic (100
IU/ml)-supplemented glucose and DMEM, and is called "complete serum
medium". Herein, the concentration of glucose is approximately 1
g/L to 4.5 g/L. The complete serum medium provides the storage and
maintenance of adipose-derived stem cells and stable basic culture
conditions in vitro and shows effective cell stabilization.
[0072] With respect to general culture conditions for the initial
culture, the most suitable conditions for cell culture are applied,
and thus the cell culture is performed in an incubator at humidity
of 90-95% and a temperature of 35-39.degree. C. under a condition
of 5-10% CO.sub.2. When the cell culture is carried out in a
condition of 5-10% CO.sub.2, a carbon source such as sodium
bicarbonate is added to a final concentration of 0.17-0.22%.
[0073] During the initial culture stage, the tissue fragments are
preferably kept attached to the bottom of the culture flask, and
the growth of the cells can be promoted through short stimulation
caused by treatment with trypsin-EDTA according to standard cell
culture techniques.
[0074] Cumulative population doubling time is maintained until the
cells being cultured in the flask reach a confluence of 75-85%.
Preferably, the cells are collected at a confluence of 80% and
subcultured in a serum-free medium for late-stage culture.
[0075] The present invention provides a serum-free medium for the
differentiation of growth factors, which stimulates the
differentiation of growth factors in adipose-derived stem
cells.
[0076] The subculture process using the serum-free medium for the
differentiation of growth factors is preferably performed by
removing the medium from the culture, washing the flask with
phosphate buffer saline, suspending the cells with trypsin-EDTA,
centrifuging the cell suspension, and then washing the resulting
pellets with buffer solution, wherein the washing process is
repeated 2-3 times. The washed pellets are suspended in the
serum-free medium developed in the present invention and are
subcultured about 3 times in a cell culture flask.
[0077] The serum-free medium developed in the present invention is
based on a DMEM that does not contain a pH indicator such as phenol
red, and a Ham's F-12 nutrient mixture (SIGMA, Cancer Research Vol
47, Issue 1, 275-280), which is added thereto at a ratio of
approximately 1:0.5-2. Herein, it is possible to add oxidation
nutrients such as L-glutamine, energy metabolites such as sodium
pyruvate, and carbon sources such as sodium bicarbonate. In
addition, it is possible to add not only other growth factors than
the growth factors targeted in the present invention, but also
growth hormones.
[0078] The inherent characteristic of the serum-free medium
developed in the present invention can be seen in the Ham's F-12
nutrient mixture. In this mixture, various inorganic substances and
amino acids, which help to maintain the growth and homeostasis of
the cells and are involved in increasing the safety and maintenance
of the cells in the late-stage culture following the initial-stage
culture of the adipose-derived stem cells, vitamin nutrients, which
can stimulate the higher production of growth factors selected from
the adipose-derived stem cells, and other factors, are mixed with
each other at a given ratio. The serum-free medium containing the
Ham's F-12 mixture, established in the present invention, do not
show any reduction or negative effect on the culture of the
adipose-derived stem cells or the production of growth factor
compared to the conditions of general serum media containing animal
serum. Also, some growth factors show a higher productivity in the
serum-free medium. All the components and contents of the
serum-free culture medium are defined, unlike serum media having
unknown components caused by the addition of serum.
[0079] This suggests that variability, which can result from animal
serum present in serum media, can be minimized, and the effects of
the present invention can be achieved at a low cost or about 50%
compared to the prior art.
[0080] Table 1 below indicates the components and contents of the
Ham's F-12 nutrient mixture contained in the serum-free medium
established in the present invention.
TABLE-US-00001 TABLE 1 Concentration Morality Components (mg/L)
(mM) Amino acid D-Pantothenic Acis 2.24 0.00895 Glycine 18.75 0.25
L-Alanine 4.45 0.05 L-Arginine hydrochloride 147.5 0.699
L-Asparagine-H.sub.2O 7.5 0.05 L-Aspartic acid 6.65 0.05 L-Cysteine
hydrochloride-H.sub.2O 17.56 0.0998 L-Cystine 2HCl 31.29 0.1
L-Glutamic Acid 7.35 0.05 L-Glutamine 365 2.5 L-Histidine
hydrochloride-H.sub.2O 31.48 0.15 L-Isoleucine 54.47 0.416
L-Leucine 59.05 0.451 L-Lysine hydrochloride 91.25 0.499
L-Methionine 17.24 0.116 L-Phenylalanine 35.48 0.215 L-Proline
17.25 0.15 L-Serine 26.25 0.25 L-Threonine 53.45 0.449 L-Tryptophan
9.02 0.0442 L-Tyrosine disodium salt dihydrate 55.79 0.214 L-Valine
52.85 0.452 Vitamins Biotin 0.0035 0.0000143 Choline chloride 8.98
0.0641 D-Calcium pantothenate 2.24 0.0047 Folic Acid 2.65 0.00601
i-Inositol 12.6 0.07 Niacinamide 2.02 0.0166 Pyridoxine
hydrochloride 2.031 0.00986 Riboflavin 0.219 0.000582 Thiamine
hydrochloride 2.17 0.00644 Vitamin B12 0.68 0.000502 Inorganic
Salts Calcium Chloride (CaCl.sub.2) (anhyd.) 116.6 1.05 Cupric
sulfate (CuSO.sub.4--5H.sub.2O) 0.0013 0.0000052 Ferric Nitrate
(Fe(NO.sub.3).sub.3''9H.sub.2O) 0.05 0.000124 Ferric sulfate
(FeSO.sub.4--7H.sub.2O) 0.417 0.0015 Magnesium Chloride (anhydrous)
28.64 0.301 Magnesium Sulfate (MgSO.sub.4) (anhyd.) 48.84 0.407
Potassium Chloride (KCl) 311.8 4.18 Sodium Chloride (NaCl) 6995.6
120.61 Sodium Phosphate dibasic 71.02 0.5 (Na.sub.2HPO.sub.4)
anhydrous Sodium Phosphate monobasic 54.3 0.45257
(NaH.sub.2PO.sub.4) anhydrous Zinc sulfate (ZnSO.sub.4--7H.sub.2O)
0.432 0.0015
[0081] The components and contents of amino acids, vitamins and
inorganic salts in the serum-free medium may be modified by one
skilled in the art, as long as this modification does not
deteriorate the object of the present invention.
[0082] To achieve the object of the present invention, it is
possible to promote the synthesis of targeted growth factors by
activating the adipose-derived adult stem cells cultured according
to the above method, through specific stimulation. Herein, the
stimulation can be performed under conditions divided into physical
conditions and chemical conditions.
[0083] Examples of physical stimulation may include exposure to UV
rays, nutrient deficiency, and oxygen deficiency. Examples of
chemical stimulation may include the addition of vitamins and other
active compounds in the composition of a medium for cell
culture.
[0084] Physical Stimulation
[0085] In order to obtain growth factors in amounts significantly
larger than those in existing methods for culturing adipose-derived
adult stem cells, it is possible to apply physical stimulation,
including low-oxygen conditions (Circulation. 2004 Mar. 16;
109(10):1292-8.), UV irradiation (FASEB J. 2003 March;
17(3):446-8), nutrient deficiency (Blood. 2004 Nov. 1;
104(9):2886-92. Epub 2004 Jun. 24), and mechanical friction. Such
physical stimulations can selectively or collectively increase
growth factors targeted in the present invention.
