WO2015166845A1 - 細胞の分化状態の評価方法 - Google Patents
細胞の分化状態の評価方法 Download PDFInfo
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- WO2015166845A1 WO2015166845A1 PCT/JP2015/062128 JP2015062128W WO2015166845A1 WO 2015166845 A1 WO2015166845 A1 WO 2015166845A1 JP 2015062128 W JP2015062128 W JP 2015062128W WO 2015166845 A1 WO2015166845 A1 WO 2015166845A1
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- the present invention relates to a method for evaluating the differentiation state of a cell.
- Patent Document 1 a method using immunostaining (see, for example, Patent Document 1) or a method for quantifying the expression level of a marker gene (see, for example, Patent Document 2) has been widely used. Yes.
- cells to be evaluated such as pluripotent stem cells
- SSEA-4 and TRA1-60 are widely used as antibodies for determining whether pluripotent stem cells are in an undifferentiated state (see, for example, Patent Document 1).
- a secondary antibody that binds to the antibody is added to the cells, and then a fluorescent label or the like previously imparted to the secondary antibody is detected. This makes it possible to evaluate whether or not an antigen for the antibody exists on the cell, that is, whether or not the cell is in an undifferentiated state.
- mRNA is extracted from pluripotent stem cells, converted into cDNA by reverse transcriptase, and then the marker gene is obtained by PCR (Polymerase Chain Reaction, polymerase chain reaction). Amplify.
- PCR Polymerase Chain Reaction, polymerase chain reaction
- Amplify At this time, NANOG and POU5F1 (OCT3 / 4) are widely used as marker genes for evaluating the undifferentiation of pluripotent stem cells (see, for example, Non-Patent Document 1).
- the PCR product is detected by electrophoresis or a real-time PCR apparatus to confirm the expression level of the marker gene in the cell, and from the result, it is evaluated whether or not the cell is in an undifferentiated state.
- any of the conventional evaluation methods described above requires invasive processing on cells. Therefore, after evaluating the differentiation state, the cells subjected to the evaluation cannot be used for another purpose, for example, as a cell source for regenerative medicine. In addition, it is impossible to evaluate changes over time for the same sample (ie, cells in the same culture dish). In order to evaluate changes over time in the differentiation state, multiple culture dishes are used in parallel. Therefore, complicated operations such as culturing were necessary.
- the present invention has been made in view of the above points, and an object of the present invention is to provide a method for non-invasively evaluating the differentiation state of cells.
- the present inventors have determined that putrescine, kynurenine, cystathionine, ascorbic acid, riboflavin, pyruvate, serine, cysteine, threonic acid, citric acid, and orotic acid in the culture supernatant depending on the differentiation state of the cells.
- the inventors have found that the abundance is different and have arrived at the present invention.
- the method for evaluating a cell differentiation state according to the present invention uses a stem cell with an unknown differentiation state or a cell that has undergone differentiation induction from a stem cell as a test cell, A method for evaluating the differentiation state of the test cell based on the abundance of the predetermined substance in the culture supernatant,
- the predetermined substance is at least one compound selected from the group consisting of putrescine, kynurenine, cystathionine, ascorbic acid, riboflavin, pyruvic acid, serine, cysteine, threonic acid, citric acid, and orotic acid.
- the method for evaluating a cell differentiation state according to the present invention includes, for example, the presence of the predetermined substance in the culture supernatant of the test cell and the presence of the predetermined substance in the culture supernatant of a control cell whose differentiation state is known. By comparing the amount, the differentiation state of the test cell can be evaluated.
- the stem cell is a pluripotent stem cell such as an ES cell (Embryonic Stem cells) or an iPS cell (Induced Puriuripotent Stem ⁇ cells, induced pluripotent stem cell).
- ES cell Embryonic Stem cells
- iPS cell Induced Puriuripotent Stem ⁇ cells, induced pluripotent stem cell
- the abundance of the predetermined substance in the culture supernatant can be quantified by mass spectrometry.
- the cell differentiation state can be evaluated non-invasively without the need to destroy the cell as in the prior art. This makes it possible to use the test cell as a cell source for regenerative medicine after evaluating the differentiation state.
