WO2012133942A1

WO2012133942A1 - Pluripotent stem cell capable of being isolated from fat tissue or umbilical cord of biological body

Info

Publication number: WO2012133942A1
Application number: PCT/JP2012/059422
Authority: WO
Inventors: 真理出澤; 正順吉田
Original assignee: 株式会社Ｃｌｉｏ
Priority date: 2011-03-30
Filing date: 2012-03-30
Publication date: 2012-10-04
Also published as: JP2014132830A; CN103459590A

Abstract

The present invention provides a method for directly obtaining a pluripotent stem cell from the fat tissue or the umbilical cord of a biological body, and the pluripotent stem cell obtained by means of the aforementioned method. A SSEA-3 positive and CD105 positive pluripotent stem cell which is capable of being isolated from the fat tissue or the umbilical cord of a biological body by using the expression of SSEA-3 as an index and which has all of the following properties: (i) has little or no telomerase activity; (ii) is capable of differentiating into any of the triploblastic cells; (iii) does not exhibit neoplastic propagation; and (iv) exerts replication competence (self-renewal abilities).

Description

Pluripotent stem cells that can be isolated from living umbilical cord or adipose tissue

The present invention relates to a pluripotent stem cell derived from a living umbilical cord or adipose tissue.

In recent years, adult stem cells or tissue stem cells that can contribute to tissue regeneration have attracted attention.
As a cell having differentiation ability obtained from an adult, for example, a bone marrow mesenchymal cell fraction having differentiation ability into various cells such as bone, cartilage, adipocyte, nerve cell, skeletal muscle by inducing differentiation ( MSC (Bone marrow stromal cell) has been reported (see Non-Patent Documents 1 and 2). However, the bone marrow mesenchymal cell fraction is a group of cells containing a plurality of cell types, and the differentiation efficiency when induction is not high. Although it has been assumed that some cells in the MSC are responsible for differentiation, the body of such cells is not clear and has been debated for a long time. In addition, in order to differentiate into a specific cell, stimulation with a specific compound, gene introduction, or the like is necessary, and a differentiation induction system has to be constructed.
Furthermore, iPS cells (induced pluripotent stem cells) artificially created from somatic cells as adult-derived pluripotent stem cells (see Patent Document 1, Patent Document 2, Non-Patent Document 3, etc.) have been reported. However, in order to establish iPS cells, an induction operation using a specific substance is required to introduce a specific gene or a specific compound into a somatic cell into a dermal fibroblast fraction (dermal fibroblast) that is a mesenchymal cell. It is.
In addition, there has been a report on stage-specific embryonic antigen-3 (SSEA-3) -positive pluripotent stem cells that can be isolated from biological tissues, and have not been known so far (Patent Document 3). And non-patent document 4).

Japanese Patent No. 4183742 JP 2008-307007 A International Publication No. WO2011 / 007900 International Publication Pamphlet

An object of the present invention is to provide a method for directly obtaining pluripotent stem cells without artificial manipulation such as gene transfer from the umbilical cord or adipose tissue of a living body, and to provide pluripotent stem cells obtained by the method. .
The present inventors isolated stem cells that are positive for SSEA-3 from dermal fibroblasts and bone marrow cells and have an antigen expression pattern that is not found in conventional stem cells, and Muse (Multilineage-differentiating Stress Ending cells) cells and (International publication pamphlet of International Publication No. WO2011 / 007900, Proc. Natl. Acad. Sci USA, 107 (19): 8639-43, 2010).
Furthermore, investigation was performed and Muse cells were isolated from the umbilical cord and adipose tissue, and the present invention was completed.
That is, the present invention is as follows.
[1] SSEA-3 positive and CD105 positive pluripotent stem cells having all of the following properties that can be isolated from living umbilical cord or adipose tissue using SSEA-3 expression as an index:
(I) low or no telomerase activity;
(Ii) has the ability to differentiate into cells of any germ layer of the three germ layers;
(Iii) no neoplastic growth; and (iv) self-replicating ability (self-renewal ability).
[2] The pluripotent stem cell according to [1], which can be isolated directly from the umbilical cord or adipose tissue of a living body using the expression of SSEA-3 as an index.
[3] The pluripotent stem cell according to [1] or [2], which can be isolated from human adipose-derived stem cells (HADSC).
[4] The pluripotent stem cell according to any one of [1] to [3], which is proliferated by culture combining suspension culture and adhesion culture.
[5] The pluripotent stem cell of any one of [1] to [4], which is CD117 negative and CD146 negative.
[6] The pluripotent stem cell of any one of [1] to [4], which is CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative and CD271 negative.
[7] Pluripotency of any of [1] to [4] of CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snail negative, Slug negative, Tyrp1 negative and Dct negative Stem cells.
[8] A cell population comprising the pluripotent stem cell according to any one of [1] to [7].
[9] A method of isolating, culturing and proliferating the pluripotent stem cell of any one of [1] to [7] using the expression of SSEA-3 as an index from umbilical cord or adipose tissue.
[10] Furthermore, the method of [9], wherein pluripotent stem cells are isolated using at least one of the following characteristics (i) to (v) as an index:
(I) CD105 positive;
(Ii) CD117 negative and CD146 negative;
(Iii) CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative and CD271 negative; and (iv) CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snail negative, Slug Negative, Tyrp1 negative and Dct negative.
[11] A method for isolating pluripotent stem cells according to any one of [1] to [7], which comprises trypsinizing umbilical cord or adipose tissue-derived mesenchymal cells and recovering surviving cells.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2011-0776635, which is the basis of the priority of the present application.

FIG. 1 is a diagram showing the results of FACS analysis using anti-SSEA-3 antibody for mesenchymal cells established from primary culture of human subcutaneous adipose tissue. A: No staining, B: Secondary antibody only, C: SSEA-3 stained.
FIG. 2 is a diagram for explaining primer sequences used in RT-PCT and positive control and negative control samples set in each.
FIG. 3 is a diagram showing the morphology of human subcutaneous adipose tissue (A) and mesenchymal cells (B) established from the tissue.
FIG. 4 is a diagram showing expression of SSEA-3 in commercially available adipose-derived mesenchymal cells (HADSC) (A) and HADSC (B) established from the subcutaneous fat tissue of FIG.
FIG. 5 is a photograph showing the morphology of Muse-derived embryoid body-like cell mass (M-cluster) collected from adipose tissue. FIG. 5A shows the morphology of a Muse-derived embryoid body-like cell cluster (M-cluster) obtained from a commercially available HADSC, and FIG. 5B shows a Muse-derived embryoid body-like cell cluster obtained from HADSC established from subcutaneous adipose tissue ( M-cluster) is shown.
FIG. 6 is a photograph of RT-PCR results showing differentiation of Muse-derived embryoid body-like cell mass (M-cluster). In the figure, M-cluster I represents the differentiation of Muse-derived embryoid body-like cell mass (M-cluster) obtained from commercially available HADSC, and M-cluster II represents the Muse-derived embryoid body-like cell mass obtained from established HADSC. (M-cluster) differentiation is shown.
FIG. 7 is a photograph showing an immunostained image showing that an embryoid body-like cell cluster (M-cluster) formed from a commercially available HADSC-derived Muse cell differentiates into a trioderm cell in gelatin culture. is there. NF (neurofilament; ectoderm), CK7 (Cytokeratin 7; endoderm), SMA (smooth muscle actin; mesoderm).
FIG. 8 is a photograph showing the expression of pluripotent factors in embryoid body-like cell mass (M-cluster) formed from commercially available HADSC-derived Muse cells.
FIG. 9 is a photograph showing that Muse cells do not have tumorigenicity. Testes transplanted with Muse cells are the same size as normal testes even after 4 months, and no signs of tumor formation are seen, but those transplanted with mouse ES cells form a huge tumor in 8 weeks.
FIG. 10 is a photograph showing an HE-stained image of mouse ES cell-derived teratoma. In the figure, I indicates intestinal epithelium (endoderm), II indicates epidermis (ectodermal), and III indicates smooth muscle (mesoderm). It is a photograph 8 weeks after transplantation.
FIG. 11 is a photograph showing a tissue image of a mouse testis transplanted with Muse cells. It is a photograph 4 months after transplantation. Normal seminiferous tubules constitute the tissue and no image showing tumor formation is seen.
FIG. 12 is a diagram showing that isolated Muse cells have a self-renewal ability. An example with fat derived Muse cells is shown.
FIG. 13 is a diagram showing the morphology of mesenchymal cells obtained from the umbilical cord.
FIG. 14 is a diagram showing expression of SSEA-3 in mesenchymal cells obtained from the umbilical cord.
FIG. 15 shows the morphology of Muse cell-derived embryoid body-like cell clusters (M-cluster) formed from SSEA-3 positive cells isolated from the umbilical cord.
FIG. 16 shows Muse cell α-fetoprotein (α-FP; endoderm), GATA6 (endoderm), MAP-2 (ectoderm) and Nkx2.5 (mesoderm) isolated from the umbilical cord. ) Shows the results of RT-PCR analysis.

