WO2021014515A1 - 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 PDF

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WO2021014515A1
WO2021014515A1 PCT/JP2019/028556 JP2019028556W WO2021014515A1 WO 2021014515 A1 WO2021014515 A1 WO 2021014515A1 JP 2019028556 W JP2019028556 W JP 2019028556W WO 2021014515 A1 WO2021014515 A1 WO 2021014515A1
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cells
abundance
cell
differentiation
pluripotent stem
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French (fr)
Japanese (ja)
Inventor
原　圭祐
慎一 五味
智久 永田
加川　健一
大島　康弘
鈴木　崇
雅俊 高橋
貴子 山本
伸 川真田
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Shimadzu Corp
Tokyo Electron Ltd
Foundation for Biomedical Research and Innovation at Kobe
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Shimadzu Corp
Tokyo Electron Ltd
Foundation for Biomedical Research and Innovation at Kobe
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Priority to JP2021534873A priority Critical patent/JP7344421B2/ja
Priority to PCT/JP2019/028556 priority patent/WO2021014515A1/ja
Priority to CN201980098491.9A priority patent/CN114450418A/zh
Priority to US17/628,023 priority patent/US20220260580A1/en
Priority to EP19938680.6A priority patent/EP4001425A4/en
Priority to TW109121789A priority patent/TWI855102B/zh
Publication of WO2021014515A1 publication Critical patent/WO2021014515A1/ja
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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    • C12N2533/50—Proteins

Definitions

  • the present invention relates to a method for evaluating the state of cell differentiation in the step of inducing differentiation of human induced pluripotent stem cells (iPS cells) into retinal pigment epithelial cells.
  • the present invention further relates to a method for evaluating the state of cell differentiation in the step of inducing differentiation of human embryonic stem cells (ES cells) into retinal pigment epithelial cells.
  • Age-related macular degeneration is a disease in which the function of the macula in the center of the retina is reduced, and is one of the main causes of blindness in the elderly in developed countries.
  • Age-related macular degeneration includes exudative and atrophic types.
  • Retinal pigment epithelial (RPE) cells form a sheet-like monolayer cell layer that exists outside the retina with macula and is important for maintaining photoreceptor cells in the retina. Further outside the RPE cell layer is the blood vessel-rich choroid. For normal vision, the RPE cells and choroids on the outside of the retina need to function normally.
  • Atrophic age-related macular degeneration is a disease in which RPE cells become inflamed due to aging, and the photoreceptor cells above them are lost together with the RPE cells, resulting in impaired vision.
  • VEGF inhibitors such as anti-VEGF (vascular endothelial cell growth factor) antibody
  • anti-VEGF antibody vascular endothelial cell growth factor
  • No effective treatment has yet been obtained for atrophic age-related macular degeneration. Since both wet and atrophic age-related macular degeneration are thought to be caused by functional deterioration or damage of RPE cells, they induce differentiation of RPE cells from pluripotent stem cells ES cells or iPS cells to induce new blood vessels. Attempts have been made to transplant the cells into the retina after removal.
  • Patent Document 1 a method for inducing differentiation from ES cells or iPS cells into RPE cells and a method for producing sheet-shaped retinal pigment epithelium from differentiated RPE cells have been developed (Patent Document 1, Non-Patent Document 1 and Non-Patent Document 1). Patent Document 2).
  • Non-Patent Document 3 since the amount of pigment can be measured after iPS cells are differentiated into RPE cells and the RPE cells have matured, RPE cells that are likely to be suitable for transplantation are selected before pigmentation. It is difficult to sort.
  • Non-Patent Documents 4 and 5 Induction of differentiation of pluripotent stem cells into RPE cells usually requires long-term culture of several tens of days (Non-Patent Documents 4 and 5). For this reason, predicting in culture whether differentiated RPE cells will be obtained is important in preparing for transplantation of RPE cells.
  • Non-Patent Document 5 reports OTX1 and OTX2 as markers of progenitor cells that may differentiate from ES cells to RPE cells.
  • OTX1 and OTX2 invasive treatment of cells by disrupting or lysing the cells is required, and cells to be transplanted cannot be evaluated.
  • Hirami Y Osakada F, Takahashi K, et al. Generation of retinal cells from mouse and human induced pluripotent stem cells. Neurosci Lett. 2009; 458: 126-131.
  • Kamao H Mandai M, Okamoto S, et al. TEXT of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application.
  • Kamao H, Mandai M Wakamiya S, et al. Objective evaluation of the degree of pigmentation in human induced pluripotent stem cell-derived RPE. Invest Ophthalmol Vis Sci. 2014; 55: 8309-8318.