- the test cell when evaluating changes in the differentiation state over time, it is not necessary to perform the complicated work of culturing in parallel in a plurality of culture dishes as in the past, and the cells in the same culture dish are targeted. It becomes possible to easily evaluate changes in the differentiation state with time.
- the schematic diagram explaining the evaluation method of the cell differentiation state in one Example of this invention The graph which shows the time-dependent change of the abundance of each substance calculated
- the cell differentiation state evaluation method comprises at least one compound selected from the group consisting of putrescine, kynurenine, cystathionine, ascorbic acid, riboflavin, pyruvate, serine, cysteine, threonic acid, citric acid, and orotic acid.
- a biomarker the differentiation state of the test cell is evaluated based on the abundance of the biomarker in the culture supernatant of the test cell.
- test cells typically pluripotent stem cells such as ES cells and iPS cells can be used.
- cells induced to differentiate from the stem cells can also be used as test cells.
- a medium used for culturing such test cells a medium generally used for culturing stem cells, for example, DMEM / F12, or a medium mainly composed of DMEM / F12 (for example, mTeSR1) is used. Can do.
- FIG. 4 shows the components of DMEM / F12.
- LC-MS liquid chromatograph mass spectrometer
- GC-MS gas chromatograph mass spectrometer
- a reagent that specifically develops or emits light from each of the biomarkers may be added to the culture supernatant, and the abundance of the biomarker may be determined based on the intensity of the developed or emitted light.
- FIG. 1 is a schematic diagram showing an execution procedure of a method for evaluating a cell differentiation state according to the present embodiment.
- the KhES-1 strain was planted and cultured on four culture dishes (diameter 60 mm) coated with Biocoat Matrigel (registered trademark, Corning International Co., Ltd.). Only one is shown).
- mTeSR1 modified Tenneille Serum Replacer 1
- the KhES-3 strain was also transferred to 4 culture dishes and cultured. Culturing is continued until the cells reach confluent, with the day of cell transplantation (passage) as day 0, and the culture supernatant collected from the culture dish at the time of medium exchange is replaced with a sample for mass spectrometry. did.
- the KhES-1 strain was transferred to four culture dishes (diameter 60 mm) coated with matrigel and cultured (FIG. 1 shows only one culture dish for simplification).
- As the medium mTeSR1 was used, and the medium was changed every day, and the culture was continued until it reached confluence.
- the KhES-3 strain was also transferred to 4 culture dishes and cultured.
- the day of passage was defined as day 0, and from the second day, differentiation induction stimulation was performed by replacing retinoic acid with mTeSR1 added to a final concentration of 0.1 ⁇ M during the medium exchange.
- the culture supernatant collected from the culture dish at the time of medium exchange on each day was used as a sample for mass spectrometry.
- mTeSR1 itself was used as a sample for mass spectrometry.
- the compound was quantified by a method of calculating the area of the mass chromatogram regarding ions characteristic of each derivatized compound in the sample according to the conditions set in the database.
- Quantification of each compound determined by performing the above-mentioned GC-MS analysis and LC-MS analysis on the culture supernatant collected on the last day of culture that is, the culture supernatant collected from the culture dish reaching confluence
- a value obtained by dividing the value (area value) by the quantitative value (area value) of the internal standard substance was calculated and used as an index value for the amount of each compound present in the culture supernatant.
- Tables 1 to 4 below show the compounds determined to have a significant difference in the abundance between the culture supernatant of the control cells and the culture supernatant of the test cells.
- E means a power of 10, for example, “1.326E-02” means “1.326 ⁇ 10 ⁇ 2 ”.
- Tables 1 and 2 below show compounds in which the abundance of the control cells in the culture supernatant is confirmed to be greater than the abundance of the test cells in the culture supernatant.
- Table 1 shows the results for the KhES-1 strain
- Table 2 shows the results for the KhES-3 strain.
- the “variation value” in these tables means A / B, that is, the ratio of “the average value of the index values in the control cells” to “the average value of the index values in the test cells”. ing.
