WO2016072519A1 - 未分化細胞が除去された分化誘導細胞集団、その利用及びその製造方法 - Google Patents
未分化細胞が除去された分化誘導細胞集団、その利用及びその製造方法 Download PDFInfo
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Definitions
- the present invention relates to a differentiation-inducing cell population from which undifferentiated cells have been removed, its use, and its production method.
- pluripotent stem cells remain unmixed in an undifferentiated state. Regardless of which purpose is used, this is problematic because the purity of the differentiation-inducing cells is preferably high.
- pluripotent stem cells are used, if they are undifferentiated pluripotent stem cells are mixed in the differentiation-inducing cell population, such as having tumorigenicity if they are undifferentiated, there is a safety risk when used as a medicine.
- Non-Patent Documents 1 and 2 For example, as an approach using a special medium, a method using a methionine ( ⁇ ) medium (Non-Patent Documents 1 and 2), a method using a sugar-free medium (Non-Patent Documents 3 and 4), and the like have been proposed.
- a survival signal inhibitor As an approach using a survival signal inhibitor, a method using a Survivin inhibitor (Non-patent Document 5) and the like have been proposed.
- FACS sorting purging with an anti-SSEA-5 antibody (Non-patent Document 6), purging with a sugar chain antibody (lectin) (Non-Patent Document 7), and the like have been proposed.
- the present invention is a cell population containing differentiated cells that can be obtained by inducing differentiation of pluripotent stem cells, wherein the content ratio of undifferentiated pluripotent stem cells is reduced more than conventional cells
- the challenge is to provide a group.
- the present inventors provide a cell population containing differentiated cells that can be obtained by inducing differentiation of pluripotent stem cells, wherein the content ratio of undifferentiated pluripotent stem cells is 0.2% or less.
- the present invention includes the following aspects.
- Item 1-1 A cell population containing differentiated cells that can be obtained by inducing differentiation of pluripotent stem cells, wherein the content ratio of undifferentiated pluripotent stem cells is 0.2% or less.
- Item 1-2. Item 11. The cell population according to Item 1-1, wherein the pluripotent stem cell is an induced pluripotent stem cell or an embryonic stem cell.
- Item 1-3. Item 13. The cell population according to Item 1-2, wherein the induced pluripotent stem cell is an iPS cell.
- Item 6. The cell population according to any one of Items 1-1 to 1-5, wherein the content ratio of the undifferentiated pluripotent stem cells is determined by an undifferentiated cell marker analysis using flow cytometry.
- Item 7. The cell population according to any one of Items 1-1 to 1-6, which is used as a medicine. Item 2-1.
- a method for producing a cell population containing differentiated cells obtainable by inducing differentiation of pluripotent stem cells (A) Decreasing the content ratio of undifferentiated pluripotent stem cells by culturing a cell population containing differentiated cells that can be obtained by inducing differentiation of pluripotent stem cells in the presence of an anti-CD30 antibody binding agent. And / or (B) culturing a cell population containing differentiated cells that can be obtained by inducing differentiation of pluripotent stem cells in the presence of a BET inhibitor.
- the manufacturing method including the process of reducing a content rate.
- the production method according to Item 2-1 wherein the pluripotent stem cell is an induced pluripotent stem cell or an embryonic stem cell.
- Item 2-3. Item 3.
- Item 2-4. Item 3.
- An anti-CD30 antibody-binding agent used for reducing the content ratio of undifferentiated pluripotent stem cells in a method for producing a cell population containing differentiated cells obtainable by inducing differentiation of pluripotent stem cells Composition.
- Item 3-2. A composition containing a BET inhibitor, which is used for reducing the content ratio of undifferentiated pluripotent stem cells in a method for producing a cell population containing differentiated cells obtainable by inducing differentiation of pluripotent stem cells. .
- the present invention it is possible to use a differentiation-inducing cell population having a purity higher than that of the conventional one, and to provide a drug with reduced side effects such as tumorigenicity, a highly accurate research tool, and the like. .
- FIG. 2 is a diagram illustrating a result of Example 1.
- FIG. 2 is a diagram illustrating a result of Example 1.
- FIG. 6 is a diagram illustrating a result of Example 2.
- 6 is a diagram illustrating a result of Example 2.