  • An object of the present invention is to differentiate cells from pluripotent stem cells into RPE cells by performing non-invasive evaluation of the cells before pigmentation, which is an index of cell maturity, is observed. It is to provide a method for early discrimination between cells that do not.
  • a method for evaluating the differentiation state of pluripotent stem cells in the step of inducing differentiation of pluripotent stem cells into retinal pigment epithelial cells (1) A step of measuring the abundance of an indicator substance in the culture supernatant of pluripotent stem cells, and (2) The step of evaluating the differentiation state of cells from pluripotent stem cells to retinal pigment epithelial cells based on the change in the abundance of the indicator substance is included.
  • the method, wherein the indicator is ornithine and / or citrulline.
  • the differentiation state of pluripotent stem cells is evaluated by analyzing the time course of the abundance of the indicator substance in the culture supernatant.
  • the period for analyzing the change over time is the period from the 0th day to the 20th day, with the day when the medium for pluripotent stem cells is replaced with the medium for cell proliferation to the medium for cell differentiation as the 0th day. 2] The method described.
  • the period for analyzing the change over time is the period from the 3rd day to the 12th day, with the day when the medium for pluripotent stem cells is replaced with the medium for cell proliferation to the medium for cell differentiation as the 0th day. 2] The method described.
  • the state of cell differentiation from pluripotent stem cells to retinal pigment epithelial cells is evaluated based on the threshold of the coefficient of variation regarding the abundance of the indicator substance.
  • the method described in Crab. When the threshold of the coefficient of variation is 0.20 or more with respect to the abundance of ornithine and / or 0.30 or more with respect to the abundance of citrulline, it is evaluated that cell differentiation from pluripotent stem cells to retinal pigment epithelial cells progresses [7]. The method described in.
  • step (3) of measuring the abundance of the indicator substance in the culture supernatant of the control cells whose differentiation state is known The abundance of the indicator substance in the culture supernatant of the pluripotent stem cell measured in the step (1) and the culture supernatant of the control cell measured or measured in the step (3).
  • the ratio or difference between the abundance of the indicator substance in the culture supernatant of the pluripotent stem cell and the abundance of the indicator substance in the culture supernatant of the control cell is equal to or greater than a predetermined threshold or a threshold.
  • control cell is a cell in which an undifferentiated state is maintained in the step of inducing differentiation of pluripotent stem cells into retinal pigment epithelial cells.
  • step of inducing differentiation of pluripotent stem cells into retinal pigment epithelial cells a step of previously measuring the abundance of an indicator substance in the culture supernatant of the control cells, using cells in which cell differentiation has been confirmed as control cells (4). Including The state of cell differentiation is evaluated by comparing the abundance of the indicator substance in the culture supernatant of the pre-pluripotent stem cell with the abundance of the indicator substance in the culture supernatant of the control cell, [1]. The method described.
  • a method for evaluating the differentiation state of ES cells in the step of inducing differentiation of embryonic stem cells (ES cells) into retinal pigment epithelial cells (1) A step of measuring the abundance of an indicator substance in the culture supernatant of ES cells, and (2) The step of evaluating the differentiation state of cells from ES cells to retinal pigment epithelial cells based on the change in the abundance of the indicator substance is included.
  • the method, wherein the indicator is at least one selected from the group consisting of glutathione, ornithine, citrulline, cysteine, pipecolinic acid, putrescine, proline, 2-aminoadipic acid, cytidine, deoxycytidine, adenosine and inosine.
  • a method for evaluating the differentiation state of ES cells in the step of inducing differentiation of embryonic stem cells (ES cells) into retinal pigment epithelial cells (1) A step of measuring the abundance of an indicator substance in the culture supernatant of ES cells, and (2) The step of evaluating the differentiation state of cells from ES cells to retinal pigment epithelial cells based on the change in the abundance of the indicator substance is included.
  • the method, wherein the indicator substance is at least one selected from the group consisting of glutathione, ornithine, citrulline, pipecolic acid, 2-aminoadipic acid, cytidine and deoxycytidine.
  • [20] A method for evaluating the differentiation state of ES cells in the step of inducing differentiation of embryonic stem cells (ES cells) into retinal pigment epithelial cells.
  • (1) A step of measuring the abundance of an indicator substance in the culture supernatant of ES cells, and (2) The step of evaluating the differentiation state of cells from ES cells to retinal pigment epithelial cells based on the change in the abundance of the indicator substance is included.
  • the method wherein the indicator is adenosine and / or inosine.