Hereinafter, the present invention will be described in detail.
The present invention relates to a pluripotent stem cell or pluripotent stem cell fraction that can be obtained directly from the umbilical cord or adipose tissue of a living body, and a method for isolating the pluripotent stem cell or the pluripotent stem cell fraction, And a pluripotent stem cell or pluripotent stem cell fraction derived from the umbilical cord or adipose tissue obtained by the method. The pluripotent stem cells of the present invention are referred to as Muse cells (multilineage differentiating stress-ending cells).
In the present invention, the term “cell fraction” refers to a cell group containing at least a certain amount of cells to be isolated. For example, the pluripotent stem cell fraction includes a group of cells containing 1% or more, 10% or more, 30% or more, 50% or more, 70% or more, 90% or more, or 95% or more of pluripotent stem cells. In addition, a cell cluster obtained by culturing pluripotent stem cells and a cell group enriched with pluripotent stem cells are included. In addition, the cell fraction may be referred to as a substantially uniform cell fraction.
The umbilical cord is a mammalian umbilical cord. The umbilical cord is composed of epithelium, blood vessels, blood, and mesenchymal tissue, among which the mesenchymal tissue is the source of umbilical cord-derived mesenchymal cells.
Adipose tissue-derived pluripotent stem cells can be suitably isolated from adipose-derived stem cells (ADSC), which are mesenchymal cells contained in adipose tissue. ADSC can use commercially available human fat-derived stem cells such as Lonza, and can also obtain adipose stem cell fraction by a known method from surgically collected subcutaneous adipose tissue or subcutaneous lipoaspirate. . For example, mesenchymal cells established from adipose tissue by the method of Estes BT et al. (Ests BT., Et al. Nat Protoc. 2010 Jul: 5 (7): 1294-1311) can be used. The cell surface antigens of adipose stem cells are CD13 positive, CD29 positive, CD44 positive, CD73 positive, CD90 positive, CD105 positive, CD166 positive, CD14 negative, CD31 negative, and CD45 negative.
The umbilical cord and adipose tissue that have been collected and cryopreserved can also be used.
Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets, etc. . The pluripotent stem cells of the present invention are clearly distinguished from embryonic stem cells (ES cells) and embryonic germ stem cells (EG cells) in that they can be obtained directly from a living umbilical cord or adipose tissue.
That cells can be obtained directly from umbilical cord or adipose tissue can be isolated directly from umbilical cord or adipose tissue, or mesenchymal cells can be once cultured from these tissues and isolated therefrom It means that it can be obtained without an artificial induction operation such as compound treatment such as introduction of a foreign gene or protein or administration of a compound. Here, the foreign gene is not limited, but refers to a gene that can initialize the nucleus of a somatic cell, for example, an Oct family gene such as an Oct3 / 4 gene, a Klf family gene such as a Klf gene, a c-Myc gene, etc. Examples include Sox family genes such as Myc family genes and Sox2 genes. Examples of foreign proteins include proteins and cytokines encoded by these genes. Furthermore, examples of the compound include a low molecular weight compound that induces the expression of a gene that can reprogram the somatic cell nucleus, DMSO, a compound that functions as a reducing agent, a DNA methylating agent, and the like. The pluripotent stem cells of the present invention are clearly distinguished from iPS (Induced Primitive Stem Cell) cells and ES cells in that they can be obtained directly from the umbilical cord or adipose tissue of a living body. In the present invention, cell culture, isolation of cells or cell fractions using cell surface markers as indicators, exposure of cells to cell stress, and physical impact on cells It is not included in typical guidance operations. The pluripotent cell of the present invention may be obtained without requiring reprogramming or induction of dedifferentiation.
A pluripotent stem cell means a cell having pluripotency, and has the following characteristics.
(1) It expresses pluripotent markers such as Nanog, Oct3 / 4, SSEA-3, PAR-4, and Sox2.
(2) It has a clonality that grows from one cell and continues to make its own clones.
(3) Has the ability to self-replicate (self-renewal).
(4) It can differentiate into three germ layers (endoderm, mesoderm and ectoderm) in vitro and in vivo.
(5) When transplanted into the mouse testis or subcutaneously, no tumor is formed.
(6) Positive by alkaline phosphatase staining.
The pluripotent stem cell of the present invention is clearly distinguished from adult stem cells such as neural stem cells and hematopoietic stem cells and tissue stem cells, which are generally known, in that they have pluripotency. In addition, the pluripotent stem cell of the present invention is isolated as a single cell or a plurality of cells having pluripotency, so that it is a bone marrow mesenchymal (stem) cell or a fat-derived mesenchymal (stem) It is clearly distinguished from general mesenchymal cell fractions such as cells.
Furthermore, the pluripotent stem cell of this invention has the following characteristics.
(I) The growth rate is relatively slow and the division cycle is 1 day or longer, for example, 1.2 to 1.5 days. However, it does not show infinite proliferation as shown by ES cells and iPS cells.
(Ii) In ES cells and iPS cells, when transplanted into immunodeficient mice, teratomas including endoderm, mesodermal, and ectoderm components are observed in a short period of time, whereas in Muse cells, more than half a year Characterized by the absence of teratoma.
(Iii) A Muse-derived embryoid body-like cell mass is formed from one cell by suspension culture.
(Iv) Embryoid body-like cell clusters are formed in suspension culture, and the growth stops in about 10 to 14 days. Thereafter, it is re-growth by moving to an adherent culture.
(V) Accompanying asymmetric division during growth.
(Vi) The karyotype is normal.
(Vii) no or low telomerase activity. Here, the absence or low telomerase activity means that it cannot be detected or is low when telomerase activity is detected using, for example, TRAPEZE XL telomerase detection kit (Millipore). Low telomerase activity means, for example, telomerase activity comparable to that of human fibroblasts, or telomerase activity of 1/5 or less, preferably 1/10 or less that of Hela cells. That means.
(Viii) Regarding the methylation status, the demethylation of the promoter region of Nanog and Oct3 / 4 is high for iPS cells derived from Muse cells.
(Ix) High phagocytic ability.
(X) Does not show neoplastic growth. Here, not exhibiting neoplastic growth means that when suspension culture is performed, the growth stops when the cell mass (cluster) of a certain size is reached, and does not grow infinitely. Also, teratomas do not form when transplanted into the testis of immunodeficient mice. The above (i) to (iv) and the like are also related to not showing neoplastic growth.
That is, the cell of the present invention is, for example, the following pluripotent stem cell.
(A) A pluripotent stem cell obtained from a living umbilical cord or adipose tissue, which can be directly obtained without introducing a chemical substance, a foreign gene or a foreign protein into the cell.
(B) The pluripotent stem cell according to (A), which can be obtained without inducing reprogramming or dedifferentiation.
(C) The pluripotent stem cell according to (A), which does not form a tumor for at least half a year when transplanted to the testis.
(D) The pluripotent stem cell of (A), which does not show infinite proliferation like ES cells and iPS cells.
(E) A pluripotent stem cell derived from a umbilical cord or adipose tissue of a living body, which survives when cells of the umbilical cord or adipose tissue of the living body are treated with a protease, and is resistant to protease.
Muse cells that are pluripotent stem cells derived from the umbilical cord or adipose tissue of the present invention can be performed using cell surface markers that are highly expressed on the surface of Muse cells. For example, expression of SSEA-3 is used as an index. It can be isolated. The pluripotent stem cell of the present invention is sometimes referred to as an SSEA-3-positive Muse cell. Furthermore, Muse cells express CD105, which is a mesenchymal marker, are positive for SSEA-3, and positive for CD105. Therefore, it can be isolated using SSEA-3 expression as an indicator. Moreover, the expression of both SSEA-3 and CD105 can be isolated using as an index. By using these cell surface markers, the pluripotent stem cell of the present invention can be isolated as a single cell, and the isolated single cell can be grown by culture. In addition, this invention shall also include the pluripotent stem cell which can be isolated from the biological tissue of mammals other than a human by the marker equivalent to SSEA-3.
On the other hand, Muse cells are negative for NG2, CD34, vWF (von Willebrand factor), c-kit (CD117), CD146, CD271 (NGFR). Furthermore, Sox10, Snai1, Slug, Tyrp1, and Dct are negative.
Whether surface antigens such as NG2, CD34, vWF, CD117, CD146, and CD271 are negative or weakly expressed is an antibody reaction against these antigens, and cells using antibodies labeled with chromogenic enzymes, fluorescent compounds, etc. Can be determined by measuring with a microscope or the like. For example, cells can be immunostained using these antibodies to determine the presence or absence of surface antigens, or can be determined using magnetic beads to which the antibodies are bound. It can also be determined whether there is a surface antigen using a FACS or flow cytometer. As the flow cytometer, for example, FACSAria (manufactured by Becton Dickinson), FACS vantage (manufactured by Becton Dickinson), FACS Calibur (manufactured by Becton Dickinson), MACS (magnetic cell separation method) and the like can be used.