- 6 is a diagram illustrating a result of Example 3.
- 10 is a diagram illustrating a result of Example 4.
- 10 is a diagram illustrating a result of Example 4.
- 10 is a diagram illustrating a result of Example 5.
- Cell population of the present invention is a cell population containing differentiated cells that can be obtained by inducing differentiation of pluripotent stem cells, and the content ratio of undifferentiated pluripotent stem cells is 0.2% or less.
- the content ratio of undifferentiated pluripotent stem cells is preferably 0.1% or less, more preferably 0.05% or less.
- Pluripotent stem cells are not particularly limited and can be widely used.
- As the pluripotent stem cell either an induced pluripotent stem cell or an embryonic stem cell can be used.
- the artificial pluripotent stem cell is not particularly limited, and for example, iPS cells can be used. Although it does not specifically limit as embryonic stem cell, For example, ES cell etc. can be used. Among these, iPS cells and ES cells are particularly preferable from the viewpoint of safety and the like, especially when considering use as a medicine.
- the present invention is characterized in that the content ratio (that is, the residual ratio) of undifferentiated pluripotent stem cells in the cell population is reduced as compared with the conventional technique. That is, the present invention has been completed on the basis of the development of a new method that makes it possible to realize the survival rate of undifferentiated pluripotent stem cells, which was impossible in the past. As will be described later, specifically, these new methods are techniques for purifying by focusing on the characteristics of pluripotent stem cells remaining in an undifferentiated state. Therefore, in the present invention, the type of differentiated cells is not particularly limited.
- differentiated cells include retinal cells such as cardiomyocytes, nerve cells, and retinal pigment epithelial cells, blood (hematopoietic) cells, hepatocytes, pancreatic beta cells, kidney cells, chondrocytes and germ cells. .
- retinal cells such as cardiomyocytes, nerve cells, and retinal pigment epithelial cells
- blood (hematopoietic) cells hematopoietic) cells
- hepatocytes hepatocytes
- pancreatic beta cells pancreatic beta cells
- kidney cells chondrocytes and germ cells.
- chondrocytes are particularly preferable.
- the cell population of the present invention is characterized by a low content ratio of undifferentiated pluripotent stem cells, and may further include cells different from differentiation-inducing cells and undifferentiated pluripotent stem cells as necessary. .
- the cell population of the present invention is not particularly limited, but the total number of cells is usually 1 ⁇ 10 4 or more, preferably 1 ⁇ 10 5 to 1 ⁇ 10 9 , more preferably 1 ⁇ 10 8. ⁇ 1 ⁇ 10 9 .
- the content ratio of undifferentiated pluripotent stem cells in the cell population of the present invention can be defined as that determined by undifferentiated cell marker analysis using flow cytometry. Specifically, this analysis can be performed as follows.
- a cell detachment solution such as Accutase is allowed to act on about 1 ⁇ 10 5 to 5 ⁇ 10 5 differentiated cells at 37 ° C. for 5 minutes, and the differentiated cells are detached and centrifuged.
- Differentiated cells are suspended in 100 ⁇ L each of PBS, fluorescence-labeled anti-human TRA-1-60 antibody and isotype control antibody are added, and reacted at 4 ° C. in the dark for 20 minutes.
- Differentiated cells are washed with PBS and collected by centrifugation, then suspended in 300 ⁇ L each of PBS, passed through a cell strainer, and then collected in a FACS tube.
- the fluorescence emission is detected and quantified by a detector corresponding to the labeled fluorescence.
- a fraction stained with an isotype control antibody is defined as a negative fraction, and a fraction with higher fluorescence intensity is defined as a positive fraction.
- the proportion belonging to the positive fraction is quantitatively expressed as a percentage.
- the cell population of the present invention is not particularly limited and can be used for a wide range of applications. For example, since the content ratio of undifferentiated pluripotent stem cells is further reduced, when used as a medicine (cellular medicine), there are fewer safety risks and it is preferable.
- the use as a medicine that is, the target disease, dose, usage and the like can be appropriately determined depending on the type of differentiated cells.
- the differentiated cells are cardiomyocytes
- the cell population of the present invention can be used for the purpose of treating severe heart failure associated with myocardial infarction or cardiomyopathy (such as dilated cardiomyopathy).
