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METHOD FOR PREVENTING INCORPORATION OF UNDIFFERENTIATED IPS CELLS POSSIBLY HAVING TUMORIGENIC POTENTIAL USING DIFFERENTIATION-CONTROLLING COMPOUND NEW_EN

Foreign code F190009993
File No. (S2018-0212-N0)
Posted date Oct 30, 2019
Country WIPO
International application number 2019JP007471
International publication number WO 2019187918
Date of international filing Feb 27, 2019
Date of international publication Oct 3, 2019
Priority data
  • P2018-064199 (Mar 29, 2018) JP
Title METHOD FOR PREVENTING INCORPORATION OF UNDIFFERENTIATED IPS CELLS POSSIBLY HAVING TUMORIGENIC POTENTIAL USING DIFFERENTIATION-CONTROLLING COMPOUND NEW_EN
Abstract [Problem] To provide a technique for producing differentiated cells induced from stem cells, without having undifferentiated stem cells incorporated.
[Solution] In the present invention, a stem cell-originated medium is provided which contains, as a differentiation-controlling compound, any one or a plurality selected from the group consisting of liarozole, piogliazone, silibinin, and chrysin. The differentiation-controlling compound has a survival-inhibiting activity specific to undifferentiated stem cells, and inhibits the survival of undifferentiated stem cells without inducing apoptosis of differentiated cells. Consequently, according to the medium of the present invention, it is possible to obtain differentiated cells with no undifferentiated stem cells incorporated.
Outline of related art and contending technology BACKGROUND ART
An induced pluripotent stem cell (iPS cells), such as embryonic stem cells (ES cells) multipotent stem cells of the playing of one of the problems is in the medical arts, multipotent stem cells to differentiate into a desired cell type are implanted in the body of the patient when after, multipotent stem cells remaining undifferentiated state, differentiated cells are implanted in the body of a patient with, the risk of cancer and tumor in the body of the patient how to prevent (see Non-Patent Document 1) in.
Obtain a risk of having a tumor cell like the iPS undifferentiated as the test system to evaluate, undifferentiated pluripotent cell-specific marker or specific marker to the cells of the differentiation (see Non-Patent Document 2) an index expression of the RT-PCR analysis and flow cytometry (qRT-PCR) quantitatively and method. However, none of the fixed frequency mixing of undifferentiated pluripotent stem cells cannot be detected. Therefore, the safety assessment of the final product, undifferentiated pluripotent stem cell culture conditions is returned to the iPS cell colony culture such as the checking does not appear necessary.
PubChem database chemical molecules (https://pubchem.ncbi.nlm.nih.gov) from, and the reagent name of the available chemical formula Liarozole in. {H} Liarozole(6-[ (3-chlorophenyl) -imidazol-1-ylmethyl]-1-benzimidazole, and reference 1) is, retinoic acid inducible cytochrome P450RAI(retinoic acid inducible=) which is a kind of enzyme inhibitors.
Cytochrome P450RAI inhibitors, currently, Ketoconazole, and R116010 (Liarozole) liarozole, further, cyclopropylaryl, cyclopropylheteroaryl, cyclopropylaminoaryl, or an enzyme having the structure (1-imidazolyl) cytochrome P450RAI inhibitory effect of methyl aryl having some compounds have been known (see Patent Document 1). In the prior art, including human mammal comprising administering an inhibitor of cytochrome P450RAI of certain endogenous occur a significant increase in the level of RA, the cytochrome P450RAI inhibitors, for example treatment liarozole, retinoid treatment with a similar effect, for example leads to an improvement in psoriasis has been pointed out (see Non-Patent Document 3).
Associated with the present invention, is disclosed in Patent Document 1, retinoid, during embryonic development gene expression can be adjusted, and, as an exemplary reactive retmoic (disorders than can be treated) disorders, dermatological disorders such as acne, autoimmune disorders, inflammatory disorders, proliferative disorders, neurological disorders, pulmonary disorders and the visually-impaired and, retinoid reactive fault as a method for treating a human cytochrome P450RAI inhibitors alone or in combination with the retinoid treatment, of the retinoid in an individual to maintain or increase the benefit level to the disclosed technique. In the embodiment of this document, the differentiation of the sebaceous glands organs in the hamster-waist in the experiment, by oral gavage of the cytochrome P450RAI inhibitors, differentiation of the sebaceous gland as described in the block. Is Patent Document 2, a cytochrome P450RAI can act as an inhibitor of some compounds are shown. However, as an additive in the medium of a specific inhibitor of cytochrome P450RAI is not discussed how to use, also for inducing differentiation of the iPS cell into a cardiomyocyte specific case is not disclosed.
PubChem database chemical molecules (https: from //pubchem.ncbi.nlm.nih.gov), Pioglitazone reagent name or chemical formula can be presented. Pioglitazone(5-[ [4-[2-(5-ethylpyridin-2-yl) ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione, 2 for reference) is, thiazolidine (Thiazolidinedione) is one type of derivative.