In addition, expression of transcription factors such as Sox10, Snai1, Slug, Tyrp1, and Dct can be examined by a technique such as RT-PCR.
When these surface antigens are negative, when analyzed using FACS as described above, it means that they are not sorted as positive cells, or expression is not observed when expression is examined by RT-PCR, Even if it is expressed to such an extent that it cannot be detected by these techniques, it is considered negative in the present invention. In addition, measurement is performed simultaneously with cells such as hematopoietic stem cells that are known to be positive for the above-mentioned marker, and compared to these positive cells, it is hardly detected or may be negative when the expression level is significantly low. .
The cells of the present invention can be isolated based on the antigenic properties of these cell surfaces.
As described above, Muse cells can be isolated using SSEA-3 positivity as an indicator. For example, 40 to 70% of cells isolated from HADSC using SSEA-3 positivity as an index are Muse cells that can form Muse-derived embryoid body-like cell clusters (M-cluster). Furthermore, although it can be isolated using the co-expression of SSEA-3 and CD105 as an index, NG2, CD34, vWF (von Willebrand factor), c-kit (CD117), CD146, CD271 (NGFR), Sox10, At least one of 11 markers selected from the group consisting of Snai1, Slug, Tyrp1 and Dct, for example 2, 3, 4, 5, 6, 7, 8, 9, Non-expression of 10 or 11 markers can be isolated using as an indicator. For example, CD117 and CD146 non-expression can be isolated as an index, and CD117, CD146, NG2, CD34, vWF and CD271 non-expression can be isolated as an index. The non-expression of the marker can be isolated using as an indicator.
In the case of isolation using a surface marker, it is possible to directly isolate one or a plurality of pluripotent stem cells of the present invention from the umbilical cord or adipose tissue of a living body without undergoing culture or the like.
In addition to the above markers, the pluripotent stem cell or pluripotent cell fraction of the present invention is also characterized by high expression of other specific factors.
Muse cells, which are the pluripotent stem cells of the present invention, are obtained from the umbilical cord or adipose tissue of a living body, and further, the Muse cell-derived embryoid body (EB) -like cell mass is obtained by culturing the Muse cells. By comparing the factors expressed in Muse cells, mesenchymal cells that are the original population of Muse cells, Muse-derived embryoid body-like cell clusters, and human ES cells, factors that are highly expressed in Muse cells Recognize. Here, factors include gene transcripts, proteins, lipids, and sugars.
In the Muse cell of the present invention, the following 18 factors are highly expressed.
(I) SSEA-3
(Ii) v-fos FBJ murine osteosarcoma virtual oncogene homolog
(Iii) solid carrier family 16, member 6 (monocarboxylic acid transporter 7)
(Iv) tyrosinase-related protein 1
(V) Calcium channel, voltage-dependent, P / Q type, alpha 1A subunit
(Vi) chromosome 16 open reading frame 81
(Vii) chitinase 3-like 1 (cartile glycoprotein-39)
(Viii) protease, serine, 35
(Ix) kynureninease (L-kynurenine hydrolase)
(X) solid carrier family 16, member 6 (monocarboxylic acid transporter 7)
(Xi) apolipoprotein E
(Xii) synaptotagmin-like 5
(Xiii) chitinase 3-like 1 (cartile glycoprotein-39)
(Xiv) ATP-binding cassette, sub-family A (ABC1), member 13
(Xv) angiopoietin-like 4
(Xvi) prostaglandin-endoperoxide synthase 2 (prostaglandin G / H synthase and cyclooxygenase)
(Xvii) stanniocalcin 1
(Xviii) coiled-coil domain containing 102B
The pluripotent stem cell or pluripotent stem cell fraction of the present invention comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve of the above factors. It is characterized by the high expression of 13, 14, 15, 16, 17 or 18, and can be isolated using as an index the high expression of at least two factors.
Moreover, in the following 20 factors, the ratio of the expression level of the Muse cell of the present invention to the human ES cell is high.
(A) matrix metallopeptidase 1 (interstitial collagenase)
(B) epiregulin
(C) chitinase 3-like 1 (cartile glycoprotein-39)
(D) Transscribed locus
(E) chitinase 3-like 1 (cartile glycoprotein-39)
(F) serlycin
(G) MRNA full length insert cDNA clone EUROIMAGE 1913076
(H) Ras and Rab interactor 2
(I) lumican
(J) CLCA family member 2, Chloride channel regulator
(K) interleukin 8
(L) Similar to LOC166075
(M) dermatopontin
(N) EGF, latrophilin and seven transmembrane domain containing 1
(O) Insulin-like growth factor binding protein 1
(P) solid carrier family 16, member 4 (monocarboxic acid transporter 5)
(Q) serlycin
(R) gremlin 2, cysteine knot superfamily, homolog (Xenopus laevis)
(S) insulin-like growth factor binding protein 5
(T) sulfide quinone reductase-like (yeast)
The pluripotent stem cell or pluripotent stem cell fraction of the present invention comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve of the above factors. It is characterized by the high expression of 13, 14, 15, 16, 17, 18, 19 or 20, and can be isolated using as an index the high expression of at least two factors.
Furthermore, in the pluripotent stem cell or pluripotent stem cell fraction of the present invention, at least two of the above factors (i) to (xviii) and at least two of the above factors (a) to (t) are simultaneously highly expressed. It can be isolated using as an indicator that these genes are highly expressed.
Furthermore, the pluripotent stem cell or pluripotent stem cell fraction of the present invention expresses factors of the odorant receptor (olfactory receptor) group and the chemokine receptor group other than the pluripotency marker. That is, it is characterized by being positive for a specific odorant receptor or chemokine receptor.
Examples of the odorant receptor expressed in the pluripotent stem cell or pluripotent stem cell fraction of the present invention include the following 22 receptors.
olfactory receptor, family 8, subfamily G, member 2 (OR8G2);
olfactory receptor, family 7, subfamily G, member 3 (OR7G3);
olfactory receptor, family 4, subfamily D, member 5 (OR4D5);
olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);
olfactory receptor, family 10, subfamily H, member 4 (OR10H4);
olfactory receptor, family 10, subfamily T, member 2 (OR10T2);
olfactory receptor, family 2, subfamily M, member 2 (OR2M2);
olfactory receptor, family 2, subfamily T, member 5 (OR2T5);
olfactory receptor, family 7, subfamily D, member 4 (OR7D4);
olfactory receptor, family 1, subfamily L, member 3 (OR1L3);
olfactory receptor, family 4, subfamily N, member 4 (OR4N4);
olfactory receptor, family 2, subfamily A, member 7 (OR2A7);
guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type (GNAL);
olfactory receptor, family 6, subfamily A, member 2 (OR6A2);
olfactory receptor, family 2, subfamily B, member 6 (OR2B6);
olfactory receptor, family 2, subfamily C, member 1 (OR2C1);
olfactory receptor, family 52, subfamily A, member 1 (OR52A1);
olfactory receptor, family 10, subfamily H, member 3 (OR10H3);
olfactory receptor, family 10, subfamily H, member 2 (OR10H2);
olfactory receptor, family 51, subfamily E, member 2 (OR51E2);
olfactory receptor, family 5, subfamily P, member 2 (OR5P2); and
olfactory receptor, family 10, subfamily P, member 1 (OR10P1)
Examples of the chemokine receptor expressed in the pluripotent stem cell or pluripotent stem cell fraction of the present invention include the following five receptors.
chemokine (C-C motif) receptor 5 (CCR5);
chemokine (C-X-C motif) receptor 4 (CXCR4);
chemokine (C-C motif) receptor 1 (CCR1);
Duffy blood group, chemokine receptor (DARC); and
chemokine (C-X-C motif) receptor 7 (CXCR7)
The pluripotent stem cell or pluripotent stem cell fraction of the present invention expresses at least one of the olfactory receptors, or expresses at least one of the chemokine receptors.
Muse cells, which are the pluripotent stem cells of the present invention, migrate to damaged tissues by the action of migration factors that bind to these odorant receptors and chemokine receptors and receptors, and differentiate according to the location. . For example, when the liver, skin, spinal cord, or muscle is damaged, the specific migratory factor and the odorant receptor expressed on the cell surface migrate to and engraft each tissue, the liver (endoderm), Differentiated into skin (ectodermal), spinal cord (ectodermal), muscle (mesoderm) cells, and tissue can be regenerated.
Further, in the Muse cell fraction rich in Muse cells that are pluripotent stem cells of the present invention, increased expression of Rex1, Sox2, KLF-4, c-Myc, DPPA2, ERAS, GRB7, SPAG9, TDGF1, etc. The expression of DAZL, DDX4, DPPA4, Stella, Hoxb1, PRDM1, and SPRY2 is increased in the cell mass of Muse cells.
Moreover, in the pluripotent stem cell or pluripotent stem cell fraction of the present invention, the expression of CD34 and CD117, which are hematopoietic stem cell markers, is not observed or the expression is extremely low.
Furthermore, the pluripotent stem cells of the present invention can be increased in content by applying cell stress to cells of the umbilical cord or adipose tissue of the living body and collecting the surviving cells. Here, cell stress refers to external stress, including protease treatment, culture under low oxygen conditions, culture under low phosphate conditions, culture under serum starvation conditions, culture under sugar starvation conditions, under radiation exposure Exposure to stress by culturing in the presence of heat shock, culturing in the presence of toxic substances, culturing in the presence of harmful substances, culturing in the presence of active oxygen, culturing under mechanical stimulation, culturing under pressure treatment, etc. That means. Among these, protease treatment, that is, culture in the presence of protease is preferable. The protease is not limited, and serine proteases such as trypsin and chymotrypsin, aspartic proteases such as pepsin, cysteine proteases such as papain and chymopapain, metalloproteases such as thermolysin, glutamate protease, N-terminal threonine protease, and the like can be used. The concentration at which protease is added to the culture is not limited, and it may be used at a concentration generally used when peeling adherent cells cultured in a petri dish or the like. The Muse cell which is the pluripotent stem cell of the present invention can be said to be a stem cell resistant to the above external stress, for example, a cell resistant to trypsin.