- the cell population of the present invention When used as a medicine, it has an effect that safety risks such as tumor formation are reduced.
- the cell population of the present invention can be used as an active ingredient in a pharmaceutical composition when used as a medicine.
- This pharmaceutical composition is not particularly limited, and may contain various other components that can be contained in so-called cell medicines.
- the cell population of the present invention may contain the cell population described above as appropriate in the cell culture medium according to the purpose of use, or may be formed into a sheet.
- the cell population of the present invention is not particularly limited, but can be obtained by the method described below. You may carry out combining these methods as needed. All of these methods belong to technology for removing undifferentiated pluripotent stem cells that are inevitably mixed, and the content ratio of undifferentiated pluripotent stem cells can be reduced to a level that could not be realized by conventional methods. It is a new way that can be done.
- an anti-CD30 antibody binding agent comprising an antibody that specifically recognizes CD30, which is known as an undifferentiated cell marker, is used.
- CD30 belongs to TNF-R superfamily and is expressed in some lymphomas. In normal tissues, it is only expressed on activated lymphocytes. NF- ⁇ B and MAPK pathways are known as downstream signals, and contribute to survival through these signals.
- this anti-CD30 antibody-binding drug is an antibody-drug conjugate (ADC) in which a drug having an activity of killing cells is bound with a linker that cleaves in the intracellular environment.
- ADC antibody-drug conjugate
- This anti-CD30 antibody-binding drug enters into the antibody-recognized cell, and the drug is released into the cell, whereby the cell can be selectively killed.
- linker is not particularly limited, and examples thereof include a linker that is cleaved by a proteolytic enzyme.
- the anti-CD30 antibody binding agent is not particularly limited, and for example, Adcetris (registered trademark) (SGN-35) (Millenium, Seattle Genetics and Takeda), which is commercially available as an antibody drug, can be used.
- Adtris registered trademark
- the concentration of the anti-CD30 antibody binding agent in the cell culture medium can be appropriately set according to the type of pluripotent stem cells, etc., but is preferably 5 ⁇ g / ml to 50 ⁇ g / ml, more preferably 10 ⁇ g / ml to 50 ⁇ g / ml. The amount is preferably 25 ⁇ g / ml to 50 ⁇ g / ml.
- the culture time can also be appropriately set according to the type of pluripotent stem cells, etc., but is preferably 24 hours to 96 hours, more preferably 48 hours to 96 hours, and even more preferably 72 hours to 96 hours.
- the BET (Bromodomain and extraterminal) protein family consists of BRD2, BRD3, BRD4 and BRDT, and is deeply involved in transcription control by RNA polymerase II. It recognizes the acetylated lysine residue of epilepsy histone tail via Bromodain (Epigenetic reader), and recruits the transcriptional control complex to epithelial acetylated chromatin.
- BET inhibitors such as low molecular weight compounds JQ1 have been reported to have a therapeutic role in cancer and inflammation (Nature. 2010; 468: pp. 1067-73, Cell. 2011; 146: pp. 904-17).
- BRD4 is said to regulate the expression of c-Myc, NK- ⁇ B and Nanog in some carcinomas, which is due to BRD4 binding to Super-enhancer.
- Undifferentiated pluripotent stem cells are killed by culturing in the presence of a BET inhibitor. Similar to the method (1) above, by culturing a cell population containing differentiated cells that can be obtained by inducing differentiation of pluripotent stem cells in the presence of a BET inhibitor, The content ratio can be reduced.
- I-BET151 and I-BET762 can be used in addition to JQ1.
- the concentration of the BET inhibitor in the cell culture medium can be appropriately set according to the type of the BET inhibitor, the type of pluripotent stem cells, etc.
- JQ1 JQ1 is used, it is preferably 0.1 ⁇ M to 10 ⁇ M, 0.2 ⁇ M to 10 ⁇ M is more preferable, and 0.5 ⁇ M to 10 ⁇ M is even more preferable.
- the culture time can also be appropriately set according to the type of BET inhibitor, the type of pluripotent stem cells, etc.
- JQ1 when used, it is preferably 24 hours to 96 hours, more preferably 48 hours to 96 hours. 72 hours to 96 hours are more preferable.
