# Patent

## Title
Method for producing pluripotent cell using bacterium having fermentation ability

## Abstract

It is an object of the present invention to provide a method for producing pluripotent cells that are free of the risk of cellular canceration and that can be applied to regenerative medicine with a high degree of safety. The present invention provides a method for producing pluripotent cells from somatic cells comprising a step of bringing bacteria having fermentation ability or a component or secretory product thereof into contact with somatic cells.

## Background
TECHNICAL FIELD
The present invention relates to a method for producing pluripotent cells using bacteria having fermentation ability.
BACKGROUND ART
ES cells are embryonic stem cells that were discovered in mouse embryos in 1981 and in human embryos in 1998. ES cells having the ability to develop into a variety of types of cells (i.e., pluripotent cells) except for the cells that constitute placenta have been primarily studied for the construction of tissues or organs therefrom. Because of the use of the fertilized eggs that would develop into new lives if they were allowed to grow smoothly, ES cells raise serious ethical questions. Another serious issue is the problem of rejection. When differentiated cells or organs prepared from ES cells are transplanted into a patient, the immune system of the patient may recognize such cells or organs as foreign substances and attack them.
In order to overcome the problems of ES cells, Professor Shinya Yamanaka et al. at Kyoto University developed cells capable of developing into various types of cells from dermal cells that are not generally differentiated into cells exerting other functions, and they designated these cells “iPS cells.” They demonstrated that introduction of four factors referred to as “Yamanaka factors;” i.e., Oct 3/4, Sox2, Klf4, and c-Myc, into mouse or human dermal cells with the use of a retrovirus vector would lead to reprogramming of cells and production of pluripotent cells, as is the case with ES cells (Non-Patent Document 1: Takahashi and Yamanaka, Cell 126, 663-676, 2006; and Non-Patent Document 2: Takahashi et al., Cell 131, 861-872, 2007). Since the cells used in such case are derived from somatic cells, such as differentiated dermal cells, of the patient him/herself, the immune system would recognize an organ prepared from the cells differentiated from the iPS cells as an autonomous organ upon transplantation thereof, and the transplant would accordingly not be rejected. As a result of the discovery of iPS cells, the issue of ethical concern regarding ES cells was overcome.
As described above, iPS cells have drawn attention worldwide as a powerful tool for regenerative medicine, although the technical problem of canceration of cells remains problematic. A cause of canceration is related to the introduction of the c-Myc gene into cells; however, iPS cells have been produced from the other 3 factors than the c-Myc gene in recent years. When introducing a gene into a cell, iPS cells were prepared with the use of the adenovirus or plasmid vector instead of the retrovirus vector. This allowed for the production of iPS cells with advanced safety and usefulness. However, this technique involves the artificial and forced expression of several genes in cells that had completed differentiation, and the risk that such cells would experience canceration in the future cannot be denied.
Meanwhile, Patent Document 1 describes a method of using Mycobacterium leprae or a component thereof so as to produce reprogrammed embryonic stem (ES)-like cells. Specifically, Patent Document 1 describes a method for producing reprogrammed ES-like cells comprising bringing Mycobacterium leprae or a component thereof into contact with a differentiated cell derived from an adult, and it also describes cells produced by such method. However, Mycobacterium leprae is a lepra bacillus, and application thereof to regenerative medicine remains problematic in terms of safety.
PRIOR ART DOCUMENTS
Non-Patent Documents


    Non-Patent Document 1: Takahashi and Yamanaka, Cell 126, 663-676, 2006
    Non-Patent Document 2: Takahashi et al., Cell 131, 861-872, 2007


Patent Documents


    Patent Document 1: US Patent Application No. 2006/0222636 A1


DISCLOSURE OF THE INVENTION
Object to be Solved by the Invention
As described above, embryonic stem (ES) cells that can be obtained during the process of the development of fertilized eggs into embryos or induced pluripotent stem (iPS) cells obtained from one's own body are pluripotent stem cells that can grow into substantially any type of tissue in the future. While the applicability of such cells to treatment of intractable diseases has been expected, these cells remain problematic in terms of ethical concerns and canceration risk. Accordingly, it is an object of the present invention to provide a method for producing pluripotent cells that are free of the risk of cellular canceration and that can be applied to regenerative medicine with a high degree of safety.
Means for Solving the Object
In order to attain the above object, the present inventor focused on bacteria having fermentation ability, such as lactic acid bacteria and Bacillus subtilis var natto, and inspected the correlations between such bacteria and cells. Specifically, the present inventor confirmed that the human dermal fibroblasts (HDFs) that had completed differentiation (Cell Applications, Inc., Cat No. 106-05a) would form cell masses, as in the case of ES cells or iPS cells, upon infection with lactic acid bacteria (i.e., Lactococcus lactis subsp. lactis (JCM20101), Streptococcus salivarius subsp. thermophilus (JCM20026), and Lactobacillus sp. (JCM20061), Japan Collection of Microorganisms, RIKEN BioResource Center), or Bacillus subtilis var. natto, and that such cell masses could be stained with alkaline phosphatase. In addition, these cell masses were found to express marker molecules (SSEA-4) that could be expressed specifically in ES cells or iPS cells. Further, these cell masses were found to differentiate into cells of the mesoderm or ectoderm. In general, pluripotent stem cells induced by lactic acid bacteria that are present in a human body may be used to overcome the problems of ethical concerns and canceration, and the use of such pluripotent stem cells enables the production of pluripotent cells that are applicable to regenerative medicine with a high degree of safety. The pluripotent cells produced by the method of the present invention can serve as useful materials for regenerative medicine in the treatment of diseases that were impossible to cure in the past. In the present invention, further, the endosymbiotic theory proposed by Margulis in 1970 (i.e., anaerobic eukaryotes ingested aerobic bacteria to realize the symbiotic conditions and then evolved into the eukaryocytes of the current conditions) was experimentally verified, and the origin of eukaryocytes having organelles that independently generate energy, such as mitochondria or chloroplast, can thereby be expected to be elucidated. The present invention had been completed on the basis of the above findings.
The present invention provides the following invention.
(1) A method for producing pluripotent cells from somatic cells comprising a step of bringing bacteria having fermentation ability or a component or secretory product thereof into contact with somatic cells.
(2) The method according to (1), wherein the somatic cells are somatic cells derived from a mammalian.
(3) The method according to (1) or (2), wherein the somatic cells are somatic cells derived from a human or mouse.
(4) The method according to any one of (1) to (3), wherein the somatic cells are cancer cells.
(5) The method according to any one of (1) to (4), wherein the bacteria having fermentation ability are lactic acid bacteria or Bacillus subtilis var. natto. 
(6) The method according to (5), wherein the lactic acid bacteria belong to the genus Lactococcus, Streptococcus, or Lactobacillus. 
(7) The method according to (6), wherein the lactic acid bacteria are Lactococcus lactic subsp. Lactis, Streptococcus salivarius subsp. thermophilus, Lactobacillus sp., or Lactobacillus acidophilus. 
(8) The method according to any one of (1) to (7), wherein the step of bringing bacteria having fermentation ability or a component or secretory product thereof into contact with the somatic cells is a step of infecting the somatic cells with bacteria having fermentation ability or a component or secretory product thereof.

(9) The method according to any one of (1) to (8), which comprises a step of treating somatic cells with trypsin before bacteria having fermentation ability or a component or secretory product thereof are brought into contact with the somatic cells.

(10) A pluripotent cell, which can be produced by the method according to any one of (1) to (9).

(11) A method for producing somatic cells which were induced to differentiate from pluripotent cells which comprises the steps of:

(a) producing pluripotent cells by the method according to any one of (1) to (9); and

(b) inducing the pluripotent cells produced in step (a) to differentiate.

(12) A somatic cell which was induced to differentiate from pluripotent cells, which can be obtained by the method according to (11).

(13) A kit used for producing pluripotent cells from somatic cells, which comprises bacteria having fermentation ability or a component or secretory product thereof.

(14) A method for producing non-cancer cells from cancer cells, which comprises a step of bringing bacteria having fermentation ability or a component or secretory product thereof into contact with cancer cells.

(15) The method according to (14), wherein the cancer cells are human cancer cells.

(16) The method according to (14) or (15), wherein the bacteria having fermentation ability are lactic acid bacteria or Bacillus subtilis var. natto. 

(17) The method according to (16), wherein the lactic acid bacteria belong to the genus Lactococcus, Streptococcus, or Lactobacillus. 

(18) The method according to (17), wherein the lactic acid bacteria are Lactococcus lactis subsp. Lactis, Streptococcus salivarius subsp. thermophilus, Lactobacillus sp., or Lactobacillus acidophilus. 

(19) The method according to any one of (14) to (18), wherein the step of bringing bacteria having fermentation ability or a component or secretory product thereof into contact with the cancer cells is a step of infecting the cancer cells with bacteria having fermentation ability or a component or secretory product thereof.