[0086] In order to examine whether physical stimulations promote
the synthesis and secretion of growth factors, in the present
invention, the adipose-derived stem cells were cultured in vitro
using the serum medium and then using the serum-free medium as
described above, the cells were then collected and the culture
media were completely removed from the cells. In this state, the
cells were subjected to each of low-oxygen culture, UV irradiation,
nutrient deficiency and mechanical friction, and then were normally
cultured, and the concentrations of growth factors secreted in the
culture medium of the adipose-derived stem cells were measured.
[0087] Specifically, the low-oxygen culture is preferably performed
in conditions of about 5% carbon dioxide and 1-5% oxygen for 36-48
hours for the highest synthesis of growth factors. The UV
irradiation is preferably performed by irradiating ultraviolet B
having a wavelength of 280-320 nm at an energy dose of 80-120
mJ/cm.sup.2. The nutrient deficiency is preferably performed by
culturing the cells in a Mg.sup.2+ and Ca.sup.2+-containing
Dulbecco's phosphate buffered saline for up to a maximum of 4 hours
immediately before the cells precipitate, and then normally
culturing the cells in a medium obtained by adding a Ham's F-12
nutrient mixture to DMEM (not containing a pH indicator such as
phenol red) at a ratio of about 1:1. The mechanical friction is
preferably performed by applying scratch stimulation in a lattice
to the cell medium.
[0088] As a result, bFGF expression was increased 1.74 times in the
case of the low-oxygen stimulation and 2.71 times in the case of
the UV stimulation. Also, when the low-oxygen stimulation and the
UV stimulation were performed in combination, the synergistic
effect thereof was shown (see FIG. 9).
[0089] VEGF expression was increased 2.53 times in the case of the
low-oxygen stimulation, 1.36 times in the case of the UV
stimulation, and 1.30 times in the case of the scratch stimulation
using mechanical friction. Also, when the low-oxygen stimulation,
the UV stimulation and the scratch stimulation were performed in
combination, the synergistic effect thereof was shown (see FIG.
10).
[0090] TGF.beta.-1 expression was increased 1.64 times in the case
of the low-oxygen stimulation, 1.75 times in the case of the UV
stimulation, 2.13 times in the case of the scratch stimulation
using mechanical friction, and 2.01 times in the case of the
nutrient deficiency stimulation. Also, when the low-oxygen
stimulation and the UV stimulation were performed in combination,
the synergistic effect thereof was shown (see FIG. 11).
[0091] Chemical Stimulations
[0092] With respect to chemical conditions, it is preferable to
expose a variety of generally widely known activating compounds
directly or indirectly to individuals.
[0093] Activating compounds, which can be added to the
adipose-derived stem cells according to the present invention,
include cell aging-related retinoic acid, vinpocetine as a
precursor thereof, picamillon serving as an assistant in this
cycle, and quinic acid and quinate, which serve as protein kinase.
In addition, carbohydrate synthesis factors, such as adenine
dinucleotide and acetyl-L-carnitine, which are involved in
metabolism, perform an important role in cell nutrition, and such
activating compounds include other additives, such as
dimethylaminoethanol, which acts to stop apoptosis, L-lipoic acid
and L-hydroxy acid, which are involved in cell proliferation, and
coenzyme Q-10, which is involved in amino acid production. As
described above, these activating compounds can be added
simultaneously or individually during the culture of the
adipose-derived stem cells.
[0094] The present invention focused on the stimulatory effects of
vitamin series among various activating compounds.
[0095] Generally, vitamin A is known to be involved in primary
immune reactions, cell development processes related thereto, and a
series of apoptosis reactions. A typical example thereof is
retinoic acid, which binds to a retinoic acid receptor (RAR) to
regulate and activate metabolic processes associated therewith.
Among them, complexes bound to RAR-alpha, RXR-alpha and RXR-beta
were reported to promote or inhibit the expression of about 128
genes, which are involved in the development of T lymphocytes as
major immune cells. In particular, it was reported that bcl2 family
genes known as typical anti-apoptotic proteins are definitely
increased in apoptosis mechanisms (Rasooly, R. et al., J. Immunol.,
175: 7916-7929 (2005); Spilianakis, C. G. et al., Eur. J. Immunol.,
35(12): 3400-4 (2005); Evans T, Exp. Hematol., 33(9): 1055-61
(2005)). This suggests that vitamin A can show the effect of
inhibiting apoptosis reactions.
[0096] Vitamin B is generally known as riboflavin and performs an
important role in maintaining human health. One research team in
Sweden reported that treatment with this substance showed an effect
on neutrophil migration, thus causing an increased primary immune
response (Verdrengh, M. and Tarkowski, A., Inflamm. Res., 54(9):
390-3 (2005)). It is expected that this substance can increase
initial immune responses, caused by the migration of primary immune
cells.
[0097] Vitamin C has important intracellular functions of promoting
the synthesis of collagen and fibroblasts, and typical examples
thereof include ascorbic acid. When it is used in combination with
other cytokines TGF-.beta. and IFN-.gamma. the effect thereof is
further increased (Chung, J. H. et al., J. Dermatol. Sci., 15(3):
188-200 (1997)). Vitamin D3 has been frequently used, because it is
known to influence cell development and differentiation, unlike
other vitamins. Vitamin D3 mediates an important signaling system
in cell growth and development processes, particularly formation
processes of epidermal keratinocytes and osteogenic cells such as
osteoblasts and osteoclasts. Also, it has an inhibitory effect
against various cytokines, such as IL-1.alpha., IL-6 and IL-8d,
which are involved in inflammatory reactions (Alper, G. et al.,
Endocr. Rev., 23: 763 (2002)).
[0098] In the present invention, any one of or mixture of vitamin
A, vitamin C and vitamin D as chemical stimulation conditions was
added to culture media in an effective amount without causing
cytotoxicity.
[0099] In order to examine whether chemical stimulation using a
vitamin promotes the synthesis and secretion of growth factors, in
the present invention, adipose-derived stem cells were cultured in
vitro using serum media and then using serum-free media as
described above. Then, the cells were collected and washed with
phosphate buffer saline to completely remove the media, and the
cells were cultured in a medium obtained by adding an Ham's F-12
nutrient mixture to a modified DMEM that did not contain a pH
indicator such as phenol red, at a ratio of about 1:1, and adding
thereto at least one selected from among oxidation nutrients such
as L-glutamine, energy metabolites such as sodium pyruvate, and
carbon sources such as sodium bicarbonate. A suitable amount of at
least one vitamin selected from vitamin A, vitamin B, vitamin C and
vitamin D was added to the culture medium, and then the
concentrations of growth factors secreted in the culture medium of
the adipose-derived stem cells were measured.
[0100] Specifically, the optimal concentration of vitamin A is 2-5
.mu.M, the optimal concentration of vitamin B2 is 50-100 .mu.M, the
optimal concentration of vitamin C is 10-100 .mu.M, and the optimal
concentration of vitamin D is 5-10 .mu.M. Preferably, after vitamin
is added to the culture medium, the culture is incubated for more
than 48 hours. When a mixture of vitamins is used, the optimal
concentrations of vitamins are the same as above.
[0101] As a result, it was seen that bFGF expression was increased
1.62 times in the case of vitamin A, 1.33 times in the case of
vitamin B, 2.33 times in the case of vitamin C, and 2.80 times in
the case of vitamin D.
[0102] Also, it was observed that VEGF expression was increased
1.59 times in the case of vitamin A, 1.68 times in the case of
vitamin B, 1.68 times in the case of vitamin C, and 1.30 times in
the case of vitamin D.