- Composition of the present invention further provides the following composition used for the above-mentioned method.
- A a composition comprising an anti-CD30 antibody binding agent
- B a composition comprising a BET inhibitor
- a composition comprising an anti-CD30 antibody binding agent and a BET inhibitor a composition comprising an anti-CD30 antibody binding agent and a BET inhibitor.
- compositions The compounding concentration of the anti-CD30 antibody binding agent and / or the BET inhibitor in these compositions is not particularly limited and can be appropriately set according to the required storage stability, purpose of use and the like. These compositions may further contain other components as long as the above methods are not inhibited.
- Examples of other components include, but are not limited to, carbohydrates such as glucose, maltose, sucrose, lactose, raffinose, trehalose, mannitol, hydroxyethyl starch and pullulan, gluconic acid, lactic acid, acetic acid, propionic acid, ⁇ - Organic acids such as hydroxybutyric acid and citric acid, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium bicarbonate, Electrolytes such as potassium bicarbonate, sodium carbonate and potassium carbonate, vitamins such as L-ascorbic acid and vitamin E, amino acids such as glycine, glutamic acid and lysine, hormones such as antidiuretic hormone and insulin, citric acid, citrate, heparin Anticoagulants such as sodium edetate, antihypertensives such as calcium antagonists, ad
- Example 1 Method for removing undifferentiated cells with anti-CD30 antibody-binding drug Human iPS cell line MYH-GIP4 was seeded in a 12-well plate (cell density: 20% confluency). 48 hours after seeding, anti-CD30 antibody-binding drug (generic name: Breutuximab vedotin, abbreviated as BV) (trade name: Adcetris (registered trademark)) was added to each well at 0, 0.05 and 5 ⁇ g / ml (every 24 hours) Replace the drug-containing medium. Thereafter, it was observed under a microscope every 24 hours. In addition, since Adcetris (registered trademark) includes additives in addition to BV, the numerical values converted based on the net weight of BV are shown. That is, “BV is 0, 0.05, and 5 ⁇ g / ml”.
- Example 2 the same experiment as in Example 1 was performed for the purpose of examining the effect of BV on human normal cells (NHDF).
- NHDF Human fibroblasts
- Example 2 Method for removing undifferentiated cells with anti-CD30 antibody-binding agent
- BV was applied to human iPS cells in vitro.
- iPS cell lines (1231A3, MYH-GIP4 and 253G1) and NHDF were seeded in 12-well plates (cell density: 20% confluency). However, the cell density of NHDF was 50% fconfluency.
- FIG. 3 After 24 hours), FIG. 4 (after 48 hours) and FIG. 5 (after 72 hours).
- the maximum cell growth inhibitory effect was observed after 72 hours, and a BV concentration-dependent effect was also confirmed.
- the BV concentration that led to almost complete suppression of the cell number in 72 hours was 5-50 ⁇ g / ml for 1231A3 and 25-50 ⁇ g / ml for MYH-GIP4 and 253G1.
- NHDF is almost unaffected by BV.
- Example 3 FIG. Method for removing undifferentiated cells with anti-CD30 antibody-binding agent
- BV is added to human iPS cells in vitro.
- a cell proliferation assay (CCK-8 assay) reflecting the number of cells was performed, and live cells were quantitatively evaluated.
- NHDF Three human iPS cell lines (253G1, MYH-GIP4 and 201B7) and NHDF were seeded in a 12-well plate (cell density: 20% confluency). However, the cell density of NHDF was 50% fconfluency.
- Example 4 Method for removing undifferentiated cells using anti-CD30 antibody-binding drug Human iPS cell-derived cardiomyocytes (iPS-CM) are targeted. iPS-CM is thought to contain undifferentiated cells having tumorigenicity in addition to cardiomyocytes.
- IPS-CM derived from human iPS cell line 253G1 was seeded in a 12-well plate (cell density: 90% confluency). 48 hours after seeding, 0, 5, and 10 ⁇ g / ml of BV were added to each well (drug-containing medium was replaced every 24 hours). Thereafter, mRNA was extracted 72 hours and 96 hours after the addition. The expression level of Lin28 was quantified by quantitative PCR.