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • RPE cells retinal pigment epithelial cells
  • the method of the present invention does not require invasive treatment of cells, that is, disruption or lysis of cells, and is non-invasive to cells because it measures indicator substances in cell supernatants, and in fact The success or failure of differentiation induction can be evaluated in advance for the cells themselves to be transplanted. Therefore, preparation for transplantation of RPE cells can be smoothly performed.
  • the results of culturing human iPS cells in independent culture vessels are shown.
  • the culture vessels 16035, 16040, 17005 and 17010 are the same as the culture vessels shown in FIG.
  • the period from the 3rd to the 12th day was the 0th day when the cell growth medium was replaced with the cell differentiation medium.
  • a marked increase in the abundance of ornithine was confirmed in, but no pigmentation was observed during that period.
  • At 16040 no significant increase in ornithine abundance was observed.
  • the results of culturing human iPS cells in independent culture vessels are shown.
  • the culture vessels 16035, 16040, 17005 and 17010 are the same as the culture vessels shown in FIG.
  • the period from the 5th to the 12th day was the 0th day when the cell growth medium was replaced with the cell differentiation medium.
  • a marked increase in the abundance of citrulin was confirmed, but no pigmentation was observed during that period.
  • iPS cells were cultured in independent culture vessels (16035, 16040, 17005 and 17010) and the abundance of ornithine (A) and citrulline (B) in each culture vessel was measured.
  • the culture vessels 16035, 16040, 17005 and 17010 are the same as the culture vessels shown in FIG.
  • the coefficient of variation was calculated based on the average value and standard deviation of the abundance measured at each measurement of the abundance of ornithine or citrulline during the cell culture period.
  • the present invention provides a method for evaluating the differentiation state of pluripotent stem cells in the step of inducing differentiation of pluripotent stem cells into retinal pigment epithelial cells.
  • the method is from pluripotent stem cells to retinal pigment epithelial cells based on (1) the step of measuring the abundance of the indicator substance in the culture supernatant of the pluripotent stem cell and (2) the abundance of the indicator substance.
  • the indicator substances are ornithine and / or citrulline.
  • the present invention further provides a method for evaluating the differentiation state of ES cells in the step of inducing differentiation of embryonic stem cells (ES cells) into retinal pigment epithelial cells.
  • the method is based on (1) a step of measuring the abundance of an indicator substance in the culture supernatant of ES cells and (2) a change in the abundance of the indicator substance, and the cells from ES cells to retinal pigment epithelial cells. Includes the step of evaluating the state of differentiation of.
  • the indicator substance is at least one selected from the group consisting of glutathione, cysteine, deoxycytidine, 2-aminoadipic acid, citrulline, pipecoric acid, putrescine, ornithine, cytidine, adenosine, inosine and proline.
  • the indicator substance may be at least one selected from the group consisting of glutathione, deoxycytidine, 2-aminoadiponic acid, citrulline, pipecolic acid, ornithine and cytidine.
  • retinal pigment epithelial cells are produced from pluripotent stem cells.
  • pluripotent stem cells undifferentiated cells such as iPS cells (Induced Pluripotent Stem cells) or ES cells (Embryonic Stem cells) can be used.
  • iPS cells are artificial stem cells and can be produced by introducing specific pluripotent inducers such as nucleic acids, proteins or low molecular weight compounds into somatic cells (Takahashi K. Yamanaka D. Cell, 2006). 126: 663-676, Takahashi K. et al. Cell, 2007; 131: 861-872, WO2007 / 069666).
  • ES cells can be produced by removing the inner cell mass from the blastocyst of a fertilized mammalian egg and culturing this inner cell mass (Suemori H. et al. Biochem Biophys Res Communi. 2006; 345: 926-932).
  • iPS cells and ES cells cells derived from mammals such as humans, monkeys, mice, rats, dogs, cats, cows, horses, pigs, sheep, goats, rabbits, hamsters and guinea pigs can be used. However, it is preferably a human-derived cell.
  • the stem cells are first cultured in a cell proliferation medium and then replaced with a cell differentiation medium.
  • a cell proliferation medium As the culture conditions and medium for differentiating the stem cells into retinal pigment epithelial cells, culture conditions and media known to those skilled in the art can be used, but those skilled in the art can also appropriately modify them.
  • a commercially available medium may be used, and examples of the cell growth medium include Essential 8 (registered trademark) medium, and examples of the cell differentiation medium include Essential 6 (registered trademark) medium.
  • differentiation into retinal pigment epithelial cells can be confirmed by confirming the expression of genes characteristic of retinal pigment epithelial cells.
  • Genes characteristic of retinal pigment epithelial cells include RPE65, CRALBP, MERTK and BEST1.