[0103] Moreover, it was observed that TGF.beta.-1 expression was
increased 1.20 times in the case of vitamin A, 1.56 times in the
case of vitamin B, 1.20 times in the case of vitamin C, and 1.16
times in the case of vitamin D.
[0104] In addition, it was observed that, when vitamin A, vitamin
B, vitamin C and vitamin D were added to the culture medium at the
above-described optimal concentrations, bFGF expression was
increased 3.62 times, VEGF was increased 2.03 times, and
TGF.beta.-1 was increased 1.68.
[0105] Combination of Stimulations
[0106] With respect to the case where physical stimulation was used
in combination with chemical stimulation, UV light was irradiated
into a culture medium, and then immediately the medium was replaced
with a DMEM medium containing a Ham's F-12 nutrient mixture and
optimized concentrations of vitamins A, B, C and D, and the medium
was cultured in a condition of low-oxygen stimulation for the
optimal culture time. In this case, bFGF expression was increased
4.11 times.
[0107] Also, VEGF expression was increased 3.92 times, when UV
light was irradiated into a culture medium followed by applying
scratch stimulation, and then immediately the medium was replaced
with a DMEM medium containing a Ham's F-12 nutrient mixture and
optimized concentrations of vitamins A, B, C and D, and the medium
was cultured in a condition of low-oxygen stimulation for the
optimal culture time.
[0108] Moreover, TGF.beta.-1 expression was increased 2.35 times,
when UV light was irradiated into a culture medium followed by
applying scratch stimulation and nutrient deficiency, and then
immediately the medium was replaced with a DMEM medium containing a
Ham's F-12 nutrient mixture and optimized concentrations of
vitamins A, B, C and D, and the medium was cultured in a condition
of low-oxygen stimulation for the optimal culture time.
[0109] Furthermore, in order to increase the expression of bFGF,
VEGF and TGF.beta.-1, it is most preferable to use a combination of
low-oxygen stimulation, UV light stimulation and vitamins A, B, C
and D. Specifically, after UV light is irradiated into a culture
medium, the medium is replaced with a medium containing Ham's F-12
nutrient mixture and optimized concentrations of vitamins A, B, C
and D, and the medium is cultured in a condition of low-oxygen
stimulation for the optimal culture time. In this case, bFGF
expression is increased 4.11 times, VEGF expression is increased
3.8 times, and TGF.beta.-1 expression is increased 1.9 times.
[0110] The second aspect of the present invention provides novel
uses of a culture medium obtained using the method according to the
first aspect, or human growth factors purified from the culture
medium.
[0111] Specifically, adipose-derived stem cell culture media or
human growth factors obtained according to the present invention
can be used in drugs, quasi drugs, drug supplements, and cosmetics,
which are used for anti-wrinkle, wound healing, and scar
removing.
[0112] The adipose-derived stem cell culture media obtained
according to the present invention include all media obtained
through the following cases: i) a case in which adipose-derived
stem cells are cultured in serum-free media; ii) a case in which
adipose-derived stem cells were stabilized in serum media, and then
cultured in serum-free media; and iii) adipose-derived stem cells
were activated through physical or chemical stimulation during a
culture process. Also, the human growth factors according to the
present invention include all human growth factors obtained by
purifying the cells or culture media obtained through the
above-described culture method.
[0113] It is preferable either to use culture media obtained by
culturing cells in serum media and then in serum-free media through
the optimized method according to the first aspect of the present
invention, or to use human growth factors purified from the said
culture media.
[0114] It is more preferable either to use culture media obtained
by culturing cells in serum media and then in serum-free media
through the optimized method according to the first aspect of the
present invention and activating the cells through physical or
chemical stimulation during the culture process, or to use human
growth factors purified from the said culture media.
[0115] The growth factors produced from adipose-derived adult stem
cells are distinguished from growth factors synthesized through
existing methods, that is, growth factors synthesized from amino
acids through chemical synthesis methods, and growth factors
synthesized through genetic recombinant methods. In comparison with
such growth factors synthesized by the genetic recombinant methods
or the chemical synthesis methods, the growth factors produced
according to the present invention have advantages in that they are
structurally similar to the original growth factors of the human
body, and thus have excellent skin compatibility and secured
safety.
[0116] In terms of functions, the growth factors according to the
present invention do not show isomerization or three-dimensional
stereospecificity and have the same form as the growth factors of
the body. Thus, the growth factors according to the present
invention have excellent activity compared to the growth factors
produced by the recombinant or chemical synthesis methods.
[0117] Specifically, fibroblast proliferation potential was
compared between the growth factors of the present invention and
the growth factors produced by the recombinant or chemical
synthesis methods. As a result, it was seen that the growth factors
produced from adipose-derived stem cells according to the present
invention had excellent activity compared to the growth factors by
the existing synthesis methods (see Table 5).
[0118] Also, either the adipose-derived stem cell culture media
according to the present invention or human growth factors purified
from the culture media have activities that increase intracellular
collagen synthesis, promote fibroblast proliferation, inhibit
keratinocyte proliferation caused by UV light, mitigate skin
hyperkeratinization and remove wrinkles (see Tables 4, 6, 7 and 8,
and FIGS. 6, 12, 13, 14, 15 and 16).
[0119] Thus, the adipose-derived stem cell culture media according
to the present invention or the human growth factors purified from
the culture media can be advantageously used as raw materials for
drugs, quasi drugs, drug supplements and cosmetics, which are used
for anti-wrinkle, wound healing, and scar removing. In particular,
according to the present invention, it is possible to produce human
growth factors in large amounts and to solve the prior problem of
producing on an industrial scale effective amounts of growth
factors from adipose-derived adult stem cells without other
operations.
EXAMPLES
[0120] Hereinafter, the present invention will be described in
further detail with reference to examples. It will however be
obvious to those skilled in the art that various modifications,
additions and substitutions are possible, without departing from
the scope and spirit of the invention as disclosed in the
accompanying claims.
Example 1
(1-1) Isolation of Adipose-Derived Stem Cells
[0121] Human liposuction material collected from Leaders Clinic,
Seoul, Korea was washed with an equal volume of phosphate buffer
saline, and only adipose tissue was separated from the liposuction
material.
[0122] The extracellular matrix of the adipose tissue was
enzymatically treated with 0.075% collagenase in a 5% CO.sub.2
incubator at 37.degree. C. for 45 minutes, and the enzymatically
treated adipose tissue was centrifuged at 1200 g for 5 minutes to
collect a stromal vascular fraction containing a high density of
stem cells. The pellets were washed with phosphate buffer saline
and passed through a 70-.mu.m nylon cell filter to remove other
tissues, and only monocyte cells and cell fragments including red
blood cells were separated from the remaining material using
Histopaque-1077 (SIGMA).
[0123] The separated monocytes were cultured in a non-inducing
medium containing Dulbecco's Modified Eagle's Medium (DMEM), 10%
fetal bovine serum (FBS), 1% penicillin streptomycin and 0.17%
sodium bicarbonate, in a 5% CO.sub.2 incubator at 37.degree. C. for
24 hours, and non-adhesive cells were removed therefrom, thus
isolating stem cells (see FIG. 1).
(1-2) Culture of Adipose-Derived Stem Cells
[0124] The initial stage culture of the stem cells isolated from
the adipose tissue was performed using DMEM containing 10% FBS.