(20) An non-cancer cell which can be produced by the method according to any one of (14) to (19).

(21) An anti-cancer agent comprising lactic acid bacteria or a component or secretory product thereof.

(22) The anti-cancer agent according to (21), wherein the lactic acid bacteria belong to the genus Lactococcus, Streptococcus, or Lactobacillus. 

(23) The anti-cancer agent according to (21) or (22), wherein the lactic acid bacteria are Lactococcus lactis subsp. Lactis, Streptococcus salivarius subsp. thermophilus, Lactobacillus sp., or Lactobacillus acidophilus. 

(24) A method for screening for an anti-cancer component derived from lactic acid bacteria, which comprises a step of bringing lactic acid bacteria or a component or secretory product thereof into contact with cancer cells and a step of measuring the extent of conversion of cancer cells into non-cancer cells.

(25) The method according to (24), wherein the lactic acid bacteria belong to the genus Lactococcus, Streptococcus, or Lactobacillus. 

(26) The method according to (24) or (25), wherein the lactic acid bacteria are Lactococcus lactis subsp. Lactis, Streptococcus salivarius subsp. thermophilus, Lactobacillus sp., or Lactobacillus acidophilus. 

Effects of the Invention
According to the present invention, bacteria having fermentation ability, such as lactic acid bacteria, that coexist with cells in the human body are allowed to infect somatic cells, and pluripotent stem cells can then be produced. Since the method of the present invention does not require any artificial gene introduction procedure, the risk of canceration occurring in the produced pluripotent cells can be substantially equivalent to that in the normal state. The method for producing pluripotent cells according to the present invention that involves the use of bacteria having fermentation ability, such as lactic acid bacteria, is useful in the medical field (including drug discovery research and testing of safety, efficacy, and side effects of pharmaceutical products), disease research (elucidation of cause and development regarding therapeutic and preventive methods for intractable diseases), regenerative medicine (restoration of neurological, vascular, and organ functions), and the food industry.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows HDF cells cultured together with lactic acid bacteria.
FIG. 2 shows the results of staining attained by infecting the HDF cells with lactic acid bacteria and staining the formed cell mass with an alkaline phosphatase coloring solution.
FIG. 3 shows the results of staining attained by infecting the HDF cells with lactic acid bacteria and staining the formed cell mass with anti-SSEA-4 antibody (MILLIPORE).
FIG. 4 shows the results obtained by infecting the HDF cells with lactic acid bacteria and subjecting cDNA derived from the formed cell mass to RT-PCR.
FIG. 5 shows the results of examination attained by infecting the HDF cells with lactic acid bacteria or Lactobacillus sp. and inspecting whether or not the formed cell mass can be maintained for a long period of time.
FIG. 6 shows the results of staining attained by infecting the HDF cells with lactic acid bacteria and staining the resultants with the anti-α-SMA antibody (a vascular marker), the anti-Desmin antibody (a mesoderm marker), the anti-Tuj1 antibody (a nerve cell marker), and the anti-GFAP antibody (a glial cell marker), respectively.
FIG. 7 shows the results of culture attained by infecting the HDF cells with lactic acid bacteria and culturing the resultants with a culture solution that induces the cells to differentiate into bone cells, fat cells, or cartilage cells.
FIG. 8 shows the results of observation attained by infecting the HDF cells with lactic acid bacteria and observing the formed cell mass under an electron microscope.
FIG. 9 shows the results of microarray-based gene expression analysis of tRNAs purified from the control HDF cells (C-HDF) and from the HDF cells infected with lactic acid bacteria (Bala-HDF).
FIG. 10 shows the results attained by infecting the HDF cells with lactic acid bacteria, administering the resultants to one of the testes of an SCID mouse, and inspecting the formation of teratoma 3 months later.
FIG. 11 shows the results attained by isolating mouse embryonic fibroblasts from E12.5 GFP mouse embryos, infecting the cells with lactic acid bacteria (JCN1021), and culturing the cells for 5 days.
FIG. 12 shows the results attained by infecting breast cancer cells (MCF7), hepatic cancer cells (HepG2), or lung cancer cells (A549) with lactic acid bacteria (JCM1021) and conducting culture for 4 days.
FIG. 13 shows the results of cell culture conducted with the addition of yogurt to hepatic cancer cells (HepG2) and breast cancer cells (MCF7) for 9 days.
FIG. 14 shows the results of RT-PCR attained by infecting hepatic cancer cells (HepG2) with lactic acid bacteria (JCM1021), recovering the cells 4, 8, and 12 days thereafter, and conducting RT-PCR with the use of c-Myc and CEA cancer cell markers.
FIG. 15 shows the results attained by preparing cell masses of lung cancer cells (A549), transplanting the resulting cell masses hypodermically to 8-week-old female nude mice, and observing tumor formation approximately 1 month later.
FIG. 16 shows the results of measurement of the weight of tumors extracted 40 days after hypodermic transplantation thereof. Control mice were subjected to tumor transplantation in the absence of lactic acid bacteria (JCM1021), and the target mice subjected to lactic acid bacteria injection 3 times were subjected to tumor transplantation in the presence of lactic acid bacteria (2×108 in 0.2 ml each), followed by further lactic acid bacteria injection on Day 3 and Day 6.
FIG. 17 shows HDF cells cultured together with Bacillus subtilis var. natto or E. coli. 