Thiazolidine derivative synthesized from a compound of a group of the thiazolidine and, for example, "ω - (carbocyclylamino) alkoxylated nonthiazolidinedione '-2,4-benzyl',-5-(±) " 2-5-2,4-'methyl' benzokisazoiru (2-naphthalenylmethyl) thiazolidinedione and the like are exemplified. Specifically, the insulin resistance-improving agent is used as rosiglitazone (Rosiglitazone: Glaxo Wellcome, smith kline), pioglitazone (Pioglitazone: Takeda), base (Lobeglitazone: Chong Kun Dang) The glitazones, troglitazone (Troglitazone: first Sankyo), (Rivoglitazone: first Sankyo) rivoglitazone, or, and ciglitazone (Ciglitazone: Takeda). Thiazolidine derivative, peroxisome proliferator-activated receptor (PPAR) γ agonists, liver and adipose tissue, skeletal muscle to increase insulin sensitivity, chronic hyperglycemia can be improved. In the prior art, insulin, transferrin, dexamethasone, biotin, ascorbic acid, glucose, epidermal growth factor or fibroblast growth factor, and the selenous acid or salt thereof, and indomethacin, prostaglandins, long chain fatty acids and at least one selected from the group consisting thiazolidine derivatives of compound 1 was added by using the nutrient medium, a state in which the serum-free or low serum to induce the differentiation of fat cells can be primary precursor can be pointed out (see Patent Document 3).
Associated with the present invention, is disclosed in Patent Document 4, non-steroidal anti-inflammatory agent or bone thiazolidine derivatives, giant soft generated to induce the expression of PPAR γ sarcoma tumor or cartilage and, thereby induce the differentiation of fat cells apoptosis or bone, chondrosarcomas tumor or giant soft generated of the prophylactic or therapeutic agent can be used as a screening method described. However, in Patent Document 4, as an additive in the medium of a specific method of use are considered Pioglitazone not, the iPS cell into a cardiomyocyte differentiation induction of the specific case is not disclosed.
PubChem database chemical molecules (https: from //pubchem.ncbi.nlm.nih.gov), the reagent name Silibinin chemical formula (Silybin) can be presented. {R} Silibinin(Silybin) ((2-, 3-{R} {R}) -3, 5, 7-trihydroxy-2-[ (2-, 3-and 3 {R}) -3-(4-hydroxy-3-methoxyphenyl) -2-(hydroxymethyl) -2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one, reference) is, the standardized seed eropteran maria (Silybum marianum) and silymarin extract is the main active ingredient (Silymarin).
Is silymarin, silibinin, (silydianin) silydianin, isosilybin, and the like (silychristin) silychristin. Is silymarin, gluco-inhibitory effect of the incorporation of cellulose, hypoxia inducible factor (HIF) acting on the inhibition of the activity, the inhibitory effect of a signaling system such as PI3/Akt/mTOR, a plurality of mechanisms of action of the effect of the cancer cells warburg have been reported (see Non-Patent Document 4).
Associated with the present invention, is disclosed in Patent Document 5, silymarin is, of the cornea and/or sclera of the eye in a mammal ophthalmic through a penetration enhancer, penetration enhancers, absorption enhancer used as functional components, an example used for the playback of the corneal epithelial cells are raised. Patent Document 6 is, silymarin is, aging treatment of immune related to aging and for the purpose of functional deterioration of the hematopoietic stem cells, and, for the purpose of anti-cancer effects as a supplement to an application example of the raised. However, as an additive in the medium of a specific method of use are considered silymarin not, the iPS cell into a cardiomyocyte differentiation induction of the specific case is not disclosed.
PubChem database chemical molecules (https: from //pubchem.ncbi.nlm.nih.gov), Chrysin reagent name or chemical formula can be presented. Chrysin(5,7-dihydroxy-2-phenylchromen-4-one, 4 for reference) is, the skin of a fruit, passion flower or propolis the like included in one of flavonoids. In addition, known as the principal active component of propolis.
Flavon is, inhibition of COX-2 gene or prostaglandin E2 an anti-inflammatory action or the like (see non-patent document 5), hypoxia inducible factor (HIF) to inhibit the activity (see Non-Patent Document 6) has been reported.
Associated with the present invention, in non-patent document 7, flavon is, has the effect of the proliferation of human cancer cells, associated with a particular leukemia cancer cell lines compared to other Free effect an especially strong induction of apoptosis has been disclosed. Also, the same effect of inhibition of the mechanism of action involving Akt signaling has been reported (see Non-Patent Document 7). However, as an additive in the medium of a specific method of use are considered flavon not, the iPS cell into a cardiomyocyte differentiation induction of the specific case is not disclosed.
Scope of claims (In Japanese)[請求項1]
分化コントロール化合物として、Liarozole、Pioglitazone、Silibinin、Chrysinのいずれか又は複数を含み、下記(a)から(c)のいずれかにおいて、培養または/および保存のために用いられる培地。(a) 幹細胞(b) (a)と幹細胞由来分化細胞(c) (b)と幹細胞由来分化細胞から作製された臓器