Also, 1% penicillin-streptomycin (100 IU/ml, GIBCO) was added as an
antibiotic agent, amphotericin B (0.5 .mu.g/ml, Amresco) was added
as an antifungal agent, tylosin (10 .mu.g/ml, Serva, Heidelberg)
was added as a mycoplasma inhibitor, and 2 mM glutamine and 1 mM
sodium pyruvate were further added. The culture was conducted in a
5% CO.sub.2 incubator at a humidity of 95% and a temperature of
37.degree. C. During the 5% CO.sub.2 culture, sodium bicarbonate
was added at a final concentration of 0.17%.
[0125] The stem cells isolated in the above section (1-1) were
suspended at a density of 10.sup.4 cells/ml, and 10 ml of the cell
suspension was transferred and cultured in a T25 flask (area: 25
cm.sup.2; volume: 50 ml) in the above-described conditions.
[0126] Cumulative doubling time was maintained until the cells
being cultured in the flask reached a confluence of 80%, and the
cells were subcultured at a confluence of 80%.
[0127] The flask, from which the medium had been removed, was
washed with PBS, the cells were suspended with 0.25% Trypsin-EDTA
(GIBCO), and the cell suspension was then centrifuged. Then, the
number and viability of the cells were measured, and the cells were
subcultured three times. A serum-free medium used in the subculture
process was based on DMEM which did not contain a pH indicator such
as phenol red, and a Ham's F-12 nutrient mixture (SIGMA) which was
added thereto at a ratio of 1:1. Also, 2 mM L-glutamine and 1 mM
sodium pyruvate were added to the medium, and then sodium
bicarbonate was added at a concentration of 0.1 wt %. The
subculture process was repeated three times.
[0128] The cell number measurement and viability examination were
performed by mixing 0.1 ml of the cell suspension with the same
amount of 0.2% trypan blue (SIGMA), counting stained dyed cells and
non-stained cells using a hemocytometer under a microscope, and
calculating the percentages of the stained and non-stained cells
relative to the total number of the cells.
(1-3) Identification of Stem Cells
[0129] Adipose-derived stem cells express a number of adhesion and
surface proteins. Regarding these proteins, SH-2, SH-3 and SH-4
monoclonal antibodies recognize the surface epitope of human
mesenchymal stem cells. As a result of the analysis of peptide base
sequences and absorbance, SH-3 and SH-4 were identified to be CD73
(ecto-5'-nucleotidase), and SH-2 was identified to be CD105
(endoglin). Such cell surface markers are shared by adipose-derived
stem cells (Barry, F. et al., Biochem Biophys Res Commun., 289(2):
519-24 (2001)).
[0130] The confirmation of stem cells was performed by carrying out
the flow cytometry of the adipose tissue-derived stem cells,
cultured in the initial culture, first subculture and second
subculture, using a fluorescence activated cell sorter (Beckman
Coulter). Specifically, the cells were collected with 0.25%
trypsin-EDTA, washed with PBS and adjusted to a cell density of
10.sup.5 cells/ml. Then, the cells were allowed to react with
mesenchymal stem cell-specific markers CD73-PE and CD105-FITC (BD
science) antibodies, and analyzed at 488 nm with an argon
laser.
[0131] In the results of the flow cytometry of the PLA cells
isolated from the adipose tissue, the initially isolated stromal
vascular fraction showed a homology of 5.27% to stem cells (see
FIG. 2), and after the second subculture, the cells showed a
homology of 92.32% to stem cells for CD73-PE and a homology of
90.67% to stem cells for CD105-FITC (see FIG. 3).
Example 2
Identification of Activated Growth Factors of Adipose-Derived Stem
Cells
[0132] In order to examine whether adipose-derived stem cells
synthesize growth factors, adipose-derived stem cells were cultured
in the same culture medium and culture conditions as in Example
(1-2), and then RNA in the stem cells was analyzed through a
reverse transcription-polymerase chain reaction.
[0133] Specifically, the total RNA of the adipose-derived stem
cells was extracted with a RNeasy Plus Mini kit (QIAGEN Corp.,
Valencia, Calif.), and the RNA was amplified by PCR using a
MMLV-reverse transferase (Promega Corp., U.S.A) at 37.degree. C.
for 45. Then, the MMLV-reverse transferase was inactivated at
65.degree. C. for 45 minutes. The PCR reaction solution had a total
volume of 50 .mu.l and contained 1.5 mM MgCl.sub.2, 0.25 mM dNTP,
and 2.5 unit Taq polymerase (QIAGEN). The PCR reaction was
performed in TGRADIENT (BIOMETRA) in the following conditions: a
cDNA denaturation of 3 min at 94.degree. C., and then 30 cycles
each consisting of 30 sec at 94.degree. C. (DNA denaturation), 30
sec at 60.degree. C. (annealing) and 30 sec at 72.degree. C.
(extension), followed by a final extension of 5 min at 72.degree.
C. The reverse transcription was performed as described above to
synthesize cDNA, which was then subjected to reverse
transcription-PCR using primers for VEGF-.beta., bFGF and
TGF.beta.-1 (see Table 2).
TABLE-US-00002 TABLE 2 Base sequences of primers used in RT-PCR
Forward primer Reverse primer VEGF 5-TACCTCCACCATGCCAAGTG-3;
5-TGATGATTCDTGCCCTCCTCC-3; SEQ ID NO: 1 SEQ ID NO: 2 bFGF
5-GACGGCAGAGTTGACGG-3; 5-CTCTCTCTTCTGCTTGAAGTTGTAGC-3; SEQ ID NO: 3
SEQ ID NO: 4 TGF.beta.-1 5-GCTGAGCGCTTTTCTGATCCT-3;
5-CGAGTGTGCTGCAGGTAGACA-3; SEQ ID NO: 5 SEQ ID NO: 6
[0134] As a result, a 482-bp bFGF product (1522 bp-2003 bp)
corresponding to SEQ ID NO: 7, a 343-bp VEGF product (1080 bp-1422
bp) corresponding to SEQ ID NO: 8, and a 212-bp TGF.beta.-1 product
(2119 bp-2330 bp) corresponding to SEQ ID NO: 9, identified the
expression of growth factors in the adipose-derived stem cells (see
FIG. 4).
[0135] Also, in order to examine whether growth factors are present
in a culture medium, an enzyme-linked immunosorbent assay was
performed.
[0136] Specifically, 1 ml of the culture medium obtained by
culturing adipose-derived stem cells to passage 3 in the conditions
of Example 1-2 was centrifuged at 1200 g for 5 minutes, and then
filtered through a 0.22 mm filter to remove cell residue. The
filtrate was serially diluted to determine optimal conditions in
which a non-specific reaction did not occur. Then, the
concentration of each of growth factors VEGF, bFGF and TGF.beta.-1
secreted in the culture medium was measured using a sandwich ELISA
kit (Quantikine Human FGF basic Immunoassay, R&D systems).
[0137] To examine the concentrations of optimally diluted growth
factors, the culture medium was serially diluted at the same
concentration ratio with a calibration dilution in the kit, the
concentration at the absorbance included in the range of the
standard curve was determined, and the values included in the range
of the standard curve were plotted as data. 100 ml of each growth
factor-containing culture media and standard solutions of growth
factors was placed in each well coated with an antibody to each of
the growth factors (Quantikine Human FGF basic Immunoassay, R&D
systems) and was cultured at room temperature for 2 hours. After
completion of the antigen-antibody binding reaction, each well was
washed four times with wash buffer, and 200 ml of a solution
(Quantikine Human FGF basic Immunoassay, R&D systems),
containing a secondary antibody to each of the growth factors, was
placed in each well, followed by culture at room temperature for 2
hours. After completion of the second antibody binding reaction,
each well was washed four times with wash buffer, 200 ml of a
color-developing reagent (tetramethylbenzidine, R&D systems)
was added to each well, and then the absorbance of the medium was
measured at 450 nm with an ELISA reader.