EMBODIMENTS FOR CARRYING OUT THE INVENTION
Hereafter, the present invention is described in greater detail.
The method for producing pluripotent cells from somatic cells according to the present invention is characterized by a step comprising bringing bacteria having fermentation ability or a component or secretory product thereof into contact with somatic cells.
In the present invention, any somatic cells can be used for reprogramming, without particular limitation. Specifically, the term “somatic cells” used in the present invention refers to any cells which constitute an organism, except for germ cells. Differentiated somatic cells or undifferentiated stem cells may be used. Somatic cells may originate from any organisms, such as mammalians, birds, fish, reptiles, or amphibians, without particular limitation, with mammalians (e.g., rodents such as mice or primates such as humans) being preferable, and humans or mice being particularly preferable. When human somatic cells are used, such somatic cells may be derived from an embryo, a newborn, or an adult. When the pluripotent cells produced by the method of the present invention are used for the treatment of a disease in the field of regenerative medicine, the use of somatic cells isolated from a patient with the disease of interest is preferable. In the present invention, cancer cells can be used as somatic cells. By bringing bacteria having fermentation ability or a component or secretory product thereof into contact with cancer cells, non-cancer cells can be produced from the cancer cells. In the present invention, a step of bringing bacteria having fermentation ability or a component or secretory product thereof into contact with somatic cells (including cancer cells) can be carried out in vitro.
The term “pluripotent cells” used in the present invention refers to cells capable of autonomous replication under particular culture conditions (specifically in the presence of lactic acid bacteria) for a long period of time and capable of differentiating into a plurality of types of cells (e.g., ectoderm, mesoderm, or endoderm cells) under particular differentiation-inducing conditions, and these cells may also be referred to as “stem cells.”
In the present invention, bacteria having fermentation ability or a component or secretory product thereof are brought into contact with somatic cells.
Bacteria having fermentation ability used in the present invention are not particularly limited. Aerobic bacteria, such as lactic acid bacteria or Bacillus subtilis var natto, or anaerobic bacteria, such as Bifidobacterium, may be used.
Lactic acid bacteria used in the present invention are not particularly limited. The term “lactic acid bacteria” is a generic term for bacteria capable of producing lactic acid from a saccharide via fermentation. Representative examples of lactic acid bacteria include those belonging to the genera Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, and Streptococcus, and such lactic acid bacteria can be used in the present invention. Use of lactic acid bacteria belonging to the genus Lactococcus, Streptococcus, or Lactobacillus is preferable. Use of Lactococcus lactis subsp. Lactis, Streptococcus salivarius subsp. thermophilus, Lactobacillus sp., or Lactobacillus acidophilus is particularly preferable.
Examples of components of bacteria having fermentation ability include, but are not limited to, a cell wall, a nucleic acid, a protein, a cell organelle, a lipid, a sugar, a carbohydrate, a glucolipid, and a glycosylated sugar.
In the present invention, culture is conducted in the presence of bacteria having fermentation ability with the use of a common medium for cell culture. Thus, pluripotent cells or non-cancer cells according to the present invention can be separated and cultured. According to need, various growth factors, cytokines, or hormones (e.g., components associated with proliferation and maintenance of human ES cells, such as FGF-2, TGFβ-1, activin A, Noggin, BDNF, NGF, NT-1, NT-2, or NT-3) may be added to a medium used for culturing the pluripotent cells of the present invention. Moreover, the differentiation potency and proliferation potency of the separated pluripotent cells can be verified by a method of confirmation known with respect to ES cells.
The applications of the pluripotent cells and the non-cancer cells produced by the method of the present invention are not particularly limited, and these cells can be used for various types of testing, research, or disease treatments, or for other purposes. For example, the pluripotent cells produced by the method of the present invention can be treated with a growth factor, such as retinoic acid or EGF, or with glucocorticoid to induce differentiation into cells of interest (e.g., nerve cells, cardiac muscle cells, hepatic cells, pancreatic cells, or blood cells). The differentiated cells thus obtained can be returned to the patient's body, so as to realize stem cell therapy by autologous cell transplantation.
Examples of central nervous system diseases that can be treated with the use of the pluripotent cells of the present invention include Parkinson's disease, Alzheimer's disease, multiple sclerosis, cerebral infarction, and spinal injury. For the treatment of Parkinson's disease, the pluripotent cells are differentiated into dopaminergic neurons and then transplanted intrastriatally to the patient with Parkinson's disease. Differentiation into dopaminergic neurons can be carried out via coculture of the mouse stroma cell line (PA6 cells) and the pluripotent cells of the present invention under serum-free conditions. For the treatment of Alzheimer's disease, cerebral infarction, and spinal injury, the pluripotent cells of the present invention may be induced to differentiate into neural stem cells and then transplanted into the site of a lesion.
Also, the pluripotent cells of the present invention can be used for the treatment of hepatic diseases, such as hepatitis, cirrhosis, or liver failure. For the treatment of such diseases, the pluripotent cells of the present invention may be differentiated into hepatic cells or hepatic stem cells and then transplanted. The pluripotent cells of the present invention may be cultured in the presence of activin A for 5 days, and culture may be further conducted for about 1 week in the presence of the hepatic cell growth factor (HGF). Thus, hepatic cells or hepatic stem cells can be obtained.
Further, the pluripotent cells of the present invention can be used for the treatment of pancreatic disorders, such as type I diabetes mellitus. In the case of type I diabetes mellitus, the pluripotent cells of the present invention may be differentiated into pancreatic β cells and transplanted into the pancreas. The pluripotent cells of the present invention can be differentiated into pancreatic β cells in accordance with a method of differentiating ES cells into pancreatic β cells.
Further, the pluripotent cells of the present invention can be used for the treatment of cardiac failure associated with ischemic heart diseases. For the treatment of cardiac failure, it is preferable that the pluripotent cells of the present invention be differentiated into cardiac muscle cells and then transplanted into the site of a lesion. By adding Noggin to a medium 3 days before an embryoid body is formed, the pluripotent cells of the present invention can be differentiated into cardiac muscle cells about 2 weeks after the embryoid body is formed.
According to the present invention, bacteria having fermentation ability (e.g., lactic acid bacteria) or a component or secretory product thereof are brought into contact with cancer cells, and non-cancer cells can then be produced from the cancer cells. Accordingly, the lactic acid bacteria, or a component or secretory product thereof, are useful as an anti-cancer agent, and the present invention can provide an anti-cancer agent which comprises lactic acid bacteria or a component or secretory product thereof.
According to the present invention, further, lactic acid bacteria or a component or secretory product thereof are brought into contact with cancer cells, the extent of conversion of cancer cells into non-cancer cells is assayed, and anti-cancer components originating from lactic acid bacteria can then be screened for. The anti-cancer components originating from lactic acid bacteria that are identified by the above-described screening method are useful as anti-cancer agents.
The present invention is described in greater detail with reference to the following examples, although the technical scope of the present invention is not limited to these examples.
EXAMPLES
Example 1
Human dermal fibroblasts (HDF cells) (Cell Applications, Inc., Cat No. 106-05a) were cultured in a fibroblast growth medium (Cell Applications, Inc.) in a 10-cm petri dish. The cells were washed with 10 ml of CMF (Ca2+ Mg2+-free buffer). A 0.25% trypsin solution (containing 1 mM EDTA) was added in an amount of 1 ml and allowed to spread across and throughout the dish. The cells were introduced into a CO2 incubator (37° C.) and allowed to stand therein for 5 minutes. A trypsin inhibitor solution (3 ml, Cell Applications, Inc.) was added to prepare a cell suspension, and the number of the cells was counted. Lactic acid bacteria (i.e., Lactococcus lactis subsp. Lactis (JCM20101), Streptococcus salivarius subsp. thermophilus (JCM20026), Lactobacillus sp. (JCM20061), or Lactobacillus acidophilus (JCM1021)) were introduced into a 6-well plate at 7×107 cells/well in advance, and the HDF cells were then added (5×105 cells/2 ml). Lactic acid bacteria purchased from the Japan Collection of Microorganisms of the RIKEN BioResource Center were used. The cells were cultured in that state in an incubator at 34° C. in the presence of 5% CO2.
As a result, cell masses were observed several days later. The photographs shown in FIG. 1 show the conditions 8 days after the initiation of culture.
Example 2
HDF cells (5×105 cells/2 ml) were infected with lactic acid bacteria (7×107 cells) (i.e., Lactococcus lactis subsp. Lactis (JCM20101), Streptococcus salivarius subsp. thermophilus (JCM20026), or Lactobacillus sp. (JCM20061)) in a 6-well plate, culture was conducted in an incubator at 34° C. in the presence of 5% CO2 for 8 days, the resulting cell masses were transferred to a 4-well plate, the plate was introduced into an alkaline phosphatase coloring solution (Roche), and color was allowed to develop at room temperature for 1 hour.
As a result, the cell masses turned purple, as shown in FIG. 2. This indicates that the HDF cells infected with lactic acid bacteria are pluripotent.
Example 3
HDF cells (5×105 cells/2 ml) were infected with lactic acid bacteria (7×107 cells) (i.e., Lactococcus lactis subsp. Lactis (JCM20101)) in a 6-well plate, culture was conducted in an incubator at 34° C. in the presence of 5% CO2 for 8 days, and the formed cell mass was fixed with 4% PFA at room temperature for 15 minutes, followed by staining thereof with a mouse anti-SSEA-4 antibody (MILLIPORE).
As a result, the cell mass was found to express the SSEA-4 antigen, which would be expressed specifically by pluripotent cells, as shown in FIG. 3.
Example 4
HDF cells (2×105/ml) were seeded on a 12-well plate, infected with lactic acid bacteria (2×107 cells) (i.e., Lactobacillus acidophilus (JCM1021)), and cultured in an incubator at 34° C. in the presence of 5% CO2 for 8 days. Half of the culture solution was exchanged every 5 days, and tRNA was purified from the 20 formed cell masses with the use of a Trizol reagent (Invitrogen) 2 weeks later.
cDNA was synthesized with the use of Oligo (dT) primer and SuperScriptTM III (Invitrogen), and RT-PCR was carried out with the use of a set of primers for several genes reported to be associated with pluripotency. The amplified DNA was subjected to 2% agarose gel electrophoresis, and a band was detected via ethidium bromide staining.
As a result, induction of the expression of c-Myc, Nanog, Oct3/4, Sox2, and TDGF1, which were not expressed in the HDF cells, was observed in the cell masses infected with lactic acid bacteria, although expression of REX1, Fgf4, GDF3 or ECAT16 was not observed.
Example 5
HDF cells (5×105 cells/2 ml) were infected with lactic acid bacteria (2×107 cells) (i.e., Streptococcus salivarius subsp. thermophilus (JCM20026) or Lactobacillus sp. (JCM20061)) in a 6-well plate, culture was conducted in an incubator at 34° C. in the presence of 5% CO2, half of the culture solution was exchanged every 5 days, and whether or not the cell masses could be maintained for a long period of time was investigated. Culture was conducted with the use of a fibroblast growth medium (Cell Applications, Inc.) to which lactic acid bacteria had been added or had not been added. In FIG. 5, the four photographs on the left show the conditions 30 days after the initiation of culture and the two photographs on the right show the conditions 50 days after the initiation of culture.
As a result, the cell masses were found to be maintained 50 days later if they had been cultured in the presence of lactic acid bacteria, while the cell masses that had been cultured in the absence of lactic acid bacteria were found to have undergone cell death, as shown in FIG. 5. This indicates that lactic acid bacteria are necessary for the maintenance of the cell masses.
Example 6
HDF cells (5×105 cells/2 ml) were infected with lactic acid bacteria (2×107 cells) (i.e., Lactococcus lactis subsp. Lactis (JCM20101)) in a 6-well plate, and the cell masses that had formed 8 days layer were subjected to culture on a glass cover coated with poly-L-lysine and laminin (Sigma, 50 μg/ml) for 7 days. The cell masses were fixed with 4% PFA at room temperature for 15 minutes, followed by staining thereof with a mouse anti-α-SMA antibody (Sigma, a vascular marker), a rabbit anti-Desmin antibody (Thermo, a mesoderm marker), a mouse anti-Tuj1 antibody (R&D, a nerve cell marker), and a rabbit anti-GFAP antibody (Dako, a glial cell marker).
As a result, it was found that the differentiated cells could be recognized by relevant antibodies, as shown in FIG. 6. This indicates that the HDF cells were differentiated into various types of cells.
Example 7
HDFs (5×105 cells/2 ml) were infected with lactic acid bacteria (2×107 cells) (i.e., Lactobacillus acidophilus (JCM1021)) in a 6-well plate, and the resulting cell masses were transferred to a 4-well plate 2 weeks later. Culture solutions that induce HDF cells to differentiate into bone cells (B: A shows a 96-well plate after staining with B), fat cells (C), and cartilage cells (D) (GIBCO; A10072-01, A10070-01, and A10071-01) were added in amounts of 500 μl each, half of the culture solution was exchanged every 3 days, and culture was conducted for an additional 2 weeks. In order to examine cell differentiation, the cells on each plate were subjected to staining with Alizarin Red S (bone cells), Oil Red O (fat cells), and Alcian Blue (cartilage cells).
As a result, the cell masses infected with lactic acid bacteria were found to be stained with Alizarin Red S (bone cells), Oil Red O (fat cells), and Alcian Blue (cartilage cells), as shown in FIG. 7. Thus, cell differentiation was confirmed.
Example 8
HDF cells (5×105 cells/2 ml) were infected with lactic acid bacteria (2×107 cells) (i.e., Lactobacillus acidophilus (JCM1021)) in a 6-well plate, half of the culture solution was exchanged every 5 days, and the formed cell masses were observed under an electron microscope in accordance with a conventional resin embedding method for ultrathin sectioning (Tokai Electron Microscopy was commissioned to perform the observation).
As a result, the presence of lactic acid bacteria was observed in the cytoplasm (the red arrow in the left diagram), as shown in FIG. 8. The right diagram is an enlarged view showing the framed region in the left diagram.
Example 9
tRNAs were purified from the control HDF cells (C-HDF) and the 20 HDF cell masses infected with lactic acid bacteria (Lactobacillus acidophilus (JCM1021)) (Bala-HDF) with the use of a Trizol reagent (Invitrogen), and microarray-based gene expression analysis was performed (Agilent Whole Genome (4×44K) Human; type: one-color). Since this experiment was performed with the addition of lactoferrin (25 μg/ml) in order to improve the efficiency for cell mass formation, the cells were indicated with the term “Bala-HDF.” Oncomics Co., Ltd. was commissioned to perform the analysis.
The results are shown in FIG. 9.
FIG. 9-1 shows the results of cluster analysis of the genes exhibiting two-or-more-fold increases/decreases in gene expression levels. The group of genes exhibiting an increased expression level in Bala-HDF compared with that in C-HDF was designated as Group I, the group of genes exhibiting substantially the same expression levels in both C-HDF and Bala-HDF was designated as Group II, and the group of genes exhibiting a decreased expression level in Bala-HDF compared with that in C-HDF was designated as Group III. FIG. 9-2 shows the results of analysis conducted while paying attention to the group of genes that had been reported to be involved with the pluripotency of stem cells.
There were 108 genes exhibiting expression levels increased by 30 or more fold in Bala-HDF compared with that in C-HDF. In contrast, there were 126 genes exhibiting the expression levels decreased by 30 or more fold in Bala-HDF compared with that in C-HDF (Table 1). Concerning the genes related to the pluripotent stem cells, the expression level of the Nanog gene was increased by 8.5 fold and that of the Oct3/4 gene was increased by 2.7 fold in Bala-HDF compared with that in C-HDF. It should be noted that 19 types of Hox genes (i.e., the Homeotic genes) which play a key role in the determination of the structure along the body axis of every animal (Nos. 1 to 4, 6, 8, 10, 13, 14, 17, 18, 22, 35, 47, 53, 59, 74, 117, and 121 in Table 1) are present in the genes which shows the expression levels decreased by 30 or more times in Bala-HDF compared with that in C-HDF.