- the expression of the undifferentiated marker Lin28 was reduced to about 50% in the BV addition group compared to the control (BV 0 ⁇ g / ml).
- Lin28 expression was reduced to about 50 to 70% in the BV addition group compared to the control (FIG. 7).
- Embodiment 5 Method for removing undifferentiated cells with BET inhibitor
- BET inhibitor name of drug used: JQ1
- human iPS cells and in vitro JQ1 was added to human normal cells (NHDF) and observed visually for cell proliferation and cell death.
- Human iPS cell line 253G1 was seeded in a 12-well plate (cell density: 20% confluency).
- Human fibroblasts (NHDF) were seeded in 12-well plates (cell density: 50% confluency).
- Example 6 Method for removing undifferentiated cells with BET inhibitors
- JQ1 was added to human iPS cells in vitro, and the number of cells was reduced.
- the reflected cell proliferation assay (CCK-8 assay) was performed 96 hours later and live cells were evaluated quantitatively.
- Human iPS cell line 253G1 was seeded in a 12-well plate (cell density: 20% confluency).
- Test example Toxicity test of JQ1
- NHDF and iPS-CM normal human differentiated cells
- NHDF and iPS-CM were seeded in a 12-well plate (cell density: 50% and 90% confluency, respectively).
- Example 7 Method for removing undifferentiated cells with BET inhibitor iPS-CM derived from human iPS cell line MYH-GIP4 was seeded in a 12-well plate (cell density: 90% confluency). 48 hours after seeding, 0, 1, and 5 ⁇ M of JQ1 were added to each well (drug-containing medium was changed every 48 hours). Thereafter, the positive rate of TRA-1-60 was quantified 96 hours after the addition. As a control, the same measurement was performed using an Isotype control antibody, and it was confirmed that there was no false positive.
- the TRA-1-60 positive rate in the control (JQ1: 0 ⁇ M) group was 2.0%, whereas the JQ1: 1 ⁇ M group was 0.2%, and JQ1: 5 ⁇ M. In the group, it decreased to 0.1%.
Abstract
Description
多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団であって、未分化多能性幹細胞の含有割合が、0.2%以下である細胞集団。
項1-2.
前記多能性幹細胞が、人工多能性幹細胞又は胚性幹細胞である、項1-1に記載の細胞集団。
項1-3.
前記人工多能性幹細胞が、iPS細胞である、項1-2に記載の細胞集団。
項1-4.
前記胚性幹細胞が、ES細胞である、項1-2に記載の細胞集団。
項1-5.
総細胞数が、1×104個以上である、項1-1~1-4のいずれか一項に記載の細胞集団。
項1-6.
前記未分化多能性幹細胞の含有割合が、フローサイトメトリーを用いた未分化細胞マーカー解析により求められるものである、項1-1~1-5のいずれか一項に記載の細胞集団。
項1-7.
医薬として用いられる、項1-1~1-6のいずれか一項に記載の細胞集団。
項2-1.
多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団の製造方法であって、
(A)抗CD30抗体結合薬剤の存在下で、多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団を培養することにより、未分化多能性幹細胞の含有割合を低減する工程;及び/又は
(B)BET阻害剤の存在下で、多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団を培養することにより、未分化多能性幹細胞の含有割合を低減する工程
を含む、製造方法。
項2-2.
前記多能性幹細胞が、人工多能性幹細胞又は胚性幹細胞である、項2-1に記載の製造方法。
項2-3.
前記人工多能性幹細胞が、iPS細胞である、項2-2に記載の製造方法。
項2-4.
前記胚性幹細胞が、ES細胞である、項2-2に記載の製造方法。
項3-1.
多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団の製造方法において、未分化多能性幹細胞の含有割合を低減するために用いられる、抗CD30抗体結合薬剤を含有する組成物。
項3-2.