[0138] As a result, bFGF protein levels showed an increase of 54.96
pg/ml after 12 hours and an increase of 76.393 pg/ml after 24
hours, and VEGF protein levels showed an increase of 87.021 pg/ml
after 12 hours and an increase of 163.52 pg/ml after 24 hours (see
FIG. 5).
Example 3
Activity of Adipose-Derived Stem Cells on Collagen Production of
Fibroblasts
[0139] One of the causes of formation of skin wrinkles is a lack of
collagen. Collagen is a major protein of skin dermis and serves to
maintain the skin structure and firmness. It is known that the
production of collagen decreases as age increases, and the
degradation thereof also increases to induce the collapse of the
dermal layer, thus producing skin wrinkles. Thus, the effect of a
substance for anti-wrinkle activity can be proven by testing the
production and degradation of collagen.
[0140] (3-1) Coculture of Adipose-Derived Stem Cells and
Fibroblasts
[0141] The effect of adipose-derived stem cells on the collagen
production of fibroblasts was estimated through the coculture of
adipose-derived stem cells and fibroblasts. In the test, an
increase in the production of collagen in fibroblasts, when
adipose-derived stem cells were cocultured with fibroblasts in a
transwell insert (Costar, Corning), was compared with the
production of collagen in the single culture of fibroblasts.
[0142] a) Coculture
[0143] Fibroblasts (primary cell line) were obtained by finely
cutting a portion of human skin tissue (Department of Laboratory
Medicine, Hangang Sungsim Hospital; 9 years old; circumcison
fragment) suspended in PBS, stirring the cut tissue with 50-100 ml
of trypsin for 20 minutes, centrifuging the stirred tissue, and
then filtering the tissue through a 7-mm nylon filter.
[0144] The filter cell suspension was seeded on the bottom of a
culture dish and a DMEM medium containing penicillin (100 IU/mL),
streptomycin (100 g/mL) and 10% FBS was added. Then, the cells were
cultured in an incubator containing 5% carbon dioxide at 37.degree.
C. The fibroblasts, which reached a confluence of 80%, were
dispensed into a 6-well plate at a density of 5.times.10.sup.4/well
and cultured for 24 hours in the same culture conditions as in the
subculture process.
[0145] The adipose-derived stem cells isolated in Example (1-1)
were cultured in a transwell insert for 24 hours in the same
conditions as the case of the fibroblasts, and then the transwell
insert was inserted into the 6-well plate in which the fibroblasts
were being cultured. The stem cells and the fibroblasts were
cocultured in the 6-well plate. At this time, the medium below the
transwell insert was discarded, the 6-well plate was washed with
PBS, a fresh medium was added thereto, and the fibroblasts and the
adipose-derived stem cells were cocultured.
[0146] 1 ml of the cocultured medium was taken at 24 hours and 72
hours after the culture, and the amount of collagen in the culture
medium was measured.
[0147] In a control group test, fibroblasts, which reached
confluence, were dispensed into a E-well plate at a density of
5.times.10.sup.4 cells/well, and then cultured for 24 hours. A
transwell insert having no adipose-derived stem cell seeded therein
was inserted into the 6-well plate.
[0148] b) Analysis of Activity for Collagen Production
[0149] 1) Measurement of Amount of Collagen by Enzyme-Linked
Immunosorbent Assay (ELISA)
[0150] The measurement of the amount of collagen was performed
using a Procollagen type I peptide EIA kit (TAKARA BIOMEDICAL Co.).
100 ml of an antibody-PoD conjugate solution was placed in each
well. Then, 20 .mu.l of fibroblasts, which were optimally diluted
with a calibration dilution so as to be included in a collagen
standard curve, and 20 .mu.l of fibroblasts cocultured with
adipose-derived stem cells, were placed in each well and left to
stand at 37.degree. C. for 3 hours. Then, each well was washed four
times with wash buffer, and 100 .mu.l of a color-developing reagent
was added to each well, followed by culture at room temperature for
15 minutes. Then, the absorbance of the culture medium was measured
at 450 nm with an ELISA reader.
[0151] 2) Measurement of Amount of Collagen in Cells by
Semi-Quantity PCR
[0152] The total RNA of fibroblasts cocultured with stem cells was
extracted using a RNeasy plus mini kit (QIAGEN), and 3 .mu.g of the
extracted RNA was amplified by PCR in a reaction solution
containing 200 unit MMLV-reverse transcriptase (Promega), 50 mM
Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl.sub.2, 10 mM DTT, 10 mM
dNTP, 25 unit RNase inhibitor and 20 pmole Oligo-dT. The PCR
reaction was performed in a T-GRADIENT (BIOMETRA) thermocylcer at
37.degree. C. for 45 minutes, and then the MMLV-reverse
transcriptase was inactivated at 65.degree. C. for 15 minutes.
Specific base sequences for the detection of collagen I type
(GenBank No. NM-000089) were constructed based on base sequences
recorded in the NCBI Genbank and are shown in Table 3 below.
TABLE-US-00003 TABLE 3 Specific base sequences for detection of
collagen type I Forward primer Reverse primer Collagen
5'-CCCTCAAGGTTTCCAAGGAC-3'; 5'-ACCAGGTTCACCCTTCACAC-3'; type I SEQ
ID NO: 10 SEQ ID NO: 11
[0153] As a result, as shown in FIG. 6, the amount of intracellular
collagen synthesis was increased 2.17 times in 1-day culture and
1.27 times in 3-day culture, compared to the control group, and the
amount of total collagen synthesis was increased 1.44 times in
1-day culture and 1.14 times in 3-day culture.
[0154] (3-2) Collagen Synthesis of Fibroblasts by Adipose-Derived
Stem Cell Culture Medium
[0155] In order to examine the effect of a culture medium
(conditioned medium) of adipose-derived stem cells on the collagen
synthesis of fibroblasts, western blot analysis (specific protein
detection) was performed.
[0156] To collect a culture medium of adipose-derived stem cells,
5.times.10.sup.5 adipose-derived stem cells were seeded into a T75
flask after subculture. Herein, serum-free DMEM was used as the
medium. The cells were cultured in a 5% CO.sub.2 incubator at
37.degree. C. for 3 days, and then the medium was harvested and
filtered through a 0.22-um syringe filter. The filtrate was used as
a serum-free conditioned medium in the following test.
[0157] Fibroblasts were subcultured, and then seeded into a 0.1%
FBS-containing DMEM in a 6-well plate at a density of
5.times.10.sup.4 cells/well. After the cells were cultured for 24
hours, the medium was replaced with the above-described serum-free
conditioned medium. For the control group, serum-free DMEM was
used. After 12 hours, serum was adjusted to 2%, and after 30 hours
of culture, the culture medium was harvested and subjected to
western blot analysis.