TABLE 1







126 Genes exhibiting expression levels decreased by 30 or more fold in Lip cells than in HDFs
























Fold change
Regulation












[Bala-HGF]
[Bala-HaF]








vs
vs
raw data
raw data
Flag
Flag


no
ProbeName
UniGeneID
GeneSymbol
GeneName
Description
[C-HGF]
[C-HGF]
[Bala-HGF]
[C-HGF]
[Bala-HGF]
[C-HGF]





















1
A_24_P124558
Hs.664500
HOXC8
homeobox C8
Homo sapiens homeobox C8 (HOXC8),
594.18225
down
4.66344
2869.18900
Compromised
Detected







mRNA [NM_022658]


2
A_23_P500998
Hs.659350
HOXA9
homeobox A9
Homo sapiens homeobox A9 (HOXA9),
592.35516
down
3.55414
2179.96880
Compromised
Detected







mRNA [NM_152739]


3
A_23_P70968
Hs.660918
HOXA7
homeobox A7
Homo sapiens homeobox A7 (HOXA7),
547.44600
down
5.21965
3525.65870
Compromised
Detected







mRNA [NM_006896]


4
A_23_P363316
Hs.654456
HOXB5
homeobox B5
Homo sapiens homeobox B5 (HOXB5),
489.89343
down
3.73538
1894.82500
Compromised
Detected







mRNA [NM_002147]


5
A_33_P3341686
Hs.529901
XIST
X (inactive)-specific
Homo sapiens X (inactive)-specific
406.94977
down
17.06895
7192.51030
Detected
Detected






transcript (non-
transcript (non-protein coding) (XIST).






protein coding)







non-coding RNA [NR_001564]


6
A_23_P66682
Hs.98428
HOXB6
homeobox B6
Homo sapiens homeobox B6 (HOXB6),
385.91574
down
44.43997
17758.20900
Detected
Detected







mRNA [NM_018952]


7
A_23_P148541
Hs.534310
CTAG1A
cancer/testis antigen
Homo sapiens cancer/testis antigen 1A
249.25270
down
93.85477
24223.07800
Detected
Detected






1A
(CTAG1A), mRNA [NM_139250]


8
A_33_P3300965
Hs.549040
HOXC6
homeobox C6
Homo sapiens homeobox C6 (HOXC6),
247.46118
down
33.13974
8491.59500
Detected
Detected







transcript variant 2. mRNA







[NM_153693]


9
A_23_P7727
Hs.2799
HAPLN1
hyaluronan and
Homo sapiens hyaluronan and
241.03157
down
2.67364
667.28190
Compromised
Detected






proteoglycan link
proteoglycan link protein 1 (HAPLN1),






protein 1
mRNA [NM_001884]


10
A_24_P77904
Hs.110637
HOXA10
homeobox A10
Homo sapiens homeobox A10
212.57760
down
2.51297
553.14400
Compromised
Detected







(HOXA10), transcript variant 1, mRNA







[NM_018951]


11
A_23_P7313
Hs.313
SPP1
secreted phospho-
Homo sapiens secreted phosphoprotein
170.12749
down
5.62395
990.71533
Compromised
Detected






protein 1
1 (SPP1), transcript variant 1, mRNA







[NM_001040058]


12
A_23_P374695
Hs.89640
TEK
TEK tyrosine
Homo sapiens TEX tyrosine kinase,
168.16338
down
4.01029
698.29730
Compromised
Detected






kinase,
endothelial (TEK), mRNA [NM_000459]






endothelial


13
A_23_P3264528
Hs.249171
HOXA11
homeobox A11
Homo sapiens homeobox A11
135.72766
down
2.86949
403.27990
Compromised
Detected







(HOXA11), mRNA [NM_005523]


14
A_33_P3300975
Hs.549040
HOXC4
homeobox C4
Homo sapiens homeobox C4 (HOXC4),
131.16129
down
7.26983
987.33136
Compromised
Detected







transcript variant 1, mRNA







[NM_014620]


15
A_24_P264943
Hs.1584
COMP
cartilage
Homo sapiens cartilage oligomeric
126.96592
down
308.84100
40602.72700
Detected
Detected






oligomeric
matrix protein (COMP), mRNA






matrix protein
[NM_000095]


16
A_23_P256956
Hs.718626
KIF20A
kinesin family
Homo sapiens kinesin family member
124.42455
down
18.31738
2359.94800
Detected
Detected






member 20A
20A (KIF20A), mRNA [NM_005733]


17
A_23_P25150
Hs.658823
HOXC9
homeobox C9
Homo sapiens homeobox C9 (HOXC9),
122.49983
down
52.65937
6679.50440
Detected
Detected







mRNA [NM_006897]


18
A_24_P218805
Hs.44276
HOXC10
homeobox C10
Homo sapiens homeobox C10
116.49500
down
2.27947
274.98307
Compromised
Detected







(HOXG10), mRNA [NM_017409]


19
A_23_P51085
Hs.421956
SPC25
SPC25, NDC80
Homo sapiens SPC25, NDC80
111.57378
down
98.13633
11337.70200
Detected
Detected






kinetochore
kinetochore complex component,






complex component
homolog (S. cerevisiae) (SPC25),






homolog
mRNA [NM_020675]






(S. cerevisiae)