  • analysis by immunostaining such as undifferentiated markers Oct3 / 4, Sox and TRA-1-60, or undifferentiated marker genes (OCT3 / 4, Nanog and It can be evaluated by the measurement of Lin28 etc.).
  • the method of the present invention can identify the differentiating cells or cell population at an early stage before the differentiation of pluripotent stem cells iPS cells or ES cells into retinal pigment epithelial cells is confirmed.
  • the abundance of the indicator substance in the culture supernatant of iPS cells or ES cells is measured.
  • the indicator substance is a compound released into the culture supernatant as a metabolite in iPS cells or ES cells, or a medium component contained in the culture medium for differentiation, and is composed of ornithine and citrulline, which are one type of amino acids. preferable.
  • ES cells in addition to ornithine and cytidine, glutathione, cysteine, and pipecholine are used as index substances for early discrimination of cells or cell populations that differentiate from ES cells to retinal pigment epithelial cells.
  • Select acid pipecolic acid
  • putrescine putrescine
  • proline 2-aminoadipic acid
  • cytidine deoxycytidine
  • adenosine and inosine can do.
  • the step (2) of the present invention is a cell from a pluripotent stem cell to a retinal pigment epithelial cell based on the abundance of the indicator substance in the culture supernatant of the pluripotent stem cell measured in the step (1) of the present invention. It is a step of evaluating the differentiation state of.
  • the differentiation state of pluripotent stem cells is evaluated by analyzing the time course of the abundance of the indicator substance.
  • the time-dependent change in the abundance of the indicator substance is a change in the amount of the indicator substance in the culture supernatant with the passage of the culture time of the pluripotent stem cells.
  • the amount of the indicator substance in the culture supernatant can be indicated by the concentration or absolute amount of the indicator substance in the culture solution, the amount of the indicator substance per unit number of cells, the total amount of the indicator substance per culture vessel, and the like. Not limited.
  • To analyze the time course of the abundance of the indicator substance in the culture supernatant detect a transient or persistent increase or decrease in the abundance in the graph or profile of the time change of the abundance of the indicator substance. Is included.
  • a transient or persistent increase or decrease in the abundance of the indicator substance in the culture supernatant is recognized as a variation from the baseline of the graph or profile of the abundance over time.
  • the period for analyzing the time course of the abundance of ornithine and / or citrulline in the culture supernatant is the day when the medium for pluripotent stem cells is replaced with a medium for cell proliferation from a medium for cell differentiation. Is preferably the 0th day, and the period is preferably from the 0th day to the 20th day.
  • the analysis of the change over time may be performed over the entire period, but as long as the change is observed, it may be analyzed in a shorter period, for example, from the 3rd day to the 12th day.
  • ornithine abundance it may be analyzed in the period from the 4th day to the 12th day, and in the case of the citrulline abundance, it may be analyzed in the period from the 8th day to the 12th day. You may.
  • the replacement of the cell growth medium with the cell differentiation medium is performed after culturing in the cell growth medium for 7 to 10 days, but the number of days is increased or decreased depending on the degree of cell growth.
  • the abundance of ornithine or citrulline in the culture supernatant of pluripotent stem cells changes significantly with time
  • cell differentiation from the pluripotent stem cells into retinal pigment epithelial cells progresses. It is evaluated that there is a high possibility of doing so.
  • the change over time is large
  • the abundance of ornithine or citrulline increases rapidly due to a large variation or deviation from the baseline of the graph or profile of the abundance of ornithine or citrulline over time. It refers to a state in which it decreases thereafter.
  • a clear peak is observed over time in the abundance of ornithine or citrulline.
  • the peak over time may be a monomodal peak or a multimodal peak.
  • iPS cells or ES cells which are pluripotent stem cells
  • ES cells which are pluripotent stem cells
  • pigmentation which is an indicator of differentiation.
  • a peak in the time course of the abundance of ornithine or citrulline in the culture supernatant is observed before the time when pigmentation occurs in the cells.
  • pigmentation which is an index of differentiation into retinal pigment epithelial cells, occurs by continuing the culture thereafter. Therefore, when the peak is observed, the pluripotent stem cells to the retinal pigment It is evaluated that there is a high possibility that cell differentiation into epithelial cells will progress.
  • cell differentiation from pluripotent stem cells to retinal pigment epithelial cells occurs when the abundance of ornithine and citrulline in the culture supernatant of pluripotent stem cells is substantially the same over time. Evaluated as it progresses. Since the evaluation is based on both the change in the abundance of ornithine and citrulline over time, the reliability of the evaluation result is higher than when the evaluation is performed using either the change in the abundance of ornithine or citrulline alone. improves. The time course of the abundance of ornithine and citrulline in the culture supernatant is substantially the same, which means that the time change satisfies the following two conditions.