- Tables 3 and 4 below show compounds that were confirmed to be present in the culture supernatant of the test cells in a larger amount than that in the culture supernatant of the control cells.
- Table 3 shows the results for the KhES-1 strain
- Table 4 shows the results for the KhES-3 strain.
- the “variation value” in these tables means the ratio of “average value of the index value in the test cell” to the above B / A, that is, “average value of the index value in the control cell”. ing.
- each of the compounds shown in Tables 1 to 4 is a biomarker for evaluating the differentiation state of a cell, in which the amount of metabolism in the cell and / or the amount of secretion outside the cell changes due to differentiation induction. It can be used as. That is, for example, any of the compounds described in Tables 1 and 2 is a stem cell that is unclear whether or not the undifferentiated state is maintained as a test cell, and a stem cell that is clearly undifferentiated is a control cell.
- the test It can be determined that the cell is not in an undifferentiated state.
- the ratio of the “abundance in the culture supernatant of the test cell” to the “abundance in the culture supernatant of the control cell” is obtained for any of the compounds described in Tables 3 and 4, and the value is determined in advance.
- the threshold value is equal to or greater than a predetermined threshold, it can be determined that the test cell is not in an undifferentiated state.
- a cell that is clearly differentiated is used as a control cell, and any of the compounds shown in Tables 1 and 2 is subjected to the The ratio of the “abundance amount of the test cell in the culture supernatant” is obtained, and when the value is equal to or greater than a predetermined threshold value, the test cell may be determined to be in an undifferentiated state.
- the ratio of the “abundance in the culture supernatant of the control cell” to the “abundance in the culture supernatant of the test cell” is obtained for any of the compounds described in Tables 3 and 4, and the value is determined in advance.
- the threshold is equal to or greater than a predetermined threshold, the test cell can be determined to be in an undifferentiated state.
- stem cell-derived differentiation-inducing cells for which it is unknown whether undifferentiated cells remain or not are used as test cells, and cells that are clearly undifferentiated are used as control cells.
- the ratio of the “abundance in the culture supernatant of the test cell” to the “abundance in the culture supernatant of the control cell” was determined for any of the compounds described, and the value was equal to or greater than a predetermined threshold. In this case, the test cell can be determined to contain a mixture of undifferentiated cells.
- the ratio of the “abundance in the culture supernatant of the control cell” to the “abundance in the culture supernatant of the test cell” is obtained for any of the compounds described in Tables 3 and 4, and the value is determined in advance.
- the threshold value is equal to or greater than a predetermined threshold, it can be determined that the test cells are mixed with undifferentiated cells.
- a differentiation-inducing cell derived from a stem cell in which it is unclear whether or not an undifferentiated cell remains as described above is used as a test cell, and it is clear that the control cell is differentiated, contrary to the above.
- Using cells determine the ratio of “abundance in the culture supernatant of the test cell” to “abundance in the culture supernatant of the control cell” for any of the compounds listed in Table 1 and Table 2. When the value is equal to or greater than a predetermined threshold, it can be determined that the test cells are mixed with undifferentiated cells.
- the ratio of the “abundance in the culture supernatant of the control cell” to the “abundance in the culture supernatant of the test cell” is obtained for any of the compounds described in Tables 3 and 4, and the value is determined in advance.
- the threshold value is equal to or greater than a predetermined threshold, it can be determined that the test cells are mixed with undifferentiated cells.
- the amount of the compound present in the culture supernatant of the control cell does not need to be measured at the same time as the test cell, and data measured in advance may be used. Good.