20
A_24_P319613
Hs.153704
NEK2
NIMA (never in
Homo sapiens NIMA (never in mitosis
107.40808
down
3.01208
334.99344
Compromised
Detected






mitosis gene a)-
gene a)-related kittens 2 (NEK2), mRNA






related kinase 2
[NM_002497]


21
A_33_P3318343
Hs.87225
CTAG2
cancer/testis
Homo sapiens cancer/testis antigen 2
104.32980
down
62.47739
6749.38800
Detected
Detected






antigen 2
(CTAG2), transcript variant 2, mRNA







[NM_020994]


22
A_23_P55281
Hs.436181
HOXB7
homeobox B7
Homo sapiens homeobox B7 (HOXB7),
98.93823
down
2.50469
256.59653
Compromised
Detected







mRNA [NM_004502]


23
A_23_P35219
Hs.153704
NEK2
NIMA (never in
Homo sapiens NIMA (never in mitosis
96.84769
down
12.15671
1219.09720
Detected
Detected






mitosis gene a)-
gene a)-related kinase 2 (NEK2), mRNA






related kinase 2
[NM_002497]


24
A_33_P3276918
Hs.592116
FAM64A
family with
Homo sapiens family with sequence
95.78413
down
21.28121
2110.68300
Detected
Detected






sequence similarity
similarity 64, member A (FAM64A),






64, member A
mRNA [NM_019013]


25
A_33_P3421243
Hs.518808
AFP
alpha-fetoprotein
Homo sapiens alpha-retoprotein (AFP),
91.34399
down
4.93107
466.39575
Compromised
Detected







mRNA [NM_001134]


26
A_23_P1118174
Hs.592049
PLK1
polo-like kinase 1
Homo sapiens polo-like kinase 1
91.00964
down
27.14974
2558.50440
Detected
Detected






(Drosophila)
(Drosophila) (PLK1), mRNA







[NM_005030]


27
A_23_P43164
Hs.409602
SULF1
sulfatase 1
Homo sapiens sulfatase 1 (SULF1),
87.87735
down
242.28316
22046.17600
Detected
Detected







transcript variant 3, mRNA







[NM_015170]


28
A_23_P118815
Hs.514527
BIRC5
baculoviral
Homo sapiens baculoviral IAP repeat-
84.09252
down
897.69434
78166.19500
Detected
Detected






IAP repeat-
containing 5 (BIRC5), transcript variant






containing 5
3, mRNA [NM_001012271]


29
A_23_P118842
Hs.534499
KRTAP1-5
keratin associated
Homo sapiens keratin associated
82.79930
down
69.21690
5934.33300
Detected
Detected






protein 1-5
protein 1-5 (KRTAP1-5), mRNA







[NM_031957]


30
A_23_P3307495
Hs.24553
STRA6
stimulated by
Homo sapiens stimulated by retinoic
81.89140
down
24.68651
2093.29790
Detected
Detected






retineic acid gene 6
acid gene 6 homolog (mouse) (STRA6),






homolog (mouse)
transcript variant 1, mRNA







[NM_001142617]


31
A_23_P79302
Hs.357567
LYPD6B
LY6/PLAUR
Homo sapiens LY6/PLAUR domain
81.38724
down
2.33069
196.41484
Compromised
Detected






domain
containing 6B (LYPD6B), mRNA






containing 6B
[NM_177964]


32
A_23_P88331
Hs.77695
DLGAP5
discs, large
Homo sapiens discs, large (Drosophila)
80.77577
down
74.09805
6197.56400
Detected
Detected






(Drosophila)
homolog-associated protein 5






homolog-associated
(DLGAP5), transcript variant 1, mRNA






protein 5
[NM_014750]


33
A_33_P3387524
Hs.445098
DEPDC1
DEP domain
Homo sapiens DEP domain containing 1
78.75519
down
2.93570
239.39984
Compromised
Detected






containing 1
(DEPDC1), transcript variant 1, mRNA







[NM_001114120]


34
A_33_P3245218
Hs.631957
ODZ2
odz, odd Oz/ten-m
Homo sapiens odz, odd Oz/ten-m
78.10015
down
5.73433
463.73224
Compromised
Detected






homolog 2
homolog 2 (Drosophila) (ODZ2), mRNA






(Drosophila)
[NM_001122679]


35
A_23_P370588
Hs.514292
HOXB8
homeobox B8
Homo sapiens homeobox B8 (HOXB8),
76.56639
down
2.33384
185.03010
Compromised
Detected







mRNA [NM_024016]


36
A_32_P96719
Hs.123253
SHCBP1
SHC SH2-domain
Homo sapiens SHC SH2-domain
75.35630
down
49.26647
3844.18380
Detected
Detected






binding protein 1
binding protein 1 (SHCBP1), mRNA







[NM_024745]


37
A_23_P141624
Hs.247934
KRTAP1-1
keratin associated
Homo sapiens keratin associated
72.33463
down
2.34718
177.01785
Compromised
Detected






protein 1-1
protein 1-1 (KRTAP1-1), mRNA







[NM_030987]


38
A_23_P65757
Hs.194698
CCNB2
cyclin B2
Homo sapiens cyclin B2 (CCNB2),
71.74918
down
487.13727
36191.06200
Detected
Detected







mRNA [NM_004701]


39
A_23_P35871
Hs.523526
E2F8
E2F transcription
Homo sapiens E2F transcription factor
71.73170
down
2.91475
216.49382
Compromised
Detected






factor 8
8 (E2F8), mRNA [NM_024680]


40
A_33_P3288159
Hs.121028
ASPM
asp (abnormal
Homo sapiens asp (abnormal spindle)
71.65447
down
38.55512
2860.60820
Detected
Detected






spindle)
homolog, microcephaly associated






homolog,
(Drosophila) (ASPM), mRNA






microcephaly
[NM_018136]






associated






(Drosophila)


41
A_24_P323598
Hs.99480
ESCO2
establishment of
Homo sapiens establishment of
89.80357
down
4.74371
342.86966
Compromised
Detected






cohesion 1
cohesion 1 homolog 2 (S. cerevisiae)






homolog 2
(ESCO2), mRNA [NM_001017420]






(S. cerevisiae)


42
A_23_P107421
Hs.515122
TK1
thymidine kinase 1,
Homo sapiens thymidine kinase 1,
68.75995
down
1940.31380
138146.73000
Detected
Detected






soluble
soluble (TK1), mRNA [NM_003258]


43
A_33_P3291831
Hs.14559
CEP55
centrosomal protein
Homo sapiens centrosomal protein
68.03040
down
19.57336
1378.80080
Detected
Detected






55 kDa
55 kDa (CEP55), transcript variant 1,







mRNA [NM_018131]


44
A_23_P155711
Hs.405467
NEIL3
nei endonuclease
Homo sapiens nei endonuclease VII-
67.63126
down
3.82752
268.03870
Compromised
Detected






VIII-like 3
like 3 (E coli) (NEIL3), mRNA






(E. coli)
[NM_018248]


45
A_23_P215634
Hs.450230
IGFBP3
Insulin-like growth
Homo sapiens insulin-like growth factor
67.36516
down
1824.75790
127283.97000
Detected
Detected






factor binding
binding protein 3 (IGFBP3), transcript






protein 3
variant 1, mRNA [NM_001013398]


46
A_23_P45011
Hs.486798
PPP1R14C
protein
Homo sapiens protein phosphatase 1,
66.81310
down
29.65725
2051.75500
Detected
Detected






phosphatase 1,
regulatory (inhibitor) subunit 14C






regulatory
(PPP1R14C), mRNA [NM_030949]






(inhibitor)






subunit 14C


47
A_23_P316511
Hs.654560
HOXB3
homeobox B3
Homo sapiens homeobox B3 (HOXB3),
65.99564
down
2.28753
156.32037
Compromised
Detected







mRNA [NM_002146]


48
A_23_P52017
Hs.121028
ASPM
asp (abnormal
Homo sapiens asp (abnormal spindle)
65.81075
down
157.07414
10703.71700
Detected
Detected






spindle) homolog,
homolog, microcephaly associated






microcephaly
(Drosophila) (ASPM), mRNA






associated
[NM_018136]






(Drosophila)


49
A_23_P126212
Hs.175613
CLSPN
claspin homolog
Homo sapiens claspin homolog
65.52806
down
2.25390
152.93062
Compromised
Detected






(Xenopus laevis)
(Xenopus laevis) (CLSPN), mRNA







[NM_022111]


50
A_23_P138507
Hs.334562
CDC2
cell division cycle 2,
Homo sapiens cell division cycle 2, G1
62.70113
down
119.53158
7760.52200
Detected
Detected






G1 to S and G2
to S and G2 to M (CDC2). transcript






to M
variant 1, mRNA [NM_001786]


51
A_23_P398854
Hs.122110
DOK7
docking protein 7
Homo sapiens docking protein 7
62.14818
down
2.55532
164.43954
Compromised
Detected







(DOK7), transcript variant 1, mRNA







[NM_173660]