[0158] The electrophoresis of the culture medium was performed
according to the SDS-PAGE method using an electrophoresis kit
(Bio-rad). In the electrophoresis, 8% polyacrylamide gel was used
and transferred to a PVDF membrane (Bio-rad). Then, the blot was
blocked with a 5% nonfat dry milk-containing TBST (50 mM Tris,
pH8.0, 138 mM NaCl, 2.7 mM KCl, and 0.1% (w/v) Tween 20). The
medium was allowed to react overnight with antibody to collagen
type I (Santacruz) as a primary antibody and allowed to react with
peroxidase-Rabbit anti-goat IgG (Zymed) as a secondary antibody for
30 minutes. Finally, the medium was allowed to react with an
antibody detection reagent (ECL, Milipore) for 1 minute, and the
test results were observed.
[0159] As a result, as shown in FIG. 16, the adipose-derived stem
cell culture medium treated with fibroblasts showed an increase of
more than 2 times the amount of collagen compared to the medium
untreated with fibroblasts (quantified with a gene tool software;
2.16-fold increased). This demonstrates that the culture medium of
adipose-derived stem cells increases the collagen synthesis of
fibroblasts, and thus suggests that the culture medium of
adipose-derived stem cells can be used as an anti-wrinkle substance
for preventing skin aging.
Example 4
Stimulation of Fibroblast Proliferation by Adipose-Derived Stem
Cells
[0160] Stem cells isolated from adipose tissue were cultured to
passage 3 as described in Example (1-2), and then 10.sup.6 cells of
the stem cells were seeded into a T175 flask (area: 175 cm.sup.2;
and volume: 500 ml) and cultured for 3 days. The stem cell culture
medium was collected, and added to concentrations of 10%, 25%, 50%
and 100% to a 6-well plate, in which passage-4 fibroblast cells
from the skin tissue of a 9 year old boy (Department of Laboratory
Medicine, Hangang Sungsim Hospital; 9 years old; circumcison
fragment) were dispensed at a density of 25,000 cells/well.
[0161] After 3 days, the proliferation of the fibroblasts was
measured with a cell viability measurement kit (cell counting
kit-8, Dojindo Molecular Technologies, Inc.), and then the
absorbance of the cells was measured at 450 nm with an ELISA
reader.
[0162] As a result, as shown in Table 4, it was demonstrated that
the growth factors secreted from the adipose-derived stem cells
stimulated the proliferation of fibroblasts. Also, it was observed
that the proliferation of fibroblasts was stimulated as the
concentration of growth factors secreted from adipose-derived stem
cells was increased (see FIG. 7).
TABLE-US-00004 TABLE 4 Number of fibroblast cells in varying
concentrations of culture media containing growth factors secreted
from adipose-derived stem cells Classification Cells Normal media
(control group) 35000 Treated with 10% ADSC 3-passage 3-day
cultured 45000 medium Treated with 25% ADSC -3passage 3-day
cultured 93000 medium Treated with 50% ADSC 3-passage 3-day
cultured 180000 medium Treated with 100% ADSC 3-passage 3-day
240000 cultured medium
Example 5
Comparison of Growth Factors Secreted from Adipose-Derived Stem
Cells with Recombinant Growth Factors Expressed in E. Coli
[0163] Stem cells isolated from adipose tissue were cultured to
passage-3 as described in Example (1-2), and then 10.sup.6 cells of
the stem cells were seeded into a T175 flask (area: 175 cm.sup.2;
volume: 500 ml) and cultured for 3 days. The culture medium was
collected, and added at a concentration of 100% to a 6-well plate
in which passage-4 fibroblast cells from the skin tissue of a 9
year old boy were dispensed at a density of 5000 cells/well. For
the control group, a medium containing the same concentrations of
recombinant growth factors VEGF and bFGF (Santa Cruz.) expressed in
E. coli was used. Cell proliferation potential was compared between
the adipose-derived stem cell culture medium and the recombinant
growth factor-containing culture medium using a cell viability
measurement kit.
[0164] As a result, when the growth factors obtained by the
overexpression and isolation of growth factor genes in E. coli
using the genetic recombinant method were compared with the growth
factors synthesized from the adult stem cells, it was demonstrated
that the growth factors synthesized from the adipose-derived adult
stem cells had excellent effects compared to the growth factors
obtained by the existing synthesis method. The results are shown in
Table 5 below.
TABLE-US-00005 TABLE 5 Comparison of cell proliferation potential
between adipose-derived stem cell culture media and recombinant
growth factor-containing media Classification Cells Normal media +
rFGF (50 ng) 8215.3 Normal media + rVEGF (500 ng) 4408.9 Normal
media + rFGF + rVEGF 6727.1 Treated with 100% ADSC 3-passage 3-day
17215 cultured medium
Example 6
Culture Method of Adipose-Derived Stem Cells Having Increased
Growth Factor Secretion
[0165] (6-1) Physical Stimulation
[0166] Adipose tissue-derived stem cells were cultured to passage 3
as described in Example (1-2), and when the cultured cells reached
a confluence of 80%, the cells were collected with trypsin/EDTA.
Then, the cells were accurately dispensed into a 6-well plate at a
density of 25,000 cells/well.
[0167] 24 hours after the cells were dispensed and had attached to
the well, the culture medium was completely removed, and the cells
were incubated in a multigas incubator containing 5% carbon dioxide
and 1% oxygen under physical conditions, and were irradiated with
an energy of 90 mJ/cm.sub.2 at a wavelength of 280-320 nm (model
BEX-800; Ultra-Lum, Inc.) corresponding to UV B. Immediately
following, in a nutrient deficient reaction, the cells were
cultured in Dulbecco's phosphate buffered saline containing
Mg.sup.2+ and Ca.sup.2+ for a maximum of 4 hours until the cells
precipitated, and then, the medium was replaced with the serum-free
medium of Example (1-2). In scratch stimulation using mechanical
friction, the cell medium, which was being cultured attached to the
plate, was scratched with a blade in the form of a lattice having a
size of 1 mm.times.1 mm.
[0168] (6-2) Chemical Conditions
[0169] To the serum-free medium of Example (1-2), prepared by
adding a Ham's F-12 nutrient mixture to DMEM, which did not contain
a pH indicator such as phenol red, at a ratio of 1:1, and adding
thereto 2 mM L-glutamine, 1 mM sodium pyruvate and 0.17% sodium
bicarbonate, vitamins A, B, C and D at effective levels that did
not cause cytotoxicity. Cells were cultured in this medium.
[0170] The cells were cultured in the above medium in a 5% carbon
dioxide incubator at 37.degree. C. for more than 48 hours, and then
200 .mu.l of the culture medium supernatant was centrifuged at
3,000 rpm and filtered through 0.22-.mu.m filter paper. Then, the
concentrations of bFGF, VEGF and TGF.beta.-1 in the filtrate were
measured by the enzyme-linked immunosorbent assay (ELISA) of
Example 2.
[0171] In the analysis results, the optimal concentration of
vitamin A was 2-10 .mu.M, the optimal concentration of vitamin B2
was 50-100 .mu.M, the optimal concentration of vitamin C was 10-100
.mu.M, and the optimal concentration of vitamin D was 5-10 .mu.M.
It was seen by MTT assay that, no cytotoxicity occurred at such
concentrations, and the synthesis of each of the growth factors
targeted in the present invention was increased.
[0172] (6-3) Culture Conditions of Control Group
[0173] As a control group, a normal medium was used, which was not
subjected to physical stimulation or chemical stimulation. The
increase in growth factors was compared between the control group
and the culture medium subjected to physical stimulation and
chemical stimulation.