  • the peak observed in the time course of the abundance of ornithine and citrulline is a peak having a shape in which the abundance increases with the lapse of the culture time and then decreases.
  • the culture time at which the main peak with the highest abundance is obtained is almost the same. What is almost common is that the difference in culture time giving the main peak position between ornithine and citrulline is within 5 days, preferably within 3 days, and more preferably within 1 day.
  • the state of cell differentiation from pluripotent stem cells to retinal pigment epithelial cells is evaluated based on the coefficient of variation with respect to the abundance of the indicator substances ornithine and / or citrulline.
  • the coefficient of variation is calculated by dividing the standard deviation of the abundance by the average value of the abundance. That is, during the culture period of pluripotent stem cells, the number of times the abundance of the index substance is measured is used as the number of data, and the average value of the abundance measured each time is calculated. Next, the positive square root of the value obtained by dividing the sum of the squares of the difference between the measured abundance value and the average value of the abundance at each time by the number of data is calculated as the standard deviation.
  • the coefficient of variation is calculated by dividing the standard deviation calculated in this way by the average value of the abundance.
  • the coefficient of variation is the ratio of the magnitude of the variation of the data, and it is shown that the larger the coefficient of variation is, the larger the variation in the abundance of the indicator substance measured during the culture period is, and conversely, the smaller the coefficient of variation is. , It is shown that the variation in the abundance of the indicator substance measured during the culture period is small.
  • pluripotent stem cells to retinal pigment epithelial cells when the coefficient of variation threshold is 0.20 or higher for ornithine abundance, preferably 0.25 or higher and / or 0.30 or higher for citrulline abundance. It is evaluated that cell differentiation into the disease progresses. That is, when the abundance of ornithine and / or citrulline in the culture supernatant shows a certain fluctuation during the culture period, it is evaluated that the cell differentiation progresses.
  • the coefficient of variation threshold for the abundance of ornithine and / or citrulline is a value empirically derived from the comparison between the calculated value of the coefficient of variation and the state of cell differentiation from pluripotent stem cells to retinal pigment epithelial cells.
  • the culture period for calculating the coefficient of variation can be arbitrarily determined as long as it is after the day when the medium for pluripotent stem cells is replaced with the medium for cell proliferation and the medium for cell differentiation.
  • the culture period is preferably a period from the 0th day to the 20th day, with the day when the medium for pluripotent stem cells is replaced with the medium for cell proliferation to the medium for cell differentiation as the 0th day, and more preferably. It is the 3rd to the 12th day. In the case of ornithine abundance, such a culture period is more preferably from day 4 to day 12, and in the case of citrulline abundance, the period from day 8 to day 12 is preferable. More preferred.
  • the step of the present invention further comprises the step (3) of measuring the abundance of an indicator substance (ornithine and / or citrulin) in the culture supernatant of a control cell whose differentiation state is known.
  • a method for evaluating the state of cell differentiation from pluripotent stem cells to retinal pigment epithelial cells is provided by comparing with the abundance of the indicator substance.
  • the step (3) for measuring the abundance of the indicator substance in the culture supernatant of the control cells may be performed at the same time as the step (1), or may be performed independently of the step (1).
  • the control cell may be a pluripotent stem cell derived from the same cell line as the cell subjected to the induction of differentiation from the pluripotent stem cell to the retinal pigment epithelial cell, or a pluripotent different from the cell subjected to the induction of differentiation. It may be a cell derived from a sex stem cell line.
  • a control cell in which the differentiation state of the cell is known is a cell in which it is known whether the cell is in an undifferentiated state or a differentiated state, and the differentiation state does not change during the culture period.
  • the undifferentiated state is confirmed by immunostaining for the presence of undifferentiated markers such as Oct3 / 4, Sox and TRA-1-60, or by measurement of undifferentiated marker genes (OCT3 / 4, Nanog and Lin28, etc.). can do.
  • the differentiated state is, for example, in the case of retinal pigment epithelial cells, by the presence of brown pigmentation (melanin) or polygonal or paving stone cell morphology, or by measuring gene expression such as RPE65, CRALBP, MERTK and BEST1. Can be confirmed by.
  • a method of assessing the state of cell differentiation from pluripotent stem cells to retinal pigment epithelial cells is provided based on whether the difference is greater than or equal to a predetermined threshold or below the threshold.