Most of the cells subjected to various stresses described above are killed, and the surviving cells include Muse cells that are the pluripotent stem cells of the present invention. It is necessary to remove dead cells after applying stress to the cells. When protease is used, these dead cells are degraded by the action of the protease.
Further, after applying stress to the cells, the cells that have been subjected to a physical impact on the cells and have become fragile may be removed. The physical impact can be applied, for example, by vigorous pipetting, vigorous stirring, vortexing or the like.
Muse cells that are pluripotent stem cells of the present invention are obtained by subjecting cells to cell stress and subjecting them to physical impact as necessary, and then centrifuging the cells to obtain and collect the surviving cells as pellets. The content of can be increased. Moreover, the pluripotent stem cell or pluripotent cell fraction of the present invention can be further isolated from the cells thus obtained using the following surface marker as an index.
As an example, a method for treating these cells with trypsin will be described. The trypsin concentration at this time is not limited. For example, in the normal culture of adherent cells, the trypsin concentration may be used within the concentration range used when peeling the adherent culture adhered to the culture vessel, and is 0.1 to 1%, preferably 0. 1 to 0.5% is exemplified. For example, cells derived from a umbilical cord or adipose tissue of a living body containing 100,000 to 500,000 cells can be exposed to external stress by incubating in 5 ml of a trypsin solution having the above concentration. The trypsin treatment time is about 5 to 24 hours, preferably about 5 to 20 hours. In the present invention, trypsin treatment for 8 hours or more, for example, treatment for 8 hours or 16 hours is referred to as long-time trypsin treatment.
After the trypsin treatment, it is desirable to give a physical impact by pipetting, stirring, vortexing or the like as described above. This is to destroy and remove dead or dying cells.
In suspension culture after trypsin treatment, in order to prevent aggregation between cells, it is desirable to incubate in a gel such as methylcellulose gel. In order to prevent the cells from adhering to the culture container and maintain the floating state, it is desirable to coat the container with Poly (2-hydroxyethyl methacrylate) or the like.
When cells exposed to external stress are collected by centrifugation and subjected to suspension culture, cell clusters (cell clusters) are formed. The size of this cell mass is about 25 μm to 150 μm in diameter. The pluripotent stem cells (Muse cells) of the present invention are contained in a concentrated state in the cell population that survived this external stress. This cell population is referred to as the Muse enriched population. The abundance of Muse cells in the rich Muse cell fraction varies depending on the stress treatment method.
Thus, the survival of the pluripotent stem cell or pluripotent stem cell fraction of the present invention even after stress is applied to the fact that the pluripotent stem cell or pluripotent stem cell fraction of the present invention is stress resistant. Show.
The medium and culture conditions used for culturing cells derived from the umbilical cord or adipose tissue of a living body may be the same as those used for normal animal cell culture. Further, a known stem cell culture medium may be used. To the medium, serum such as fetal bovine serum, antibiotics such as penicillin and streptomycin, and various physiologically active substances may be appropriately added.
Furthermore, the present invention also includes pluripotent stem cells that are derived or derived cells of pluripotent stem cells that can be obtained directly from the umbilical cord or adipose tissue of the living body of the present invention. Derived cells or induced cells refer to cells or cell groups obtained by culturing the pluripotent stem cells, or cells obtained by performing artificial induction operations such as introduction of foreign genes into the pluripotent stem cells, and progeny Includes cells. The iPS cells reported at the time of the present invention are said to be cells induced by pluripotent stem cells as a result of reprogramming by introducing a foreign gene into a differentiated cell of biological tissue such as skin fibroblasts. The cells that can be directly obtained from the umbilical cord or adipose tissue of the present invention and have already been subjected to an artificial induction operation such as introduction of a foreign gene into cells having the properties as pluripotent stem cells are iPS cells. Distinguished from
Embryoid body-like (Embody) -like cell clusters can be obtained by suspension culture of the pluripotent stem cells of the present invention, and the present invention relates to these embryoid body-like cell clusters and embryoid body-like cells. The cells contained in the mass are also included. The embryoid body is formed as a cell mass by suspension culture of the pluripotent stem cells of the present invention. At this time, in the present invention, an embryoid body obtained by culturing the pluripotent stem cell of the present invention may be referred to as a Muse cell-derived embryoid body-like cell mass (referred to as an M cluster). Sometimes). As a suspension culture method for forming embryoid body-like cell clusters, culture using a medium containing a water-soluble polymer such as methylcellulose (Nakahata, T. et al., Blood 60, 352-361 (1982)). And hanging drop culture (Keller, J. Physiol. (Lond) 168: 131-139, 1998). The present invention also includes an embryoid body-like cell cluster obtained by self-renewal from the embryoid body-like cell cluster, cells contained in the embryoid body-like cell cluster, and pluripotent stem cells. Here, self-renewal refers to culturing cells contained in an embryoid body-like cell cluster to form an embryoid body-like cell cluster again. The self-renewal may be repeated one to several times. The present invention also includes cells and tissues differentiated from any of the embryoid body-like cell clusters and cells contained in the embryoid body-like cell cluster.
The present invention includes not only Muse cells but also cell populations enriched with Muse cells, cell populations with expanded Muse cells, and cell populations with differentiated Muse cells, and further include Muse cells and cells derived from Muse cells. Includes research kits, cell chips, and therapeutic devices.
The pluripotent stem cell of the present invention has pluripotency, and can differentiate into any tissue. The pluripotent stem cell or pluripotent cell fraction can be used for regenerative medicine and the like. For example, it can be used for regeneration of various tissues and various organs. Specific examples include skin, cerebral spinal cord, liver, and muscle. By administering the pluripotent stem cell or pluripotent stem cell fraction of the present invention directly or nearby to a damaged or damaged tissue, organ, etc., the pluripotent stem cell enters the tissue, organ, It can differentiate into cells peculiar to the tissue and contribute to regeneration and reconstruction of the tissue and organs. Further, systemic administration may be performed by intravenous administration or the like. In this case, for example, the pluripotent stem cell is directed to a damaged tissue or organ by homing or the like, reaches / invades, and then differentiates into a cell of the tissue or organ to regenerate the tissue or organ. , Can contribute to reconstruction.
Administration can be performed, for example, by parenteral or oral administration such as subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, or intrauterine injection into an embryo. Moreover, local administration or systemic administration may be sufficient. Local administration can be performed using a catheter, for example. The dose can be appropriately determined depending on the type and size of the organ or tissue to be regenerated.
The organ to be regenerated is not limited, but bone marrow, spinal cord, blood, spleen, liver, lung, intestinal tract, eye, brain, immune system, circulatory system, bone, connective tissue, muscle, heart, blood vessel, pancreas, central nervous system , Peripheral nervous system, kidney, bladder, skin, epithelial appendages, breast-mammary gland, adipose tissue, cornea, and mucous membranes including mouth, esophagus, vagina, anus, and the like. Diseases to be treated include cancer, cardiovascular disease, metabolic disease, liver disease, diabetes, hepatitis, hemophilia, blood system disease, degenerative or traumatic neurological diseases such as spinal cord injury, autoimmune disease, genetic Examples include defects, connective tissue diseases, anemia, infections, transplant rejection, ischemia, inflammation, and skin and muscle damage.
The cells may be administered with a pharmaceutically acceptable substrate. The substrate is made of, for example, collagen, a highly biocompatible substance, or a biodegradable substance, and may be in the form of particles, plates, cylinders, containers, and the like. What is necessary is just to couple | bond with a material or to accommodate in this base material and to administer.