- FIG. 2 shows the result by GC-MS analysis
- FIG. 3 shows the result by LC-MS analysis
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Abstract
Description
前記所定物質が、プトレシン、キヌレニン、シスタチオニン、アスコルビン酸、リボフラビン、ピルビン酸、セリン、システイン、トレオン酸、クエン酸、及びオロト酸から成る群から選ばれる少なくとも一つの化合物であることを特徴としている。
バイオコートマトリゲル(登録商標、コーニングインターナショナル株式会社)がコートされた4枚の培養皿(直径60 mm)に前記KhES-1株を植え継いで培養を行った(図1では簡略化のため培養皿1枚のみを示している)。培地としてはmTeSR1(modified Tenneille Serum Replacer 1)を使用し、毎日培地の交換を行った。mTeSR1の構成成分を図5及び図6に示す。同様にKhES-3株についても4枚の培養皿に植え継いで培養を行った。細胞の植え継ぎ(継代)を行った日を0日目としてコンフルエント(confluent)に達するまで培養を継続し、各日の培地交換時に培養皿から回収した培養上清を質量分析用のサンプルとした。なお、培養0日目については、mTeSR1そのものを質量分析用サンプルとした。
マトリゲルがコートされた4枚の培養皿(直径60 mm)に前記KhES-1株を植え継いで培養を行った(図1では簡略化のため培養皿1枚のみを示している)。培地としてはmTeSR1を使用し、毎日培地の交換を行ってコンフルエントに達するまで培養を継続した。同様にKhES-3株についても4枚の培養皿に植え継いで培養を行った。継代を行った日を0日目とし、2日目からは前記培地交換の際に、レチノイン酸を終濃度0.1 μMとなるよう添加したmTeSR1に交換することにより、分化誘導刺激を行った。各日の培地交換時に培養皿から回収した培養上清を質量分析用のサンプルとし、培養0日目については、mTeSR1そのものを質量分析用サンプルとした。
前記サンプルにそれぞれ内部標準物質としてイソプロピルリンゴ酸を添加し、抽出溶液(メタノール:クロロホルム:水=2.5:1:1)で処理して除蛋白を行った。抽出後の上清を回収し、乾燥させた。
上述の前処理を行った各サンプルをメトキシアミン塩酸塩を含むピリジン溶液中でインキュベートすることにより、サンプル中の化合物のメトオキシム化を行った。更に、各サンプルにMSTFA(N-メチル-N-トリメチルシリルトリフルオロアセトアミド)を添加することでサンプル中の化合物をトリメチルシリル化した。そして、これらの誘導体化処理を施したサンプルをGC-MSによる分析に供した。分析結果の解析には、島津製作所製の「GCMS代謝成分データベースVer. 2」を使用した。該データベースは前記と同様の誘導体化処理を施した種々の化合物標準品をGC-MSで分析したデータが集約されたものである。化合物の同定は、前記データベースで設定された保持指標(保持時間を相対化した数値)とサンプル中の誘導体化化合物の保持指標との差が±5以内であるか否か、及び前記データベースで設定された定量イオン及び確認イオンの両者がサンプル中の誘導体化化合物について検出されているか否かを指標に行った。一方、化合物の定量は、前記データベースで設定された条件に従い、サンプル中の各誘導体化化合物に特徴的なイオンに関するマスクロマトグラムの面積を算出する方法により実施した。
上述の前処理を行った各サンプルに適当量の超純水(Milli-Q(登録商標)水、メルク株式会社)を加えて溶解させ、LC-MSによる分析に供した。LC-MS分析では、各サンプル中の化合物を逆相分離カラムを用いた勾配溶出によって時間的に分離した後、多重反応モニタリング(MRM、Multiple Reaction Monitoring)モードによる質量分析を行った。MRMモードにおける分析条件の設定は、化合物標準品を用いて実施した。化合物の同定は、標準品の保持時間とサンプル中の化合物の保持時間との差が±0.1分以内であるか否かを基準に行った。また、化合物の定量は、サンプル中の各化合物に特徴的なイオンについてマスクロマトグラムの面積を算出する方法により実施した。
Claims (8)
- 分化状態が未知の幹細胞あるいは幹細胞より分化誘導を行った細胞を被検細胞とし、該被検細胞の培養上清における所定物質の存在量に基づいて該被検細胞の分化状態を評価する方法であって、
前記所定物質が、プトレシン、キヌレニン、シスタチオニン、アスコルビン酸、リボフラビン、ピルビン酸、セリン、システイン、トレオン酸、クエン酸、及びオロト酸から成る群から選ばれる少なくとも一つの化合物であることを特徴とする細胞分化状態の評価方法。 - 前記被検細胞の培養上清における前記所定物質の存在量と、分化状態が既知である対照細胞の培養上清における前記所定物質の存在量とを比較することにより、前記被検細胞の分化状態を評価することを特徴とする請求項1に記載の細胞分化状態の評価方法。