  • the above threshold increased the number of cell culture samples for the culture supernatants of cells confirmed to differentiate from pluripotent stem cells to retinal pigment epithelial cells and cells not confirmed to differentiate, and the amount of ornithine and citrulin in each sample. It can be derived by repeatedly measuring the abundance. That is, the threshold value is an empirically derived value and can be easily set by those skilled in the art.
  • the time course of the abundance of the indicator substance (ornitin and / or citrulin) in the culture supernatant of the pluripotent stem cells and the time course of the abundance of the indicator substance in the culture supernatant of the control cells is provided.
  • the time-dependent change in the abundance of the indicator substance is a change in the amount of the indicator substance in the culture supernatant with the lapse of the culture time for both the pluripotent stem cells and the control cells.
  • the amount of the indicator substance can be indicated by the concentration or absolute amount of the indicator substance in the culture solution, the amount of the indicator substance per unit number of cells, the total amount of the indicator substance per culture vessel, and the like, but is not limited thereto. Comparing the time course of the abundance of the indicator substance in the culture supernatant is to grasp the similarity or dissimilarity of the graph or profile of the time change of the abundance of the indicator substance.
  • a cell whose undifferentiated state is maintained in the step of inducing differentiation from pluripotent stem cells to retinal pigment epithelial cells may be used.
  • maintaining an undifferentiated state means that pluripotent stem cells have not differentiated into retinal pigment epithelial cells, and pigmentation (melanin) is deposited. Is not observed, or the expression of genes characteristic of retinal pigment epithelial cells such as RPE65, CRALBP, MERTK and BEST1 is not observed. Maintaining pluripotent stem cells in an undifferentiated state is achieved by subculturing the pluripotent stem cell medium as it is, without replacing it with a cell differentiation medium.
  • cells in which cell differentiation has been confirmed are used as control cells, and indicator substances (ornithine and ornithine) in the culture supernatant of the control cells are used.
  • the abundance of the indicator substance in the culture supernatant of the pluripotent stem cell and the presence of the indicator substance in the culture supernatant of the control cell further include the step (4) of measuring the abundance of (/ or citrulin) in advance.
  • a method of assessing the state of differentiation of cells is provided by comparing with the amount.
  • the abundance of the indicator substance in the culture supernatant is measured in advance for control cells in which differentiation of pluripotent stem cells into retinal pigment epithelial cells has been confirmed, and the measured values are used to induce differentiation. It is used as a reference value or an index for determining that the product has been used. According to this method, it is possible to determine whether or not the cultured pluripotent stem cells differentiate into retinal pigment epithelial cells based on the reference value or the index.
  • the comparison between the abundance of the indicator substance in the culture supernatant of the pluripotent stem cell and the abundance of the indicator substance in the culture supernatant of the control cell is performed by comparing the abundance at a specific culture time. Can be done.
  • the state of differentiation of pluripotent stem cells into retinal pigment epithelial cells can be evaluated based on a threshold value determined based on the abundance of the indicator substance in the culture supernatant of the control cells.
  • information on the differentiation state of a plurality of types of control cells obtained in the step (4) and the abundance of an indicator substance (ornithine and / or citrulline) in the culture supernatant of the control cells is provided. It can be recorded as a library for each control cell.
  • the abundance of the indicator substance in the culture supernatant of the pluripotent stem cells in the differentiation induction step can be determined in the library corresponding to the same cell line as the pluripotent stem cells in the differentiation induction step. It is also possible to evaluate the differentiation state of cells by collating with the information on the abundance of cells.
  • the comparison between the abundance of the indicator substance in the culture supernatant of the pluripotent stem cell and the abundance of the indicator substance in the culture supernatant of the control cell is a comparison of the abundance at a plurality of culture times, that is, the abundance. It is also done by comparing graphs or profiles of changes over time. This is because the accuracy of determining whether or not differentiated is improved by comparing the abundances in a plurality of culture times. Whether or not they are differentiated can be determined based on the similarity of the graph or profile.
  • the similarity means that the shape of the graph or profile is similar with the passage of the culture time.
  • a gas chromatograph method GC
  • LC liquid chromatograph method
  • MS mass spectrometry method
  • LC-MS liquid chromatograph mass spectrometry
  • GC-MS gas chromatograph mass spectrometry
  • the analysis by LC-MS can be performed as follows. To 100 ⁇ L of the culture supernatant sample, add 20 ⁇ L of 0.5 mM isopropylmalic acid aqueous solution as an internal standard, mix, and then add 200 ⁇ L of acetonitrile or methanol to remove protein. The obtained sample was centrifuged at 15,000 rpm for 15 minutes at room temperature using a centrifuge manufactured by Tomy Seiko, and then the supernatant was recovered to obtain ultrapure water (MiLLi-Q® water, Merck Co., Ltd.). ) Is diluted 10-fold and subjected to LC-MS analysis.