[請求項2]
前記幹細胞が誘導性多能性幹細胞である請求項1記載の培地。

[請求項3]
前記分化細胞が心筋細胞である請求項1又は2に記載の培地。

[請求項4]
前記幹細胞がヒト由来である請求項1~3のいずれか一項に記載の培地。

[請求項5]
分化コントロール化合物を濃度10~500μMで含む請求項1~4のいずれか一項に記載の培地。

[請求項6]
前記培地が無血清培地である、請求項1~5のいずれか一項に記載の培地。

[請求項7]
前記請求項1から6のいずれか一項に記載の培地を作製するための培地作製用組成物。

[請求項8]
前記請求項1から6のいずれか一項に記載の培地を用いて作製した分化細胞。

[請求項9]
前記分化細胞が、心筋細胞である請求項8記載の分化細胞。

[請求項10]
分化コントロール化合物として、Liarozole、Pioglitazone、Silibinin、Chrysinのいずれか又は複数を有効成分とする幹細胞生存抑制剤。

[請求項11]
前記幹細胞が誘導性多能性幹細胞である請求項10記載の幹細胞生存抑制剤。

[請求項12]
前記幹細胞がヒト由来である請求項10又は11に記載の幹細胞生存抑制剤。

[請求項13]
幹細胞由来の分化細胞を含む細胞医薬組成物の生体内での腫瘍化を抑制するための医薬組成物であり、請求
項12記載の幹細胞生存抑制剤を含む医薬組成物。

[請求項14]
幹細胞から分化細胞を製造する方法であって、分化誘導後の細胞を分化コントロール化合物として、Liarozole、Pioglitazone、Silibinin、Chrysinのいずれか又は複数により処理する工程を含む方法。

[請求項15]
前記幹細胞が誘導性多能性幹細胞である請求項14記載の方法。

[請求項16]
前記分化細胞が心筋細胞である請求項14又は15記載の方法。

[請求項17]
前記幹細胞がヒト由来である請求項14~16のいずれか一項に記載の方法。

[請求項18]
幹細胞を培養する工程と、幹細胞を分化誘導する工程と、をさらに含む請求項14~17のいずれか一項に記載の方法。

[請求項19]
幹細胞由来の分化細胞を含む細胞医薬組成物を製造する方法であって、前記幹細胞を分化誘導する工程と、分化誘導後の細胞を分化コントロール化合物として、Liarozole、Pioglitazone、Silibinin、Chrysinのいずれか又は複数により処理する工程と、を含む方法。

[請求項20]
前記幹細胞が誘導性多能性幹細胞である請求項19記載の方法。

[請求項21]
前記分化細胞が心筋細胞である請求項19又は20記載の方法。

[請求項22]
前記幹細胞がヒト由来である請求項19~21のいずれか一項に記載の方法。

[請求項23]
幹細胞を培養する工程と、をさらに含む請求項19~22のいずれか一項に記載の方法。

[請求項24]
幹細胞及び分化細胞を含む細胞混合物から分化細胞のみを分離する方法であって、前記細胞混合物を分化コントロール化合物として、Liarozole、Pioglitazone、Silibinin、Chrysinのいずれか又は複数により処理する手順を含む方法。

[請求項25]
前記幹細胞が誘導性多能性幹細胞である請求項24記載の方法。

[請求項26]
前記分化細胞が心筋細胞である請求項24又は25記載の方法。

[請求項27]
前記幹細胞及び前記分化細胞がヒト由来である請求項24~26のいずれか一項に記載の方法。

[請求項28]
幹細胞を培養する工程と、幹細胞を分化誘導する工程と、をさらに含む請求項24~27のいずれか一項に記載の方法。

[請求項29]
請求項1記載の培地、請求項7記載の培地作製用組成物、請求項10記載の幹細胞生存抑制剤、請求項13記載の医薬組成物の製造のための分化コントロール化合物としてのLiarozole、Pioglitazone、Silibinin、Chrysinの単体もしくは組み合わせでの使用。

[請求項30]
幹細胞由来の分化細胞を含む細胞医薬組成物の製造のための分化コントロール化合物としてのLiarozole、Pioglitazone、Silibinin、Chrysinの使用。

[請求項31]
幹細胞由来の分化細胞を含む細胞医薬組成物の生体内での腫瘍化を抑制するための分化コントロール化合物としてのLiarozole、Pioglitazone、Silibinin、Chrysinの使用。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • UNIVERSITY OF THE RYUKYUS
  • Inventor
  • NAKASHIMA, Yoshiki
  • OMASA, Takeshi
  • NOGUCHI,Hirofumi
  • SHIOHIRA,Chika
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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