- 前記対照細胞として分化していることが明らかな細胞を使用し、プトレシン、キヌレニン、シスタチオニン、アスコルビン酸、及びリボフラビンから成る群から選ばれる少なくとも一つの化合物の、前記対照細胞の培養上清における存在量に対する前記被検細胞の培養上清における存在量の比が予め定めた閾値以上である場合に、前記分化状態が未知の幹細胞は未分化状態である、又は前記幹細胞より分化誘導を行った細胞には未分化状態の細胞が混在していると判定することを特徴とする請求項2に記載の細胞分化状態の評価方法。
- 前記対照細胞として分化していることが明らかな細胞を使用し、ピルビン酸、セリン、システイン、トレオン酸、クエン酸、及びオロト酸から成る群から選ばれる少なくとも一つの化合物の、前記被検細胞の培養上清における存在量に対する前記対照細胞の培養上清における存在量の比が予め定めた閾値以上である場合に、前記分化状態が未知の幹細胞は未分化状態である、又は前記幹細胞より分化誘導を行った細胞には未分化状態の細胞が混在していると判定することを特徴とする請求項2に記載の細胞分化状態の評価方法。
- 前記対照細胞として未分化であることが明らかな細胞を使用し、プトレシン、キヌレニン、シスタチオニン、アスコルビン酸、及びリボフラビンから成る群から選ばれる少なくとも一つの化合物の、前記対照細胞の培養上清における存在量に対する前記被検細胞の培養上清における存在量の比が予め定めた閾値以上である場合に、前記分化状態が未知の幹細胞は未分化状態である、又は前記幹細胞より分化誘導を行った細胞には未分化状態の細胞が混在していると判定することを特徴とする請求項2に記載の細胞分化状態の評価方法。
- 前記対照細胞として未分化であることが明らかな細胞を使用し、ピルビン酸、セリン、システイン、トレオン酸、クエン酸、及びオロト酸から成る群から選ばれる少なくとも一つの化合物の、前記被検細胞の培養上清における存在量に対する前記対照細胞の培養上清における存在量の比が予め定めた閾値以上である場合に、前記分化状態が未知の幹細胞は未分化状態である、又は前記幹細胞より分化誘導を行った細胞には未分化状態の細胞が混在していると判定することを特徴とする請求項2に記載の細胞分化状態の評価方法。
- 前記幹細胞が多能性幹細胞であることを特徴とする請求項1~6のいずれかに記載の細胞分化状態の評価方法。
- 前記培養上清中の前記所定物質の存在量を質量分析法により定量することを特徴とする請求項1~7のいずれかに記載の細胞分化状態の評価方法。
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AU2015254267A AU2015254267B2 (en) | 2014-05-01 | 2015-04-21 | Method for assessing state of differentiation of cells |
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US15/307,948 US11022600B2 (en) | 2014-05-01 | 2015-04-21 | Method for assessing state of differentiation of cells |
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JP6332440B2 (ja) | 2018-05-30 |
KR101931420B1 (ko) | 2018-12-20 |
EP3138922A4 (en) | 2017-04-26 |
AU2015254267A1 (en) | 2016-11-24 |
US11022600B2 (en) | 2021-06-01 |
EP3138922B1 (en) | 2020-12-09 |
SG11201609091YA (en) | 2016-12-29 |
CN106460028B (zh) | 2020-03-10 |
JP6536711B2 (ja) | 2019-07-03 |
KR20160143804A (ko) | 2016-12-14 |
CN106460028A (zh) | 2017-02-22 |
JP2018121659A (ja) | 2018-08-09 |
AU2015254267B2 (en) | 2018-03-08 |
US20170052171A1 (en) | 2017-02-23 |
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