  • the LC-MS analysis follows the analysis conditions recorded in "LC / MS / Method Package Cell Culture Profiling" (hereinafter abbreviated as "MP") manufactured by Shimadzu Corporation.
  • MP is a collection of analytical condition parameters for analyzing compounds contained in a medium and metabolites secreted from cells by LC-MS. To identify the compound, the difference between the retention time of the standard product registered in MP and the retention time of the compound in the sample is within ⁇ 0.3 minutes, and both peaks of quantitative ions and confirmed ions are detected. , And the strength value is 1000 or more.
  • the quantification of the compound is performed by a method of calculating the area of the mass chromatogram for the ion (quantitative ion) characteristic of each compound in the sample.
  • the analysis by GC-MS can be performed as follows. To 100 ⁇ L of the culture supernatant sample, add 10 ⁇ L of 0.5 mg / mL isopropylmalic acid aqueous solution as an internal standard, mix, and then add 200 ⁇ L of acetonitrile to remove protein. The obtained sample is centrifuged at 15,000 rpm for 15 minutes at room temperature, 100 ⁇ L of the supernatant is collected, and the sample is dried under reduced pressure. Each sample is incubated in a pyridine solution containing methoxyamine hydrochloride to methoxyminate the compounds in the sample.
  • MSTFA N-methyl-trimethylsilyltrifluoroacetamide
  • DB Smart Metabolites Database
  • the DB is a collection of data obtained by analyzing various standard products subjected to the same treatment as the above-mentioned derivatization treatment by GC-MS.
  • Compound identification is determined by whether or not the difference between the retention index set in the DB (a value obtained by relativizing the retention time) and the retention index of the derivatized compound in the sample is within ⁇ 5, and whether the difference is within ⁇ 5.
  • the quantification of the compound is carried out by a method of calculating the area of the mass chromatogram for the ion characteristic of each derivatized compound in the sample according to the conditions set in the DB.
  • the peak area value (Area) of the indicator substance in these measurement methods or the index substance
  • concentration (Concentration) of the indicator substance in the culture supernatant calculated from the peak area value (Area) can be used.
  • Concentration / Cell Number may be used.
  • cells By using a value corrected (standardized) by the cell density (Confluency) or the number of cells (Cell Number or Cell Count), cells can be placed between the pluripotent stem cells in the differentiation induction step and the control cells. Even if there is a difference in the proliferation rate of the above cells, it is possible to compare the peak area value (Area / Confluency) of the indicator substance or the concentration (Concentration / Confluency) of the indicator substance per number of cells among the cells. it can. Therefore, it is possible to evaluate the differentiation state of cells with high accuracy.
  • the present invention provides a method for producing retinal pigment epithelial cells from pluripotent stem cells, including a method for evaluating the differentiation state of the pluripotent stem cells of the present invention.
  • Pluripotent stem cells include iPS cells and ES cells.
  • the method for producing retinal pigment epithelial cells of the present invention can discriminate between cells that differentiate into retinal pigment epithelial cells and cells that do not differentiate in the step of producing retinal pigment epithelial cells from pluripotent stem cells.
  • the method for producing retinal pigment epithelial cells of the present invention can include a step of removing cells or cell populations that are determined not to differentiate into retinal pigment epithelial cells.
  • a monomodal peak in ornithine abundance was observed during the period from the 3rd day to the 12th day.
  • a monomodal peak in citrulline abundance was observed during the period from the 5th day to the 15th day, with the day when the medium for cell proliferation was replaced with the medium for cell differentiation as the 0th day.
  • the abundance of ornithine did not fluctuate much, and the cell growth medium was replaced with the cell differentiation medium. No clear peak was observed in the time course of ornithine abundance in the period up to the 40th day, with the day 0 as the day 0.
  • the amount of citrulline in the culture supernatant of cells in the culture vessel 16040 tended to gradually increase from around the 25th day, with the day when the cell growth medium was replaced with the cell differentiation medium as the 0th day, but there was a clear peak. was not recognized.
  • the coefficient of variation of ornithine abundance in the culture supernatants of cells in culture vessels 16035, 17005 and 17010 differentiated into retinal pigment epithelial cells peaks over time in ornithine abundance during the period from day 0 to day 20 A value of 0.20 or more was given in at least the period from the 4th day to the 12th day. On the other hand, in the culture supernatant of the cells of the culture vessel 16040, which was shown to remain undifferentiated, the coefficient of variation was less than 0.20 in the period from the 6th day to the 20th day.