Alternatively, the pluripotent stem cell of the present invention may be induced to differentiate in vitro, a tissue may be constructed using the differentiated cell, and the differentiated cell or the tissue may be transplanted. Since the pluripotent stem cell of the present invention does not become a tumor, even if the transplanted differentiated cell or the tissue contains the pluripotent stem cell of the present invention undifferentiated, the possibility of canceration is low and safe. is there. In these regenerative medicine, in order to avoid rejection of the transplanted cells or tissues by the recipient, mesoderm tissue or mesenchymal tissue is collected from a patient who is going to receive regenerative medicine, and the present invention is obtained from the tissue. It is desirable to isolate and utilize pluripotent stem cells or pluripotent cell fractions. Furthermore, the pluripotent stem cell or pluripotent stem cell fraction of the present invention can be used for the treatment of diseases caused by tissue degeneration or dysfunction. In this case, for example, the pluripotent stem cell or pluripotent stem cell fraction of the present invention may be concentrated ex vivo, proliferated, or differentiated and returned to the body. What is necessary is just to differentiate into a cell and to transplant this cell to the tissue which is going to be treated. In situ cell therapy can also be performed by cell transplantation. In this case, examples of target cells include liver cells, nervous cells such as nerve cells and glial cells, muscle cells such as skin cells and skeletal muscle cells, and the pluripotent stem cells of the present invention are differentiated into these cells. Can be transplanted and treated in situ. By this treatment, for example, Parkinson's disease, cerebral infarction, spinal cord injury, muscle degenerative disease and the like can be treated. Since the pluripotent stem cell of the present invention does not become a tumor, even if it is used for such treatment, the possibility of canceration is low and it is safe.
In addition, blood and blood components can be formed ex vivo and in vitro by differentiating the pluripotent stem cells of the present invention to form blood and blood components. As blood components, red blood cells, white blood cells, and platelets Etc. The blood and blood components formed in this way can be used for autologous blood transfusion or transfusion.
As described above, when the pluripotent stem cell or pluripotent stem cell fraction of the present invention is used for treatment, it may be differentiated ex vivo, in vivo, or in vitro. The pluripotent stem cells of the present invention include, for example, osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, myocardium, eyes, endothelium, epithelium, liver, pancreas, hematopoiesis, glia Differentiate into neurons, oligodendrocytes, etc. Differentiation of the pluripotent stem cell of the present invention can be achieved by culturing in the presence of a differentiation factor. Differentiation factors include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), dimethyl sulfoxide (DMSO) and isoproterenol; or fibroblast growth factor 4 (FGF4), hepatocyte growth factor ( HGF) and the like. The present invention also includes cells differentiated from the pluripotent stem cells of the present invention.
When the pluripotent stem cell of the present invention is used for treatment, a gene encoding a proteinaceous anticancer substance or physiologically active substance may be introduced. Thereby, the pluripotent stem cell of this invention also has the delivery function of a therapeutic agent. An example of such a substance is an anti-angiogenic drug.
The present invention relates to a cell transplantation treatment material comprising a Muse cell, an embryoid body-like cell mass made of Muse cells, and a cell or tissue / organ obtained by differentiation from the Muse cell or the embryoid body-like cell mass Alternatively, it includes a composition for cell transplantation treatment, or a material for regenerative medicine or a composition for regenerative medicine. The composition is pharmaceutically acceptable in addition to Muse cells, embryoid body-like cell masses made from Muse cells, or cells or tissues / organs obtained by differentiation from Muse cells or the embryoid body-like cell masses. Including buffer solution and diluent.
Furthermore, the Muse cell of the present invention can be collected from the umbilical cord or adipose tissue of the patient, the Muse cell can be isolated, and used for various diagnoses using the Muse cell. For example, it is possible to collect a patient gene from Muse cells, obtain genetic information, and perform an accurate diagnosis reflecting the information. For example, by differentiating Muse cells derived from a subject's cells, cells of each tissue / organ having the same genetic background as the subject can be obtained. And diagnosis of side effects, etc., it is possible to make an appropriate diagnosis according to the characteristics of each subject. That is, Muse cells, embryoid body-like cell masses made from Muse cells, and cells or tissues / organs obtained by differentiation from Muse cells or the embryoid body-like cell masses can be used as diagnostic materials, For example, the present invention diagnoses a disease or the like of a subject using a tissue or organ having the same genetic background as the subject obtained by isolating a Muse cell from the subject and differentiating the Muse cell or the Muse cell. To include a method.
In addition, because somatic cells can be obtained in large quantities by differentiating Muse cells, basic research such as elucidation of disease mechanisms, therapeutic drug development, screening of drug effects and toxicity, drug evaluation, etc. can be performed. . That is, Muse cells, embryoid body-like cell masses made from Muse cells, and cells or tissues / organs obtained by differentiation from Muse cells or the embryoid body-like cell masses are used as materials for drug evaluation and drug screening. be able to. For example, the present invention includes a method for screening and evaluating drugs by differentiating and proliferating Muse cells, obtaining somatic cells, administering a candidate drug to the somatic cells, and examining the response of the somatic cells. .
In addition, by constructing a Muse cell bank in which various (for example, various HLA types) Muse cells are made into a library, a system capable of providing cells in the above Muse cell utilization scene as needed can be realized. In addition to the above-mentioned purposes, it is possible to provide (without) rejection-free cells for urgent cell transplantation treatment. That is, the present invention includes a method for producing a library of Muse cells having different gene characteristics, that is, a Muse cell bank, by isolating and collecting Muse cells having various gene characteristics. In addition to Muse cells, embryoid body-like cell masses made from Muse cells, and cells or tissues / organs obtained by differentiation from Muse cells or the embryoid body-like cell masses are obtained in libraries or banks. Can also be built. In the present invention, an embryoid body-like cell mass made from these Muse cells, and cells or tissues / organs obtained by differentiating from the Muse cells and the embryoid body-like cell mass are also used as libraries and banks. It is called a cell library or cell bank. The present invention includes the cell library or cell bank thus prepared. The cell library or cell bank is composed of, for example, a container such as a plurality of tubes in which cells having different genetic characteristics are stored, and the cells may be frozen. For example, when it becomes necessary to transplant or regenerate a tissue or organ in a subject, a cell suitable for the genetic background or the like is selected from the cell library or cell bank. Can be used for transplantation and regenerative treatment.
The present invention provides administration of a therapeutically effective amount of the pluripotent stem cells of the present invention, the cell fraction, derivative cells derived from the cells, or induced cells to a patient in need of treatment for the treatment of a disease. A therapeutic method comprising: Here, the effective amount can be specified by, for example, the number of cells to be administered, and can be appropriately determined depending on the type and severity of the disease. In the above treatment method, the pluripotent stem cells of the present invention do not form teratomas (teratomas), and thus teratomas are not formed in patients. In addition, when administering Muse cells derived from autologous cells, it is not necessary for the patient to lose bone marrow function by treatment such as irradiation or chemotherapy, but when Muse cells that are not autologous cells are used, the above treatment is performed. Good.
Furthermore, Muse cells can be a source of iPS cells (induced pluripotent stem cells). The production efficiency of iPS cells using Muse cells as a source is much higher (at least 25 times or more than when using other cells (for example, skin fibroblasts not fractionated using SSEA-3 expression as an index)). )high.
An iPS cell can be produced by introducing a specific gene into a Muse cell or changing a cytoplasm by introducing a specific compound. The change in cytoplasm includes reprogramming, infinite growth ability and tumorigenicity, and any currently known method or any method established in the future can be used.
For example, a gene can be introduced into Muse cells as described in Japanese Patent No. 4182742, or iPS cells can be established from Muse cells as described in FIG. In addition to the method shown in FIG. 27, it can be said that iPS cells can be established by introducing a chemical substance, a foreign gene or a foreign protein. Establishment of iPS cells from Muse cells can be performed, for example, by the method described in Examples described later.
Thus, the iPS cell obtained from the Muse cell may be referred to as a Muse-derived iPS cell (Muse-iPSC), and the present invention also includes the Muse-derived iPS cell. Muse-derived iPS cells can be referred to as Muse cell-derived pluripotent stem cells having infinite proliferative properties.
EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to the following Example.

