Patent Grant
9587224
The present invention relates to a method for producing pluripotent cells using bacteria having fermentation ability.
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.
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.
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.
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.
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.
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.
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
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
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 SuperScript™ 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.
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
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
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
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
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
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
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.
Homo sapiens homeobox C8 (HOXC8),
Homo sapiens homeobox A9 (HOXA9),
Homo sapiens homeobox A7 (HOXA7),
Homo sapiens homeobox B5 (HOXB5),
Homo sapiens X (inactive)-specific
Homo sapiens homeobox B6 (HOXB6),
Homo sapiens cancer/testis antigen 1A
Homo sapiens homeobox C6 (HOXC6),
Homo sapiens hyaluronan and
Homo sapiens homeobox A10
Homo sapiens secreted phosphoprotein
Homo sapiens TEX tyrosine kinase,
Homo sapiens homeobox A11
Homo sapiens homeobox C4 (HOXC4),
Homo sapiens cartilage oligomeric
Homo sapiens kinesin family member
Homo sapiens homeobox C9 (HOXC9),
Homo sapiens homeobox C10
Homo sapiens SPC25, NDC80
Homo sapiens NIMA (never in mitosis
Homo sapiens cancer/testis antigen 2
Homo sapiens homeobox B7 (HOXB7),
Homo sapiens NIMA (never in mitosis
Homo sapiens family with sequence
Homo sapiens alpha-retoprotein (AFP),
Homo sapiens polo-like kinase 1
Homo sapiens sulfatase 1 (SULF1),
Homo sapiens baculoviral IAP repeat-
Homo sapiens keratin associated
Homo sapiens stimulated by retinoic
Homo sapiens LY6/PLAUR domain
Homo sapiens discs, large (Drosophila)
Homo sapiens DEP domain containing 1
Homo sapiens odz, odd Oz/ten-m
Homo sapiens homeobox B8 (HOXB8),
Homo sapiens SHC SH2-domain
Homo sapiens keratin associated
Homo sapiens cyclin B2 (CCNB2),
Homo sapiens E2F transcription factor
Homo sapiens asp (abnormal spindle)
Homo sapiens establishment of
Homo sapiens thymidine kinase 1,
Homo sapiens centrosomal protein
Homo sapiens nei endonuclease VII-
Homo sapiens insulin-like growth factor
Homo sapiens protein phosphatase 1,
Homo sapiens homeobox B3 (HOXB3),
Homo sapiens asp (abnormal spindle)
Homo sapiens claspin homolog
Homo sapiens cell division cycle 2, G1
Homo sapiens docking protein 7
Homo sapiens LY6/PLAUR domain
Homo sapiens homeobox B2 (HOXB2),
Homo sapiens kinesin family member
Homo sapiens odz, odd Oz/ten-m
Homo sapiens cell division cycle 25
Homo sapiens centrosomal protein
Homo sapiens homeobox A4 (HOXA4),
Homo sapiens Holliday junction
Homo sapiens G-2 and S-phase
Homo sapiens ribonucleotide reductase
Homo sapiens TTK protein kinase
Homo sapiens ubiquitin-conjugating
Homo sapiens transforming, acidic
Homo sapiens antigen identified by
Homo sapiens integrin, alpha 6 (ITGA6),
Homo sapiens hyaluronan synthase 2
Homo sapiens spindle and kinetochore
Homo sapiens sarcoglycan, gamma
Homo sapiens dickkopf homolog 1
Homo sapiens sphingomyelin
Homo sapiens homeobox B4 (HOXB4),
Homo sapiens shugoshin-like 1 (S. pombe)
Homo sapiens arachidonate 5-
Homo sapiens centromere protein M
Homo sapiens anillin, actin binding
Homo sapiens paired box 6 (PAX6),
Homo sapiens T-box 5 (TBX5),
Homo sapiens stathmin-like 2
Homo sapiens aurora kinase B
Homo sapiens pregnancy specific beta-
Homo sapiens cyclin B1 (CCNB1),
Homo sapiens cancer susceptibility
Homo sapiens HOXA11 antisense RNA
Homo sapiens NUF2, NDC80
Homo sapiens heart and neural crest
Homo sapiens hypothetical LOC283392
Homo sapiens forkhead box M1
Homo sapiens kinesin family member
Homo sapiens TPX2, microtubule-
Homo sapiens sorbin and SH3 domain
Homo sapiens budding uninhibited by
Homo sapiens hyaluronan-mediated
Homo sapiens diaphanous homolog 3
Homo sapiens microfibrillar associated
Homo sapiens excision repair cross-
Homo sapiens stimulator of
Homo sapiens sorbin and SH3 domain
Homo sapiens elastin (ELN), transcript
Homo sapiens KIAA0101 (KIAA0101),
Homo sapiens serum deprivation
Homo sapiens family with sequence
Homo sapiens establishment of
Homo sapiens scinderin (SCIN),
Homo sapiens cell division cycle
Homo sapiens NDC80 homolog,
Homo sapiens PDZ binding kinase
Homo sapiens growth differentiation
Homo sapiens budding uninhibited by
Homo sapiens kinesin family member
Homo sapiens DEP domain containing 1
Homo sapiens cyclin A2 (CCNA2),
Homo sapiens homeobox A3 (HOXA3),
Homo sapiens unc-13 homolog C (C. elegans)
Homo sapiens centromere protein M
Homo sapiens apolipoprotein B mRNA
Homo sapiens homeobox B2 (HOXB2),
Homo sapiens neural precursor cell
Homo sapiens keratin 17 (KRT17),
Homo sapiens kinesin family member
Homo sapiens Nbla00301
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
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
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
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
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
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 (
The results are shown in
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
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-152479 | Jul 2011 | JP | national |
| 2012-107210 | May 2012 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2012/067544 | 7/10/2012 | WO | 00 | 5/5/2014 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/008803 | 1/17/2013 | WO | A |
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