52
A_24_P37253
Hs.21929
LYPD6
LY6/PLAUR
Homo sapiens LY6/PLAUR domain
62.11988
down
2.63021
169.18219
Compromised
Detected






domain
containing 6 (LYPD6), mRNA






containing 6
[NM_194317]


53
A_33_P3271273
Hs.514289
HOXB2
homeobox B2
Homo sapiens homeobox B2 (HOXB2),
60.37445
down
23.25287
1453.65990
Detected
Detected







mRNA [NM_002145]


54
A_33_P3313075
Hs.714179
LOC100129619
hypothetical
PREDICTED: Homo sapiens
59.50954
down
5.94992
366.63214
Compromised
Detected






LOC100129619
hypothetical LOC100129619







(LOC100129619), mRNA







[XM_001717266]


55
A_23_P34788
Hs.720061
KIF2C
kinesin family
Homo sapiens kinesin family member
53.41870
down
273.92862
16853.63900
Detected
Detected






member 2C
2C (KIF2C), mRNA [NM_006845]


56
A_24_P299474
Hs.631957
ODZ2
odz. odd Oz/ten-m
Homo sapiens odz, odd Oz/ten-m
58.33110
down
17.59517
1062.73900
Detected
Detected






homolog 2
homolog 2 (Drosophila) (ODZ2), mRNA






(Drosophila)
[NM_001122679]


57
A_23_P70249
Hs.856
CDC25C
cell division cycle
Homo sapiens cell division cycle 25
58.22949
down
30.77745
1855.70400
Detected
Detected






25 homolog C
homolog C (S. pombe) (CDC25G),






(S. pombe)
transcript variant 1, mRNA







[NM_001790]


58
A_23_P115872
Hs.14559
CEP55
centrosomal protein
Homo sapiens centrosomal protein
57.81258
down
178.33205
10675.41700
Detected
Detected






55 kDa
55 kDa (CEP55), transcript variant 1,







mRNA [NM_018131]


59
A_33_P3377529
Hs.533357
HOXA4
homeobox A4
Homo sapiens homeobox A4 (HOXA4),
57.78450
down
6.40620
383.30520
Compromised
Detected







mRNA [NM_002141]


60
A_33_P3807062
Hs.532968
HJURP
Holliday junction
Homo sapiens Holliday junction
56.66154
down
274.61868
16112.07700
Detected
Detected






recognition protein
recognition protein (HJURP), mRNA







[NM_018410]


61
A_23_P57588
Hs.386189
GTSE1
G-2 and S-phase
Homo sapiens G-2 and S-phase
56.62428
down
44.89710
2632.41300
Detected
Detected






expressed 1
expressed 1 (GTSE1), mRNA







[NM_016426]


62
A_24_P225616
Hs.226390
RRM2
ribonucleotide
Homo sapiens ribonucleotide reductase
56.30852
down
70.34309
4101.36700
Detected
Detected






reductase M2
M2 (RRM2), transcript variant 2, mRNA







[NM_001034]


63
A_23_P259586
Hs.169340
TTK
TTK protein kinase
Homo sapiens TTK protein kinase
56.15061
down
25.58659
1487.64700
Detected
Detected







(TTK). transcript variant 1, mRNA







[NM_003318]


64
A_24_P297539
Hs.93002
UBE2C
ubiquitin-
Homo sapiens ubiquitin-conjugating
55.08201
down
533.38605
30421.77100
Detected
Detected






conjugating
enzyme E2C (UBE2CX transcript






enzyme E2C
variant 6, mRNA [NM_181803]


65
A_23_P212844
Hs.104019
TACC3
transforming, acidic
Homo sapiens transforming, acidic
53.95437
down
57.68809
3222.89280
Detected
Detected






coiled-coil
coiled-coil containing protein 3






containing
(TACC3), mRNA [NM_006342]






protein 3


66
A_24_P346855
Hs.80976
MKI67
antigen identified
Homo sapiens antigen identified by
53.93493
down
12.88017
718.32430
Compromised
Detected






by monoclonal
monoclonal antibody Ki-67 (MKI67),






antibody Ki-67
transcript variant 1, mRNA







[NM_002417]


67
A_23_P210176
Hs.133397
ITGA6
integrin, alpha 6
Homo sapiens integrin, alpha 6 (ITGA6),
53.34168
down
21.47094
1185.90750
Detected
Detected







transcript variant 2, mRNA







[NM_000210]


68
A_33_P3258627



Putative uncharacterized protein
52.59529
down
2.31702
126.18560
Compromised
Detected







ENSP00000387024







[Source: UniProtKB/TrEMBL; Acc; B8ZZ63]







[ENST00000409162]


69
A_23_P10206
Hs.159226
HAS2
hyaluronan
Homo sapiens hyaluronan synthase 2
51.20701
down
50.60540
2683.23930
Detected
Detected






synthase 2
(HAS2), mRNA [NM_005328]


70
A_33_P3216008
Hs.88523
SKA3
spindle and
Homo sapiens spindle and kinetochore
50.93999
down
12.93789
682.42560
Detected
Detected






kinetochore
associated complex subunit 3 (SKA3),






associated complex
transcript variant 1, mRNA






subunit 3
[NM_145061]


71
A_23_P423237
Hs.37167
SGCG
sarcoglycan, gamma
Homo sapiens sarcoglycan, gamma
50.23103
down
2.61723
136.12785
Compromised
Detected






(35 kDa dystrophin-
(35 kDa dystrophin-associated






associated
glycoprotein) (SGCG), mRNA






glycoprotein)
[NM_000231]


72
A_23_P24129
Hs.40499
DKK1
dickkopf homolog 1
Homo sapiens dickkopf homolog 1
49.39177
down
971.48020
49684.58200
Detected
Detected






(Xenopus laevis)
(Xenopus laevis) (DKK1), mRNA







[NM_012242]


73
A_23_P163567
Hs.368421
SMPD3
sphingomyelin
Homo sapiens sphingomyelin
49.28304
down
18.40829
939.38586
Detected
Detected






phosphodiesterase 3,
phosphodiesterase 3, neutral






neutral membrane
membrane (neutral sphingomyelinase II)






(neutral
(SMPD3), mRNA [NM_018667]






sphingo-






myelinase II)


74
A_24_P416370
Hs.664706
HOXB4
homeobox B4
Homo sapiens homeobox B4 (HOXB4),
48.99025
down
21.78193
1104.94100
Detected
Detected







mRNA [NM_024015]


75
A_24_P225970
Hs.105153
SGOL1
shugoshin-like 1
Homo sapiens shugoshin-like 1 (S. pombe)
48.72279
down
10.13061
511.09410
Compromised
Detected






(S. pombe)
(SGOL1), transcript variant A1,







mRNA [NM_001012409]


76
A_24_P347378
Hs.507658
ALOX5AP
arachidonate
Homo sapiens arachidonate 5-
48.64382
down
2.26967
114.32042
Compromised
Detected






5-lipoxygenase-
lipoxygenase-activating protein






activating protein
(ALOX5AP), mRNA [NM_001629]


77
A_33_P3387831
Hs.208912
CENPM
centromere
Homo sapiens centromere protein M
47.62641
down
238.83578
11778.23800
Detected
Detected






protein M
(CENPM), transcript variant 1, mRNA







[NM_024053]


78
A_23_P356684
Hs.62180
ANLN
anillin, actin
Homo sapiens anillin, actin binding
46.97221
down
89.14799
4335.96440
Detected
Detected






binding protein
protein (ANLN), mRNA [NM_018685]


79
A_33_P3330149
Hs.270303
PAX6
paired box 6
Homo sapiens paired box 6 (PAX6),
46.52534
down
2.62064
126.24968
Compromised
Detected







transcript variant 1, mRNA







[NM_000280]


80
A_24_P30557
Hs.381715
TBX5
T-box5
Homo sapiens T-box 5 (TBX5),
45.04174
down
4.14714
193.41812
Compromised
Detected







transcript variant 1, mRNA







[NM_000192]


81
A_23_P146274
Hs.521651
STMN2
stathmin-like 2
Homo sapiens stathmin-like 2
43.26243
down
251.80971
11280.19100
Detected
Detected







(STMN2), mRNA [NM_007029]


82
A_33_P3303956
Hs.658061
LOC100133311
similar to
PREDICTED: Homo sapiens similar to
43.11292
down
2.59771
115.96621
Compromised
Detected






hCG1644697
hCG1644697 (LOC100133311), mRNA







[XM_002344295]


83
A_23_P130182
Hs.44265B
AURKB
aurora kinase B
Homo sapiens aurora kinase B
42.91836
down
24.55805
1091.36500
Detected
Detected







(AURKB), mRNA [NM_004217]


84
A_23_P56347
Hs.654413
PSG3
pregnancy
Homo sapiens pregnancy specific beta-
42.86491
down
93.47945
4149.07400
Detected
Detected






specific beta-1-
1-glycoprotein 3 (PSG3), mRNA






glycoprotein 3
[NM_021016]