多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団の製造方法において、未分化多能性幹細胞の含有割合を低減するために用いられる、BET阻害剤を含有する組成物。
本発明は、多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団であって、未分化多能性幹細胞の含有割合が、0.2%以下である細胞集団である。
本発明の細胞集団は、未分化多能性幹細胞の含有割合が、好ましくは0.1%以下であり、より好ましくは0.05%以下である。
本発明の細胞集団は、特に限定されないが、以下に説明する方法により得ることができる。必要に応じて、これらの方法を組み合わせて行ってもよい。これらの方法は、いずれも不可避的に混入する未分化多能性幹細胞を除去する技術に属し、従来の方法では実現できなかったレベルにまで未分化多能性幹細胞の含有割合を低減することができる新しい方法である。
この方法では、未分化細胞マーカーとして知られるCD30を特異的に認識する抗体からなる抗CD30抗体結合薬剤を用いる。
また、リンカーとしては、特に限定されないが、例えば、蛋白質分解酵素により開裂するリンカー等が挙げられる。
この方法では、ヒストンアセチル化阻害剤であるBET阻害剤を用いる。
3.本発明の組成物
本発明はさらに、上記の方法のために用いられる、以下の組成物を提供する。
(a)抗CD30抗体結合薬剤を含有する組成物;
(b)BET阻害剤を含有する組成物;及び
(c)抗CD30抗体結合薬剤及びBET阻害剤を含有する組成物。
これらの組成物における、抗CD30抗体結合薬剤及び/又はBET阻害剤の配合濃度は、特に限定されず、要求される保存安定性や使用目的等に応じて適宜設定することができる。
これらの組成物は、上記の方法を阻害しない限りにおいて、さらに他の成分を含有していてもよい。その他の成分としては、特に限定されないが、例えば、グルコース、マルトース、シュークロース、ラクトース、ラフィノース、トレハロース、マンニトール、ヒドロキシエチル澱粉及びプルラン等の糖質、グルコン酸、乳酸、酢酸、プロピオン酸、β-ヒドロキシ酪酸及びクエン酸等の有機酸、塩化ナトリウム、塩化カリウム、塩化マグネシウム、塩化カルシウム、リン酸二水素ナトリウム、リン酸二水素カリウム、リン酸水素二ナトリウム、リン酸水素二カリウム、炭酸水素ナトリウム、炭酸水素カリウム、炭酸ナトリウム及び炭酸カリウム等の電解質、L-アスコルビン酸及びビタミンE等のビタミン、グリシン、グルタミン酸及びリジン等のアミノ酸、抗利尿ホルモン及びインスリン等のホルモン、クエン酸、クエン酸塩、ヘパリン及びエデト酸ナトリウム等の抗凝固剤、カルシウム拮抗剤、アドレナリンβ受容体拮抗剤及びアンギオテンシン変換酵素阻害剤等の降圧剤、アデノシン三リン酸等の核酸塩基、凍結防止蛋白質等の凍結防止剤;並びに活性酸素消去剤、細胞賦活剤、抗生物質、抗血小板因子、肝障害抑制剤、賦形剤、結合剤、崩壊剤、分散剤、粘性剤、再吸収促進剤、界面活性剤、溶解補助剤、保存剤、防腐剤、乳化剤、等張化剤、安定化剤、緩衝剤及びpH調整剤等が挙げられ、これらの少なくとも一種を必要に応じて配合することができる。
ヒトiPS細胞株MYH-GIP4を12穴プレートへ播種した(細胞密度:20% confluency)。播種48時間後、各ウェルに抗CD30抗体結合薬剤(一般名:Breutuximab vedotin、BVと略す)(商品名:Adcetris(登録商標))を0、0.05及び5μg/mlずつ添加(24時間毎に薬剤含有培地を交換)。以後、24時間毎に顕微鏡下に観察した。なお、Adcetris(登録商標)には、BVの他、添加物が含まれる為、BV正味の重量をもとに換算した数値を記載した。すなわち「BVとして0、0.05及び5μg/ml」となる。
種々のBV濃度、BV処理時間で、ヒトiPS細胞の細胞数がどのように変化するかを明らかにする目的で、in vitroでヒトiPS細胞にBVを添加し、細胞数を反映する細胞増殖アッセイ (CCK-8アッセイ)を行い、生細胞の評価を定量的に行った。
種々のBV濃度・BV処理時間で、ヒトiPS細胞の細胞数がどのように変化するかを明らかにする目的で、invitroでヒトiPS細胞にBVを添加し、細胞数を反映する細胞増殖アッセイ(CCK-8アッセイ)を行い、生細胞の評価を定量的に行った。
ヒトiPS細胞由来心筋細胞(iPS-CM)を対象とする。iPS-CMは心筋細胞の他、造腫瘍性を有する未分化細胞も含有すると考えられる。
BET阻害剤 (使用薬剤名:JQ1)がヒトiPS細胞およびヒト正常細胞(NHDF;Normal human dermal fibroblast)に及ぼす影響を調べる目的で、in vitroでヒトiPS細胞及びヒト正常細胞(NHDF)にJQ1を添加し、細胞増殖及び細胞死に関して肉眼的に観察した。