[0174] In a culture process, cells were added to a serum-free
medium prepared by adding a Ham's F-12 nutrient mixture to DMEM not
containing a pH indicator such as phenol red, at a ratio of 1:1,
and adding thereto 2 mM L-glutamine, 1 mM sodium pyruvate and 0.17%
sodium bicarbonate. Then, the cells were cultured in a 5% carbon
dioxide incubator at 37.degree. C. for more than 48 hours. Then,
200 .mu.l of the culture medium supernatant was centrifuged at
3,000 rpm, and then filtered through 0.22 .mu.m filter paper. Then,
the concentrations of bFGF, VEGF and TGF.beta.-1 were measured by
the enzyme-linked immunosorbent assay (ELISA) of Example 2.
[0175] (6-4) Combination of Stimulations
[0176] The case where physical stimulation is used in combination
with chemical stimulation will now be described. For bFGF, UV light
was irradiated into a culture medium, and then immediately the
medium was replaced with a DMEM medium containing a Ham's F-12
nutrient mixture and optimized concentrations of 2 .mu.M vitamin A,
50 .mu.M vitamin B, 10 .mu.M vitamin C and 10 .mu.M vitamin D. The
medium was cultured at low-oxygen stimulation conditions of 1%
oxygen and 5% carbon dioxide for 48 hours.
[0177] For VEGF, a medium was irradiated with UV light and then
subjected to scratch stimulation. Immediately after this, the
medium was replaced with a DMEM medium containing a Ham's F-12
nutrient mixture and optimized concentrations of 2 .mu.M vitamin A,
50 .mu.M vitamin B, 10 .mu.M vitamin C and 10 .mu.M vitamin D. The
medium was cultured low-oxygen stimulation conditions of 1% oxygen
and 5% carbon dioxide for 48 hours.
[0178] For TGF.beta.-1, a medium was irradiated with UV light and
then subjected to scratch stimulation and nutrient deficiency
stimulation. Then, the medium was replaced with a DMEM medium
containing a Ham's F-12 nutrient mixture and optimized
concentrations of 2 .mu.M vitamin A, 50 .mu.M vitamin B, 10 .mu.M
vitamin C and 10 .mu.M vitamin D. The medium was cultured at
low-oxygen stimulation conditions of 1% oxygen and 5% carbon
dioxide for 48 hours.
[0179] To obtain the highest total amount of bFGF, VEGF and
TGF.beta.-1, a medium was irradiated with UV light, and then
replaced with a DMEM medium containing a Ham's F-12 nutrient
mixture and optimized concentrations of 2 .mu.M vitamin A, 50 .mu.M
vitamin B, 10 .mu.M vitamin C and 10 .mu.M vitamin D. The medium
was cultured low-oxygen stimulation conditions of 1% oxygen and 5%
carbon dioxide for 48 hours.
[0180] (6-5) Results
[0181] As shown in FIG. 9, in comparison with the control group,
bFGF was increased 1.74 times in the case of low-oxygen
stimulation, and 2.71 times in the case of UV light stimulation. In
the case of chemical stimulation, bFGF was increased 1.62 times for
vitamin A, 1.33 times for vitamin B, 2.33 times for vitamin C and
2.80 times for vitamin D.
[0182] When low-oxygen stimulation and UV light stimulation, which
among the above stimulations have excellent effects on growth
factor expression, were performed in combination, the synergistic
effect thereof was shown. In the case of chemical stimulation, bFGF
protein levels in a medium containing optimized concentrations of
vitamins A, B, C and D were increased 3.62 times. In case where
physical stimulation was applied in combination with chemical
stimulation, bFGF protein levels were increased 4.11 times, when
low-oxygen stimulation was optimized, UV light was irradiated and
the concentrations of vitamins A, B, C and D were optimized.
[0183] As shown in FIG. 10, in comparison with the control group,
vascular endothelial growth factor (VEGF) protein levels were
increased 2.53 times in the case of low-oxygen stimulation, 1.36
times in the case of UV light stimulation, and 1.33 times in the
case of scratch stimulation caused by mechanical friction. In the
case of chemical stimulation, VEGF protein levels were increased
1.59 times for vitamin A, 1.56 times for vitamin B, 1.68 times for
vitamin C, and 1.30 times for vitamin D.
[0184] When low-oxygen stimulation and UV light stimulation, which
among the above stimulations have excellent effects on the
expression of growth factors, were performed in combination, the
synergistic effect thereof was shown. In the case of chemical
stimulation, VEGF protein levels in a medium containing optimized
concentrations of vitamins A, B, C and D were increased 2.03
times.
[0185] In the case where physical stimulation was applied in
combination with chemical stimulation, VEGF protein levels could be
increased 3.92 times, when low-oxygen stimulation was optimized, UV
light stimulation and scratch stimulation were used and the
concentrations of vitamins A, B, C and D were optimized.
[0186] As shown in FIG. 11, in comparison with the control group,
transforming growth factor beta-1 (TGF.beta.-1) protein levels were
increased 1.64 times in the case of low-oxygen stimulation, 1.75
times in the case of UV light stimulation, 2.13 times in the case
of scratch stimulation caused by mechanical friction, and 2.01
times in the case of nutrient deficiency stimulation. In the case
of chemical stimulation, TGF.beta.-1 protein levels were increased
1.20 times for vitamin A, 1.56 times for vitamin B, 1.20 times for
vitamin C, and 1.16 times for vitamin D.
[0187] When low-oxygen stimulation and UV light stimulation, which
among the above stimulations have excellent effects on the
expression of growth factors, were performed in combination, the
synergistic effect thereof was shown. In the case of chemical
stimulation, TGFb-1 protein levels in a medium containing optimized
concentrations of vitamins A, B, C and D were increased 1.68
times.
[0188] In the case where physical stimulation was applied in
combination with chemical stimulation, TGF.beta.-1 protein levels
could be increased 2.35 times, when low-oxygen stimulation was
optimized, UV light stimulation, scratch stimulation and low-oxygen
stimulation were used and the concentrations of vitamins A, B, C
and D were optimized.
[0189] Also, in order to obtain the highest total amount of bFGF,
VEGF and TGF.beta.-1, it is most preferable to subject a medium to
low-oxygen stimulation and UV light stimulation and add vitamins A,
B, C and D to the medium. Specifically, a medium is irradiated with
UV light, and then replaced with a medium containing a Ham's F-12
nutrient mixture and optimized concentrations of vitamins A, B, C
and D, and the medium is cultured in a condition of low-oxygen
stimulation for the optimized culture time. In this case, bFGF
protein levels are increased 4.11 times compared to the control
group, VEGF protein levels are increased 3.8 times, and TGF.beta.-1
protein levels are increased 1.9 times.
Example 7
Defensive Effect of Adipose-Derived Stem Cells Against Exposure of
Keratinocytes to UV Light
[0190] The defensive effect of growth factors secreted from
adipose-derived stem cells against the exposure of keratinocytes to
UV light was tested in the following manner.
[0191] Keratinocytes were dispensed into a 6-well plate at a
suitable cell concentration in KGM (karatinocyte growth medium;
Clonetics.) and incubated in a 5% carbon dioxide incubator at
37.degree. C. To the medium, 2.5 cc of 100% ADSC 3-passage 3-day
cultured medium was added to adjust the KGM to 5 cc. Also, 2 uM
retinol was added to 5 cc of KGM. Also, as a negative control
group, 5 cc of KGM alone was administered to keratinocytes. Each of
the test samples was stabilized for 4 hours, such that the raw
materials were sufficiently dispersed. After that, each of the test
samples was irradiated with UV light using a 40 W double lamp for 8
minutes in an aseptic laboratory. After 24 hours, the UV defense
function of the test sample was measured by determining the
viability of the cells in comparison with the negative control
group, which was untreated with the culture medium or retinol and
irradiated with UV light for 8 minutes.