85
A_23_P122197
Hs.23960
CCNB1
cyclin B1
Homo sapiens cyclin B1 (CCNB1),
42.74203
down
517.57600
22906.70500
Detected
Detected







mRNA [NM_031966]


86
A_23_P100127
Hs.181855
CASC5
cancer susceptibility
Homo sapiens cancer susceptibility
42.70049
down
13.65018
603.63723
Detected
Detected






candidate 5
candidate 5 (CASC5), transcript variant







1, mRNA [NM_170589]


87
A_24_P280983
Hs.587427
HOXA11AS
HOXA11
Homo sapiens HOXA11 antisense RNA
41.97556
down
34.17975
1485.58810
Detected
Detected






antisense RNA
(non-protein coding) (HOXA11AS),






(non-protein coding)
antisense RNA [NR_002795]


88
A_23_P74349
Hs.851950
NUF2
NUF2, NDC80
Homo sapiens NUF2, NDC80
41.75897
down
109.18320
4720.18600
Detected
Detected






kinetochore
kinetochore complex component,






complex
homolog (S. cerevisiae) (NUF2),






component,
transcript variant 1, mRNA






homolog
[NM_145697]






(S. cerevisiae)


89
A_23_P373521
Hs.388245
HAND2
heart and neural
Homo sapiens heart and neural crest
41.60090
down
13.50888
581.90924
Detected
Detected






crest derivatives
derivatives expressed 2 (HAND2),






expressed 2
mRNA [NM_021973]


90
A_33_P3311498
Hs.363603
LOC283392
hypothetical
Homo sapiens hypothetical LOC283392
41.49494
down
5.94969
255.63634
Compromised
Detected






LOC283392
(LOC283392), transcript variant 1,







non-coding RNA [NR_026837]


91
A_23_P151150
Hs.239
FOXM1
forkhead box M1
Homo sapiens forkhead box M1
41.37134
down
121.89368
5221.72360
Detected
Detected







(FOXM1), transcript variant 1, mRNA







[NM_202002]


92
A_23_P148475
Hs.548326
KIF4A
kinesin family
Homo sapiens kinesin family member
41.30457
down
83.02992
3551.12330
Detected
Detected






member 4A
4A (KIF4A), mRNA [NM_012310]


93
A_23_P68610
Hs.244580
7PX2
TPX2, microtubule-
Homo sapiens TPX2, microtubule-
41.25013
down
243.59319
10404.55600
Detected
Detected






associated, homolog
associated, homolog (Xenopus laevis)






(Xenopus laevis)
(TPX2), mRNA [NM_012112]


94
A_24_P140475
Hs.655143
SORBS2
sorbin and
Homo sapiens sorbin and SH3 domain
41.04034
down
6.06472
257.72375
Compromised
Detected






SH3 domain
containing 2 (SORBS2), transcript






containing 2
variant 2, mRNA [NM_021069]


95
A_23_P124417
Hs.469649
BUB1
budding uninhibited
Homo sapiens budding uninhibited by
40.92328
down
63.01636
2670.28000
Detected
Detected






by benzi-
benzimidazoles 1 homolog (yeast)






midazoles 1
(BUB1), mRNA [NM_004336]






homolog (yeast)


96
A_23_P70007
Hs.720052
HMMR
hyaluronan-
Homo sapiens hyaluronan-mediated
40.85090
down
116.10930
4911.35940
Detected
Detected






mediated motility
motility receptor (RHAMM) (HMMR),






receptor
transcript variant 2, mRNA






(RHAMM)
[NM_012484]


97
A_32_P150891
Hs.283127
DIAPH3
diaphanous
Homo sapiens diaphanous homolog 3
40.27765
down
13.45490
561.14830
Detected
Detected






homolog 3
(Drosophila) (DIAPH3), transcript






(Drosophila)
variant 1, mRNA [NM_001042517]


98
A_33_P3708413
Hs.512842
MFAP5
microfibrillar
Homo sapiens microfibrillar associated
39.62058
down
356.31840
14618.14000
Detected
Detected






associated protein 5
protein 5 (MFAP5), mRNA







[NM_003480]


99
A_23_P96325
Hs.47558
ERCC6L
excision repair
Homo sapiens excision repair cross-
39.58863
down
20.08920
823.50500
Detected
Detected






cross-
complementing rodent repair






complementing
deficiency, complementation grasp 6-






rodent
like (ERCC6L), mRNA [NM_017669]






repair






deficiency,






complementation






group 6-like


100
A_23_P167159
Hs.7122
SCRG1
stimulator of
Homo sapiens stimulator of
39.33724
down
2.66140
108.40463
Compromised
Detected






chondrogenesis 1
chondrogenesis 1 (SCRG1), mRNA







[NM_007281]


101
A_23_P121795
Hs.655143
SORBS2
sorbin and
Homo sapiens sorbin and SH3 domain
38.97343
down
13.70106
552.91187
Detected
Detected






SH3 domain
containing 2 (SORBS2), transcript






containing 2
variant 2, mRNA [NM_021069]


102
A_23_P215454
Hs.647061
ELN
elastin
Homo sapiens elastin (ELN), transcript
37.86731
down
51.50940
2019.68640
Detected
Detected







variant 1, mRNA [NM_000501]


103
A_23_P117852
Hs.81892
KIAA0101
KIAA0101
Homo sapiens KIAA0101 (KIAA0101),
36.88694
down
540.27780
20635.85700
Detected
Detected







transcript variant 1, mRNA







[NM_014736]


104
A_23_P72668
Hs.26530
SDPR
serum deprivation
Homo sapiens serum deprivation
36.05926
down
4.32851
161.66243
Compromised
Detected






response
response (SDPR), mRNA [NM_004657]


105
A_23_P323751
Hs.472716
FAM83D
family with
Homo sapiens family with sequence
35.86057
down
68.57602
2546.37650
Detected
Detected






sequence
similarity 83, member D (FAM83D),






similarity 83,
mRNA [NM_030919]






member D


106
A_33_P3326210
Hs.99480
ESCO2
establishment of
Homo sapiens establishment of
35.85314
down
2.66392
98.89648
Compromised
Detected






cohesion 1
cohesion 1 homolog 2 (S. cerevisiae)






homolog 2
(ESCO2), mRNA [NM_001017420]






(S. cerevisiae)


107
A_23_P157136
Hs.655515
SCIN
scinderin
Homo sapiens scinderin (SCIN),
35.78193
down
2.38629
88.41367
Compromised
Detected







transcript variant 2, mRNA







[NM_033128]


108
A_23_P375
Hs.524571
CDCA8
cell division cycle
Homo sapiens cell division cycle
35.73556
down
524.05695
19391.53300
Detected
Detected






associated 8
associated 8 (CDCA8), mRNA







[NM_018101]


109
A_23_P50108
Hs.414407
NDC80
NDC80 homolog,
Homo sapiens NDC80 homolog,
35.48090
down
492.26477
18085.33000
Detected
Detected






kinetochore
kinetochore complex component (S. cerevisiae)






complex
(NDC80), mRNA






component






(S. cerevisiae)
[NM_006101]


110
A_32_P62997
Hs.104741
PBK
PDZ binding kinase
Homo sapiens PDZ binding kinase
35.46259
down
272.00460
9988.02700
Detected
Detected







(PBK), mRNA [NM_018492]


111
A_32_P140489
Hs.492277
GDF6
growth
Homo sapiens growth differentiation
34.81978
down
3.73760
134.75726
Compromised
Detected






differentiation
factor 5 (GDF6), mRNA






factor 6
[NM_001001557]


112
A_33_P3272957
Hs.658061
LOC100133311
similar to
PREDICTED: Homo sapiens similar to
34.80172
down
2.87419
103.57362
Compromised
Detected






hCG1644697
hCG1644697 (LOC100133311), mRNA







[XM_002344295]


113
A_23_P163481
Hs.513645
BUB1B
budding uninhibited
Homo sapiens budding uninhibited by
34.57169
down
88.89745
3182.31400
Detected
Detected






by benzimidazoles 1
benzimidazoles 1 homolog beta (yeast)






homolog beta
(BUB1B), mRNA [NM_001211]






(yeast)


114
A_33_P3311755
Hs.270845
KIF23
kinesin family
Homo sapiens kinesin family member
34.17050
down
84.03610
2973.37900
Detected
Detected






member 23
23 (KIF23), transcript variant 1, mRNA







[NM_138555]


115
A_23_P200310
Hs.445098
DEPDO1
DEP domain
Homo sapiens DEP domain containing 1
34.04056
down
70.18784
2473.95400
Detected
Detected






containing 1
(DEPDC1), transcript variant 2, mRNA







[NM_017779]


116
A_23_P58321
Hs.58974
CCNA2
cyclin A2
Homo sapiens cyclin A2 (CCNA2),
33.80436
down
59.81775
2093.80300
Detected
Detected







mRNA [NM_001237]