以後、96時間後に顕微鏡下に観察し、細胞を可視化するためにクリスタル・バイオレット染色を行った(図9)。
種々のJQ1濃度で、ヒトiPS細胞の細胞数がどのように変化するかを明らかにする目的で、in vitroでヒトiPS細胞にJQ1を添加し、細胞数を反映する細胞増殖アッセイ(CCK-8アッセイ)を96時間後に行い、生細胞の評価を定量的に行った。
以後、添加96時間後に細胞増殖アッセイを行った(図10)。
ヒト正常分化細胞(NHDF及びiPS-CM)に対してJQ1が毒性を有することがないかを調べる目的で、実施例6と同様の細胞増殖アッセイを行った。
ヒトiPS細胞株MYH-GIP4から誘導したiPS-CMを12穴プレートへ播種した(細胞密度:90% confluency)。播種48時間後、各ウェルにJQ1を0、1及び5μMlずつ添加(48時間毎に薬剤含有培地を交換)。以後、添加96時間後にTRA-1-60陽性率を定量化した。コントロールとして、Isotypecontrol抗体を用いて同様に測定を行い、擬陽性がないことを確認した。
Claims (10)
- 多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団であって、未分化多能性幹細胞の含有割合が、0.2%以下である細胞集団。
- 前記多能性幹細胞が、人工多能性幹細胞及び胚性幹細胞である、請求項1に記載の細胞集団。
- 前記人工多能性幹細胞が、iPS細胞である、請求項2に記載の細胞集団。
- 前記胚性幹細胞が、ES細胞である、請求項2に記載の細胞集団。
- 総細胞数が、1×104個以上である、請求項1~4のいずれか一項に記載の細胞集団。
- 前記未分化多能性幹細胞の含有割合が、フローサイトメトリーを用いた未分化細胞マーカー解析により求められるものである、請求項1~5のいずれか一項に記載の細胞集団。
- 医薬として用いられる、請求項1~6のいずれか一項に記載の細胞集団。
- 多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団の製造方法であって、
(A)抗CD30抗体結合薬剤の存在下で、多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団を培養することにより、未分化多能性幹細胞の含有割合を低減する工程;及び/又は
(B)BET阻害剤の存在下で、多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団を培養することにより、未分化多能性幹細胞の含有割合を低減する工程
を含む、製造方法。 - 多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団の製造方法において、未分化多能性幹細胞の含有割合を低減するために用いられる、抗CD30抗体結合薬剤を含有する組成物。
- 多能性幹細胞を分化誘導することにより得られうる分化細胞を含有する細胞集団の製造方法において、未分化多能性幹細胞の含有割合を低減するために用いられる、BET阻害剤を含有する組成物。
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Also Published As
Publication number | Publication date |
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SG10201903910RA (en) | 2019-05-30 |
EP3216859A1 (en) | 2017-09-13 |
EP3216859B1 (en) | 2020-03-11 |
JPWO2016072519A1 (ja) | 2017-08-17 |
JP6938154B2 (ja) | 2021-09-22 |
CA2966576C (en) | 2021-07-20 |
EP3216859A4 (en) | 2017-09-13 |
AU2015344125B2 (en) | 2022-03-24 |
CN107075473A (zh) | 2017-08-18 |
US20170335284A1 (en) | 2017-11-23 |
US20210388320A1 (en) | 2021-12-16 |
KR20170074996A (ko) | 2017-06-30 |
KR102639313B1 (ko) | 2024-02-20 |
EP3567100A1 (en) | 2019-11-13 |
CA2966576A1 (en) | 2016-05-12 |
SG11201703544QA (en) | 2017-06-29 |
AU2015344125A1 (en) | 2017-05-25 |
CN107075473B (zh) | 2021-07-13 |
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