Method and material for isolation of pluripotent stem cells from adipose tissue Cell materials As mesenchymal cells derived from human adipose tissue, two commercially available cultured cells and mesenchymal cells established from primary culture of human subcutaneous adipose tissue were used. Commercially available cells are Lonza's Human Adipose-Derived Stem Cell (HADSC: 3 lots of Lot. 7F4308, 7F4089, and 7F4205), and Dulbecco's modified Eagle EM medium-H liquid (1 ×); Invitrogen Cat. 1965-092] to 15% (vol / vol) FBS [ES cell grade; HyClone], 0.1 mg / mL Kanamicin sulfate [liquid (100 ×); Invitrogen Cat. 15160-054] was added and cultured at 37 ° C.
Establishing adipose tissue-derived mesenchymal cells from subcutaneous adipose tissue using the adipose tissue provided with the consent of the patient from Tohoku University Hospital, Japan National University Corporation, using the method of Estes BT et al. BT., Et al. Nat Proto. 2010 Jul: 5 (7): 1294-1311). First, aseptically collected subcutaneous adipose tissue was finely cut. In addition, Phosphate-Buffered Saline [PBS, magnesium chloride, calcium chloride-free] 1 mg / mL collagenase Type I [100 mg; Worthington Biochemical Cat. LS004194], 1% (wt / vol) BSA [nacalai Cat. 15111-45] was added to make an enzyme solution. The cut adipose tissue and an equal amount of enzyme solution were mixed in a 50 mL centrifuge tube and reacted at 37 ° C. for 2 hours to digest the extracellular matrix. After 2 hours, centrifugation was performed at 300 g for 5 minutes to suck the liquid phase between the precipitate and the suspended matter, and the undigested extracellular substrate was removed from the precipitate and the suspended matter using a 100 μm cell strainer. Thereafter, DMEM [15% (vol / vol) FBS, 0.1 mg / mL kanamycin sulfate] was added, centrifuged at 300 g for 5 minutes, the supernatant was removed, the precipitate was suspended in the medium and seeded. The medium was changed 24 hours after seeding. Both commercially available cells and established cells were changed medium every two days, and were passaged 1: 2 when 90% confluent was reached. Mouse ES cells (TT2 cells) were prepared by using 15% (vol / vol) FBS [ES cell grade; HyClone], 0.1 mg / mL kanamycin sulfate [liquid (100 ×); Invitrogen Cat. 15160-054], 0.1 mM MEM Non-Essential Amino Acids Solution [NEAA, liquid 10 mM (100 ×); Invitrogen Cat. 11140-050], sodium pyruvic acid solution 1 mM [SP, liquid 100 mM (100 ×); Invitrogen Cat. 11360-070], 1000 U / mL Leukemia Inhibitory Factor [LIF, ESGRO], medium supplemented with 100 μM 2-mercaptoethanol, and mouse embryo fibroblasts (MEF) treated with mitomycin C were used as feeder cells. The MEF prepared from a 12.5 day-old C57BL / 6 mouse fetus was used.
2. FACS
1. FACS analysis was performed on cultured cells commercially available as human adipose tissue-derived mesenchymal cells and mesenchymal cells established from primary cultures of human subcutaneous adipose tissue. Cells used for FACS were used when they reached 100% confluence. First, the medium was removed from the dish, 3 mL of trypsin-EDTA was added, and the whole cell was spread, followed by incubation at 37 ° C. for 10 minutes. After confirming that the cells were detached from the dish, 1 mL of FBS was added to form a single cell by pipetting, and the number of cells was collected and counted. FACS buffer [0.02M PBS, 0.5% (wt / Vol) BSA, 2 mM EDTA] was used to suspend cells to 1 million cells per 100 μL, and incubated with 10% inactivated human serum for 20 minutes (on ice) to perform FcR blocking. Subsequently, using anti-SSEA-3 antibody (1:50, Millipore) as the primary antibody and FITC-labeled anti-rat IgM antibody (1: 100, Jackson Immunoresearch) as the secondary antibody, each was incubated for 1 hour (on ice) and stained. It was. After staining, analysis and sorting were performed using Special Order Research Products FACS Aria II (Becton Dickinson). The results are shown in FIG. FIG. 1 shows the results of mesenchymal cells established from primary cultures of human subcutaneous adipose tissue. At this time, the peaks of the secondary antibody-only sample (FIG. 1B) and the sample stained with SSEA-3 (FIG. 1C) were adjusted so as to be in the same position as the FITC peak of the unstained sample (FIG. 1A). After that, a cell population having almost the same FITC intensity as that of the unstained sample was obtained as a non-Muse cell (non-Muse) (P6 area in FIG. 1C), and a cell population having a FITC intensity higher than that of the secondary antibody-only sample was designated as a Muse cell (FIG. (P3 area).
3. Single cell suspension culture 2. From the cells commercially available as human adipose tissue-derived mesenchymal cells and mesenchymal cells established from primary cultures of human subcutaneous adipose tissue. Cells sorted using FACS by the above method were stained with trypan blue, and the number of viable cells was counted. Limit dilution was performed so that one cell per well was added, and 15% (vol / vol) FBS was added to α MEM using a poly-HEMA-coated 96-well dish, which was suspended and seeded. The suspension culture was performed for 7 to 10 days, and the formation rate of Muse-derived embryoid body-like cell mass (M-cluster) was measured.
4). 2. Differentiation of Muse-derived embryoid body-like cell mass (M-cluster) in vitro. Muse-derived embryoid body-like cell mass (M-cluster) cultured for 7 to 10 days in a single cell suspension culture of 0.1% gelatin and a culture dish coated in a 24 well dish, α MEM 300 μL [15% ( vol / vol) FBS] was added and transferred to a cell cluster per well. After several hours, 200 μL of medium was added. The cells were cultured for 1 to 2 weeks and analyzed by immunocytochemistry or RT-PCR.
5. 2. Immunocytochemistry The Muse-derived embryoid body-like cell mass (M-cluster) obtained in Step 3 was fixed with 4% (vol / vol) paraformaldehyde / 0.01 M PBS, and the O.D. C. T.A. The sample was embedded in a compound, and a frozen section was prepared and used for immunostaining. In the case of a Muse-derived embryoid body-like cell mass (M-cluster) differentiated on a gelatin-coated cover glass, the cover glass and the cells are fixed with 4% (vol / vol) paraformaldehyde / 0.01 M PBS, and immunized. Used for staining. As primary antibodies, SSEA-3 [1:50, Millipore], Nanog (1: 100, Millipore), Oct3 / 4 [1: 100, Santa Cruz], Sox2 (1: 1000, Millipore), PAR4 (1: 100) , Santa Cruz), Smooth Muscle Actin (SMA, 1: 100, Lab Vision), Neurofilament-M (1: 100, Millipore), Cytokeratin 7 (CK7, 1: 100, Millipore), and using these primary antibodies. FITC-, Alexa-488- or Alexa-568-labeled anti-rabbit IgG, anti-mouse IgG, anti-mouse IgM antibody (1: 500, Molle Was detected in the ular Probes).
6). RT-PCR
4). Total RNA was extracted from Muse-derived embryoid body-like cell clusters (M-cluster) differentiated in vitro by the method described in (1) above using Nucleo Spin RNA XS (Macherey-Nagel). From the extracted total RNA, cDNA was obtained by reverse transcription using Super Script VILO cDNA Synthesis Kit (Invitrogen). For the PCR, Ex Taq DNA polymerase (TaKaRa Bio, Inc.) was used, α-fetoprotein (AFP, endoderm marker), GATA6 (endoderm marker), micro tube-associated protein-2 (MAP-2, MAP-2, Expression of ectoderm marker) and Nkx2.5 (mesoderm marker) was confirmed. The various primer sequences used are shown in FIG. As controls, human whole embryo, human fetus river, human adult brain, and human adult river were used, and the details are shown below.
7. 1. Transplant to immunodeficient mouse testis SSEA-3 positive cells sorted using FACS by the above method were stained with trypan blue, and the number of viable cells was counted. After the measurement, the suspension was suspended in PBS so as to be 1.5 × 10 ⁵ cells / μL, and 1 × 10 ⁵ cells were transplanted into one testis of an 8-week-old CB17 / Icr-Prkdc scid / CrlCrlj (SCID) male. Mouse ES cells were administered at 1 × 10 ⁶ cells as a positive control, and PBS was administered as a negative control. The SSEA-3 positive cell group was fixed by perfusion 4 months after transplantation, and the mouse ES cell and PBS groups were fixed by perfusion 8 weeks after transplantation.
8). 1. Study of self-renewal ability (self-replicating ability) 3. SSEA-3 positive cells isolated from mesenchymal cells established from primary cultures of human subcutaneous adipose tissue sorted using FACS by the method of The first generation Muse-derived embryoid body-like cell mass (M-cluster) obtained as a result of the single cell suspension culture described in 1) is added to 0.1% gelatin-coated dish and αMEM 300 μL [15% (vol / vol) FBS ] Is used to grow cells by adhesion culture. Once the cells increased, trypsin-EDTA was added and detached, and single cell suspension cells were again formed to form a second generation Muse-derived embryoid body-like cell mass (M-cluster). An overview of the method is shown in FIG.
Results In this example, two types of sources were used, cells commercially available as human adipose tissue-derived mesenchymal cells and mesenchymal cells established by the present inventors from human subcutaneous adipose tissue. In the method of this example, mesenchymal cells obtained from human subcutaneous adipose tissue (FIG. 3A) having a volume of 15.4 cm ³ were cultured for 16 days, and 1960 was obtained as human adipose tissue-derived mesenchymal cells (FIG. 3B). Ten million established cells could be obtained.
As a result of FACS analysis, it was confirmed that Muse cells capable of being isolated using SSEA-3 as a marker were inherent in each cell population. In each cell population, it was confirmed that 4.3 ± 0.36% of Muse cells were inherent in commercially available cells and 7.3 ± 0.37% of Muse cells were established in cells established from primary culture ( FIG. 4). FIG. 4A shows the results of commercially available HADSC, and FIG. 4B shows the expression of SSEA-3 in HADSC established from human subcutaneous adipose tissue.
Muse cells form a Muse-derived embryoid body-like cell mass (M-cluster) resembling the embryoid body of human ES cells from one cell by suspension culture. Therefore, SSEA-3-positive Muse cells and SSEA-3-negative non-Muse cells were collected and subjected to single cell suspension culture. As a result, it was confirmed that a Muse-derived embryoid body-like cell mass (M-cluster) was formed from 1 cell only in Muse cells. It was not formed from non-Muse cells. 65 to 66% of SSEA-3 positive cells isolated from commercially available HADSCs and 44.2% of SSEA-3 positive cells isolated from HADSCs established from human subcutaneous adipose tissue formed clusters. FIG. 5 shows the morphology of the Muse-derived embryoid body-like cell cluster (M-cluster). FIG. 5A shows the morphology of a Muse-derived embryoid body-like cell mass (M-cluster) obtained from a commercially available HADSC, and FIG. 5B shows a Muse-derived embryoid body-like cell mass obtained from HADSC established from human subcutaneous adipose tissue. The form of (M-cluster) is shown.
In addition, the formed Muse-derived embryoid body-like cell mass (M-cluster) is cultured in vitro on a gelatin-coated dish, so that microtubule-associated protein-2 (MAP-2, ectoderm), GATA6 ( Endoderm), α-feto protein (AFP, endoderm), Nkxr2.5 (mesoderm) expression was confirmed by RT-PCR, and HADSC-derived Muse-derived embryoid body-like cell mass (M-cluster) Spontaneous differentiation into germinal cells was confirmed (FIG. 6). In FIG. 6, M-cluster I was obtained from differentiation of Muse-derived embryoid body-like cell mass (M-cluster) obtained from commercially available HADSC, and M-cluster II was obtained from HADSC established from human subcutaneous adipose tissue. The differentiation of Muse-derived embryoid body-like cell mass (M-cluster) is shown.
Furthermore, in the immunostaining using the antibodies of Neuro filament-M (NF, ectoderm nerve), Cytokeratin 7 (CK7, endoderm biliary system), and Smooth Muscle Actin (SMA, mesoderm smooth muscle), it is derived from HADSC-derived Muse. It was proved that the embryoid body-like cell cluster (M-cluster) differentiated into tridermal cells (FIG. 7). In FIG. 7, the blue reaction that looks like an ellipse indicates the nucleus of the DAPI-stained cell, the red fluorescence indicates the anti-NF antibody or anti-SMA antibody, and the green fluorescence indicates the anti-CK7 antibody.
The formed Muse-derived embryoid body-like cell mass (M-cluster) may also express pluripotent stem cell markers such as Nanog, Oct3 / 4, PAR4, Sox2, SSEA-3, alkaline phosphatase (ALP), etc. Muse cells isolated from HADSC were confirmed (Figure 8), and from these facts, they express pluripotency markers and can differentiate into trioderm cells, and thus have pluripotency. Proved. In FIG. 8, the DAPI-stained image in the left panel shows the nucleus and is stained blue, and the center panel is an immunostained image using antibodies against Nanog, Oct3 / 4, PAR4, Sox2 and SSEA-3 from the top. The localization of these markers is stained green. The right panel is an image obtained by superimposing a DAPI stained image and a stained image of each antibody. The ALP in the bottom panel shows a red positive reaction in the alkaline phosphatase stained image.
Furthermore, in cell transplantation into mouse testis, teratomas were formed in mouse ES cells 8 weeks after transplantation, whereas Muse cells did not form teratomas even 4 months after transplantation. It was demonstrated that HADSC-derived Muse cells are non-neoplastic (FIG. 9). In FIG. 9, A shows the result of control (Intact, no transplantation) and the result of transplantation of Muse cells (4 months), and B shows the result of transplanting mouse ES cells (8 weeks) and PBS. The results are shown (8 weeks).
Moreover, it was confirmed by the HE-stained image that mouse ES cells formed a teratoma containing a trioderm tissue (FIG. 10). In FIG. 10, I indicates the digestive tract (endoderm), II indicates the nervous system (ectodermal), and III indicates smooth muscle (mesoderm). On the other hand, in testis transplanted with Muse cells, no tumor was formed even with HE staining, and normal seminiferous tubules were confirmed (FIG. 11).
Method 8 When the self-renewal ability was performed by the method shown in FIG. 2, the formation rate of the first generation Muse-derived embryoid body-like cell mass (M-cluster) was 35.5%, and the second generation Muse-derived embryoid body The formation rate of the cell-like mass (M-cluster) was 29.7% (FIG. 12). This result indicates that the obtained Muse cells have a self-renewal ability.