117
A_23_P501538
Hs.659337
HOXA3
homeobox A3
Homo sapiens homeobox A3 (HOXA3),
33.35369
down
2.40679
83.12165
Compromised
Detected







transcript variant 2, mRNA







[NM_153631]


118
A_33_P3423585
Hs.657273
UNC13C
unc-13 homolog C
Homo sapiens unc-13 homolog C (C. elegans)
33.21877
down
2.73679
94.13646
Compromised
Detected






(C. elegans)
(UNC13C), mRNA







[NM_001080534]


119
A_24_P399888
Hs.208912
CENPM
centromere
Homo sapiens centromere protein M
32.49764
down
30.25951
1018.23220
Detected
Detected






protein M
(CENPM), transcript variant 2, mRNA







[NM_001002876]


120
A_24_P66027
Hs.226307
APOBEC3B
apolipoprotein B
Homo sapiens apolipoprotein B mRNA
31.39611
down
82.84677
2693.29900
Detected
Detected






mRNA editing
editing enzyme, catalytic polypeptide-






enzyme,
like 3B (APOBEC3B), mRNA






catalytic
[NM_004900]






polypeptide-






like 3B


121
A_23_P107283
Hs.514289
HOXB2
homeobox B2
Homo sapiens homeobox B2 (HOXB2),
31.19959
down
256.95248
8301.08400
Detected
Detected







mRNA [NM_002145]


122
A_33_P3368358
Hs.37982
NEDD9
neural precursor
Homo sapiens neural precursor cell
31.14883
down
5.83776
188.28748
Compromised
Detected






cell expressed,
expressed, developmentally down-






developmentally
regulated 9 (NEDD9), transcript variant






down-regulated 9
2, mRNA [NM_182966]


123
A_33_P3255824




31.06368
down
4.28859
137.94330
Compromised
Detected


124
A_23_P96158
Hs.2785
KRT17
keratin 17
Homo sapiens keratin 17 (KRT17),
30.95973
down
2.61022
83.67714
Compromised
Detected







mRNA [NM_000422]


125
A_33_P3242649
Hs.301052
KIF18A
kinesin family
Homo sapiens kinesin family member
30.24299
down
21.10955
661.05370
Detected
Detected






member 18A
18A (KIF18A), mRNA [NM_031217]


126
A_33_P3270514
Hs.61435
NBLA00301
Nbla00301
Homo sapiens Nbla00301
30.17527
down
4.17646
130.49452
Compromised
Detected







(NBLA00301), non-coding RNA







[NR_003679]









Example 10
HDF cells (5×105 cells/2 ml) were infected with lactic acid bacteria (2×107 cells) (i.e., Lactobacillus acidophilus (JCM1021)) in a 6-well plate, and the cell masses were collected 2 weeks later, followed by treatment with trypsin. The cells (5×105 cells/30 μl) were administered to one of the testes of a male SCID mouse (9-to-10 week-old), and the formation of teratoma was examined 3 months later.
As a result, the testis that had been infected with lactic acid bacteria (above) were found to have become somewhat larger than the control testis (below, another testis of the same mouse), as shown in the photograph of FIG. 10, but teratoma formation was not observed. Paraffin sections (6 μl) were prepared and subjected to HE staining. No differences were observed in the structure of the testis into which the JCM1021-infected HDFs had been transplanted or the control testis.
Example 11
Mouse embryonic fibroblasts (MEF cells) were sampled in accordance with the sampling method developed by the RIKEN Center for Developmental Biology. A 12.5-day-old GFP mouse embryo was extracted from the uterus, and the head, the caudal portion, the extremities, and the visceral organ were removed. The remaining tissue was cut into small pieces with the use of surgical scissors, and the resultant was incubated in a 0.25% trypsin-EDTA solution at 37° C. for 15 minutes. After the incubation product had been filtered through a cell strainer, the remnant was suspended in a cell culture solution, and the cells constituting one embryo were seeded in a 10-cm petri dish. After the cells reached confluence, the cells were infected with lactic acid bacteria (JCM1021), as with the case of the HDF cells, and culture was then conducted for 5 days.
As a result, the MEF cells that had been infected with lactic acid bacteria were found to have formed cell masses, as shown in the photograph of FIG. 11.
Example 12
Breast cancer cells (MCF7; RBRC-RCB1904), lung cancer cells (A549; RBRC-RCB0098), and hepatic cancer cells (HEP G2; RBRC-RCB1648) were obtained from the RIKEN BioResource Center. In the same manner as in Example 1, lactic acid bacteria (i.e., Lactococcus lactis subsp. Lactis (JCM20101)) were introduced into a 6-well plate at 1×108 cells/well in advance, and 5×105 cancer cells were added thereto. Culture was conducted in such state in an incubator at 34° C. in the presence of 5% CO2.
The results are shown in FIG. 12. Cell masses were observed several days later, as shown in FIG. 12. These photographs show the conditions 4 days after the initiation of culture.
Example 13
The experiment was carried out in the same manner as in Example 1, except that commercially available yogurt was introduced into a 6-well plate at 50 μl/well in advance, and 5×105 cancer cells were added thereto. Culture was conducted in an incubator at 34° C. in the presence of 5% CO2.
The results are shown in FIG. 13. Cell masses were observed several days later, as shown in FIG. 13. These photographs show the conditions 9 days after the initiation of culture.
Example 14
The experiment was carried out in the same manner as in Example 12 with the use of hepatic cancer cells (HEP G2) and lactic acid bacteria (JCM20101). The cells were recovered 4, 8, and 12 days after infection, and then RT-PCR was carried out by using c-Myc and the carcino embryonic antigen (CEA) as cancer cell markers.
The results are shown in FIG. 14. While both marker molecules were expressed on Day 0, the c-Myc expression level was decreased from Day 4, and the CEA expression level was decreased from Day 8, as shown in FIG. 14.
Example 15
According to the hanging drop method, cells are treated with trypsin, the treated cells are suspended in a culture solution at 1×105 cells/20 μl, the cell suspension is added dropwise onto the lid of a petri dish, the lid is overturned, and the petri dish is then allowed to stand overnight. On the following day, a cell mass is observed at the tip of a drop, and the resulting cell mass is transplanted into a mouse. The hanging drop method was carried out using lung cancer cells (A549) to form cell masses. The resulting 5 cell masses were transplanted hypodermically to an 8-week-old female nude mouse. Tumor formation was observed approximately 1 month later (FIG. 15). The tumors were extracted and trimmed to the size of 4×4 mm each. Control tumor masses were soaked in a PBS solution. The test tumor masses were soaked in a solution of lactic acid bacteria (JCM20101) (1×108/ml) at room temperature for 20 minutes. Thereafter, a single tumor mass was transplanted hypodermically to an 8-week-old female nude mouse. A solution containing lactic acid bacteria was injected into the target mouse of lactic acid bacteria test on Day 3 and Day 6. The tumor was extracted and weighed 40 days later.
The results are shown in FIG. 16. In comparison with the mouse into which the tumor had been transplanted, a reduction in tumor size was observed in the mouse which was infected with lactic acid bacteria and further injected with lactic acid bacteria.
Example 16
In the same manner as in Example 1, Bacillus subtilis var. natto or E. coli (XLI-blue, Stratagene) cells were introduced into a 6-well plate at 1×108 cells/well in advance, and 5×105 HDF cells (Cell Applications, Inc., Cat No. 106-05a) were added thereto. Culture was conducted in an incubator at 34° C. in the presence of 5% CO2.
The results are shown in FIG. 17. While a cell mass was observed several days later in the presence of Bacillus subtilis var. natto, formation of a cell mass was not observed in the presence of E. coli, as shown in FIG. 17. These photographs show the conditions 8 days after the initiation of culture.

## Claims

1. A method for altering the expression profile of fibroblasts, comprising treating fibroblasts with trypsin, bringing whole and live bacteria having fermentation ability into contact with fibroblasts in vitro, and culturing the fibroblasts, wherein the expression profile of the fibroblasts is altered and the altered expression comprises increased expression of Nanog and Oct3/4, and wherein the bacteria having fermentation ability are Lactococcus lactis, Streptococcus salivarius, Lactobacillus sp., or Lactobacillus acidophilus.

2. The method according to claim 1, wherein the fibroblasts are derived from a mammal.

3. The method according to claim 1, wherein the fibroblasts are derived from a human or mouse.

4. The method according to claim 1, wherein the bacteria having fermentation ability are Lactococcus lactis subsp. Lactis, Streptococcus salivarius subsp. thermophilus, Lactobacillus sp., or Lactobacillus acidophilus.

5. The method according to claim 1, wherein bringing whole and live bacteria having fermentation ability into contact with fibroblasts in vitro comprises infecting the fibroblasts with the bacteria in vitro.

6. A method for producing somatic cells which comprises:
(a) producing cells by the method according to claim 1; and
(b) inducing the cells produced in (a) to differentiate.