[0192] As a result, as shown in Table 6 below, the cell group,
which was cultured as described in Example (1-2) and contained
VEGF, bFGF and TGF.beta.-1, showed a defensive effect of 67%, the
cell group administered with retinol showed a defensive effect of
54%, and the negative control group showed a defensive effect of
55%. This indicates that retinol has a low defensive effect against
UV lights, whereas the growth factors secreted from adipose-derived
stem cells has an increased defensive effect against UV light.
TABLE-US-00006 TABLE 6 Measurement results of anti-aging effects
Tested materials Defensive effect 100% ADSC 3-passage 3-day
cultured 67% medium Retinol 54% Negative control group 55%
Example 8
Effects of Adipose-Derived Stem Cell Growth Factors on Skin
Photoaging Caused by UV Light Irradiation in Nude Mice
[0193] In order to assess the activity of a culture medium, which
was cultured as described in Example (1-2) and contained VEGF, bFGF
and TGF.beta.-1, the following test was performed using thirty
15-20-week-old nude mice.
[0194] Irradiating the backs of nude mice with UV light at a dose
of 2 mJ/cm.sup.2 using an UV simulator was performed two times a
week for 4 weeks, thus inducing the abnormal hyperkeratinization of
the skin. Then, growth factors synthesized by a chemical synthesis
method, growth factors produced by a genetic recombination method,
and growth factors produced by adipose-derived adult stem cells,
were applied on one side of the backs of the nude mice in an amount
of 1 cc two times a day for 2 weeks, while the other side was
untreated for comparison. After 2 weeks, the anti-aging effects of
the growth factors were observed visually and assessed (Jin Ho
Chung et al. Archives of Dermatology, 137-8 (2001)). The
measurement results are shown in Table 7 below.
TABLE-US-00007 TABLE 7 Measurement results of anti-aging effects
Remarkable effect Moderate effect No effect (number of mice)
(number of mice) (number of mice) 100% culture 23 7 0 medium
Negative 4 8 18 control group
[0195] When each of the growth factors was applied to the nude mice
whose aging was artificially induced, the growth factors
synthesized from adipose-derived adult stem cells showed the effect
of reducing abnormal skin hyperkeratinization in all of the 30
animals, and this effect was excellent compared to that of the
growth factors synthesized by the chemical synthesis method or the
genetic recombination method.
Example 9
Anti-Wrinkle Effect of Growth Factors Secreted from Autologous
Adipose-Derived Stem Cells in Human Beings
[0196] In order to verify the wrinkle-reducing effect of growth
factors synthesized from adipose-derived adult stem cells on
30-50-year old women, 20 persons per test group were selected and
the following test was conducted.
[0197] 1 cc of a adipose-derived stem cell culture medium purified
to have a VEGF content of 30 ng, a bFGF content of 30 ng and a
TGF.beta.-1 content of 70 ng was applied on the brow or around one
eye of each of the women subjects two times a day for 8 weeks, and
a negative control group (1 cc of physiological saline as a basic
solution) was applied around the other eye for comparison. After 4
weeks and 8 weeks, the wrinkle-reducing effect of the test sample
was visually observed and the measurement results are summarized in
Table 8 below.
TABLE-US-00008 TABLE 8 Measurement results Remarkable Moderate No
effect effects (number effects (number (number of of persons) of
persons) persons) Growth factor- 8 12 0 containing culture medium
Negative 2 4 14 control group
[0198] As can be seen from the results of Table 8 and FIGS. 12 to
15, the application of the growth factors synthesized from adult
stem cells showed an excellent wrinkle-reducing effect.
[0199] The human growth factors produced according to the method of
the present invention are substances having secured stability and
physiological activity, and thus can be used to develop drugs,
quasi drugs, cosmetics and the like for anti-wrinkles, wound
healing, and scar removing.
[0200] Also, the adipose-derived stem cell culture medium according
to the present invention contains various growth factors in large
amounts, and thus can be used by themselves in drugs, quasi drugs,
cosmetics and the like for anti-wrinkles, wound healing, and scar
removal.
SEQUENCE LIST TEXT
[0201] SEQ ID NOS: 1-6 and 10-11 according to the present invention
are amplification primer pairs for the detection of specific
proteins, and SEQ ID NOS: 7-9 are amplification products encoding
human growth factors.
Sequence CWU 1
1
11120DNAArtificial SequenceForward primer for VEGF 1tacctccacc
atgccaagtg 20221DNAArtificial SequenceReverse primer for VEGF
2tgatgattcd tgccctcctc c 21317DNAArtificial SequenceForward primer
for bFGF 3gacggcagag ttgacgg 17426DNAArtificial SequenceReverse
primer for bFGF 4ctctctcttc tgcttgaagt tgtagc 26521DNAArtificial
SequenceForward primer for TGFb-1 5gctgagcgct tttctgatcc t
21621DNAArtificial SequenceReverse primer for TGFb-1 6cgagtgtgct
gcaggtagac a 217482DNAHomo sapiens 7tgctggtgat gggagttgta
ttttcagtct tcgccaggtc attgagatcc atccactcac 60atcttaagca ttcttcctgg
caaaaattta tggtgaatga atatggcttt aggcggcaga 120tgatatacat
atctgacttc ccaaaagctc caggatttgt gtgctgttgc cgaatactca
180ggacggacct gaattctgat tttataccag tctcttcaaa aacttctcga
accgctgtgt 240ctcctacgta aaaaaagaga tgtacaaatc aataataatt
acacttttag aaactgtatc 300atcaaagatt ttcagttaaa gtagcattat
gtaaaggctc aaaacattac cctaacaaag 360taaagttttc aatacaaatt
ctttgccttg tggatatcaa gaaatcccaa aatattttct 420taccactgta
aattcaagaa gcttttgaaa tgctgaatat ttctttggct gctacttgga 480gg
4828343DNAHomo sapiens 8tacctccacc atgccaagtg gtcccaggct gcacccatgg
cagaaggagg agggcagaat 60catcacgaag tggtgaagtt catggatgtc tatcagcgca
gctactgcca tccaatcgag 120accctggtgg acatcttcca ggagtaccct
gatgagatcg agtacatctt caagccatcc 180tgtgtgcccc tgatgcgatg
cgggggctgc tgcaatgacg agggcctgga gtgtgtgccc 240actgaggagt
ccaacatcac catgcagatt atgcggatca aacctcacca aggccagcac
300ataggagaga tgagcttcct acagcacaac aaatgtgaat gca 3439212DNAHomo
sapiens 9gctgagcgct tttctgatcc tgcatctggt cacggtcgcg ctcagcctgt
ctacctgcag 60cacactcgat atggaccagt tcatgcgcaa gaggatcgag gcgatccgcg
ggcagatcct 120gagcaagctg aagctcacca gtcccccaga agactatcct
gagcccgagg aagtcccccc 180ggaggtgatt tccatctaca acagcaccag gg
2121020DNAArtificial SequenceForward primer for Collagen type I
10ccctcaaggt ttccaaggac 201120DNAArtificial SequenceReverse primer
for Collagen type I 11accaggttca cccttcacac 20
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