Isolation of pluripotent stem cells from human umbilical cord ~ 3. Muse cells were isolated using SSEA-3 expression as an index in the same manner as described in 1.
More specific description will be given below. The umbilical cord obtained with the consent of the patient was used. After the umbilical cord was disinfected, only the mesenchymal tissues in the middle were removed except for blood vessels and surface epithelium, and they were cut into 5 mm squares. Mesenchymal cells were obtained by adherent culture of fine umbilical cord mesenchymal tissue. As in the case of adipose-derived mesenchymal cells, MUSE cells are collected as SSEA-3 positive cells by FACS, and embryoid body-like cell masses are made as single cell suspension cells. -Analysis was performed by PCR or immunostaining. FIG. 13 shows the morphology of mesenchymal cells obtained from the umbilical cord. FIG. 14 shows the expression of SSEA-3 in mesenchymal cells obtained from the umbilical cord. This result is the same as that of Example 1. This is a result of FACS analysis performed by the same method as described in 1. FIG. 15 shows the morphology of Muse cell-derived embryoid body-like cell clusters (M-cluster) formed from SSEA-3 positive cells (Muse cells) isolated from the umbilical cord. Further, FIG. Α-fetoprotein (AFP, endoderm marker), GATA6 (endoderm marker), micro tube-associated protein-2 (MAP-2, ectoderm) measured by RT-PCR in the same manner as described in 1. Marker), Nkx2.5 (mesoderm marker) expression.
It was confirmed that Muse cells, which are pluripotent stem cells, are inherent in human adipose tissue-derived mesenchymal cells. Muse cells express a pluripotent stem cell marker and have the ability to differentiate from one cell to a three germ layer cell, so human adipose tissue is very useful as a cell source in autologous cell transplantation therapy. . Considering actual medical care, it is easier to collect adipose tissue from the recipient than to collect bone marrow or skin, and the establishment method used in this study has a volume of 15. When cells were collected from 4 cm ³ of human adipose tissue and cultured for 16 days, 19.6 million cells were obtained as mesenchymal cells, and when the SSEA-3 positive rate was assumed to be 5%, 1960 obtained from adipose tissue It can be estimated that 980,000 cells out of 10,000 cells are Muse cells. It is very desirable in autologous cell transplantation treatment to secure the number of cells in a short culture period even from a relatively small amount of tissue. Muse cells isolated from human adipose tissue-derived mesenchymal cells are also cells derived from living bodies, unlike human ES cells and human iPS cells. Therefore, special operations such as gene transfer are not required to acquire pluripotency. In addition, we can fully expect the possibility of solving problems such as the effects of gene transfer and the risk of tumorigenesis, which are currently faced by other pluripotent stem cell research. The fact that adipose tissue-derived Muse cells also have no tumorigenicity is inherent in mesenchymal cells obtained from adult human adipose tissue, and human adipose tissue-derived mesenchymal cells are mainly This is also a convincing result because it is a cell source 10) for turning over adipose tissue and its vascular system. Already, the practicality and future potential of human adipose tissue has been widely recognized, and this research result that natural human pluripotent stem cells Muse cells exist among them is considered to have great significance in regenerative medicine.
The same can be said for Muse cells isolated from umbilical cord-derived mesenchymal cells.

According to the present invention, pluripotent stem cells are obtained from umbilical cord or adipose tissue without using germ cells or early embryos and without undergoing artificial induction operations such as introduction of foreign genes or introduction of specific compounds. Can do. Since no artificial manipulation such as introduction of a foreign gene is required, the pluripotent stem cell of the present invention can be efficiently produced, and can be used safely even when used for treatment. Furthermore, the pluripotent stem cells of the present invention can be used for regenerative medicine, treatment of dysfunctional tissues, and the like, and further can be used for studies of cell differentiation and tissue regeneration.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

1-10 primer

Claims

SSEA-3 positive and CD105 positive pluripotent stem cells having all of the following properties that can be isolated from living umbilical cord or adipose tissue using SSEA-3 expression as an indicator:
(I) low or no telomerase activity; (ii) capable of differentiating into cells of any germ layer of the three germ layers;
(Iii) no neoplastic growth; and (iv) self-replicating ability (self-renewal ability).
The pluripotent stem cell according to claim 1, which can be isolated directly from the umbilical cord or adipose tissue of a living body using the expression of SSEA-3 as an index.
The pluripotent stem cell according to claim 1 or 2, which can be isolated from human adipose-derived stem cells (HADSC).
The pluripotent stem cell according to any one of claims 1 to 3, which is proliferated by culture combining suspension culture and adhesion culture.
The pluripotent stem cell according to any one of claims 1 to 4, which is CD117 negative and CD146 negative.
The pluripotent stem cell according to any one of claims 1 to 4, which is CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative and CD271 negative.
The pluripotent stem cell according to any one of claims 1 to 4, which is CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snail negative, Slug negative, Tyrp1 negative and Dct negative. .
A cell population comprising the pluripotent stem cells according to any one of claims 1 to 7.
A method for isolating, culturing and proliferating the pluripotent stem cell according to any one of claims 1 to 7, using SSEA-3 expression as an index from umbilical cord or adipose tissue.
The method according to claim 9, further comprising isolating pluripotent stem cells using at least one of the following characteristics (i) to (v) as an index:
(I) CD105 positive;
(Ii) CD117 negative and CD146 negative;
(Iii) CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative and CD271 negative;
(Iv) CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snail negative, Slug negative, Tyrp1 negative and Dct negative; and (vi) Low or no telomerase activity.
The method for increasing the content of pluripotent stem cells according to any one of claims 1 to 7, which comprises trypsinizing umbilical cord or adipose tissue-derived mesenchymal cells and recovering the surviving cells.