  • the coefficient of variation of citrulline abundance in the culture supernatants of cells in culture vessels 16035, 17005 and 17010 differentiated into retinal pigment epithelial cells peaks over time in citrulline abundance during the period from day 0 to day 20 In the period from at least the 8th day to the 12th day, a value of 0.60 or more was given. On the other hand, in the culture supernatant of the cells in the culture vessel 16040, which was shown to remain undifferentiated, the coefficient of variation was less than 0.30 during the period from the 8th day to the 12th day.
  • FIGS. 6a to 6c The time course of the abundance of each indicator substance after the start of culture is shown in FIGS. 6a to 6c: (A) ornithine, (B) citrulline, (C) glutathione, (D) cysteine, (E) pipechoric acid, (F). ) Putrescine, (G) proline, (H) 2-aminoadipic acid, (I) cytidine, (J) deoxycytidine, (K) adenosine and (L) inosine.
  • a peak abundance was given at a culture time of 1 to 10 days.
  • glutathione produced almost no human iPS cells at a culture time of 1 to 10 days, which was significantly different from that of human ES cells.
  • deoxycytidine (Fig. 6J) rapidly increased in abundance by 8 days after culturing and then decreased.
  • a peak abundance was given at a culture time of 5 to 10 days.
  • Deoxycytidine also gave a peak abundance during the culture time of 20-30 days.
  • Ornithine (Fig. 6A) and citrulline Fig.

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007069666A1 (ja) 2005-12-13 2007-06-21 Kyoto University 核初期化因子
WO2014030749A1 (ja) 2012-08-24 2014-02-27 独立行政法人理化学研究所 網膜色素上皮細胞シートの製造方法
WO2015166845A1 (ja) * 2014-05-01 2015-11-05 株式会社島津製作所 細胞の分化状態の評価方法
JP2017504311A (ja) * 2013-12-11 2017-02-09 ファイザー・リミテッドＰｆｉｚｅｒ Ｌｉｍｉｔｅｄ 網膜色素上皮細胞を生成する方法
WO2017068727A1 (ja) * 2015-10-23 2017-04-27 株式会社島津製作所 細胞の分化状態の評価方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2209888T3 (da) * 2007-10-12 2020-01-20 Astellas Inst For Regenerative Medicine Forbedrede fremgangsmåder til fremstilling af rpe-celler og sammensætninger af rpe-celler
WO2012056997A1 (ja) * 2010-10-28 2012-05-03 国立大学法人熊本大学 多能性幹細胞の分化誘導効率を改善するための方法及び培地
KR101357851B1 (ko) * 2011-11-02 2014-02-06 제일약품주식회사 낭성구조물로부터 망막색소상피세포의 분화를 유도하는 방법
US10519422B2 (en) * 2012-02-29 2019-12-31 Riken Method of producing human retinal pigment epithelial cells
SG11201801770VA (en) * 2015-09-08 2018-04-27 Sumitomo Dainippon Pharma Co Ltd Method for producing retinal pigment epithelial cells
CN106609256B (zh) * 2015-10-22 2020-04-07 同济大学 体外诱导人胚胎干细胞分化为视网膜色素上皮细胞的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007069666A1 (ja) 2005-12-13 2007-06-21 Kyoto University 核初期化因子
WO2014030749A1 (ja) 2012-08-24 2014-02-27 独立行政法人理化学研究所 網膜色素上皮細胞シートの製造方法
JP2017504311A (ja) * 2013-12-11 2017-02-09 ファイザー・リミテッドＰｆｉｚｅｒ Ｌｉｍｉｔｅｄ 網膜色素上皮細胞を生成する方法
WO2015166845A1 (ja) * 2014-05-01 2015-11-05 株式会社島津製作所 細胞の分化状態の評価方法
WO2017068727A1 (ja) * 2015-10-23 2017-04-27 株式会社島津製作所 細胞の分化状態の評価方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
HIRAMI YOSAKADA FTAKAHASHI K ET AL.: "Generation of retinal cells from mouse and human induced pluripotent stem cells", NEUROSCI LETT, vol. 458, 2009, pages 126 - 131
KAMAO HMANDAI MOKAMOTO S ET AL.: "Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application", STEM CELL REPORTS, vol. 2, 2014, pages 205 - 218, XP055144196, DOI: 10.1016/j.stemcr.2013.12.007
KAMAO HMANDAI MWAKAMIYA S ET AL.: "Objective evaluation of the degree of pigmentation in human induced pluripotent stem cell-derived RPE", INVEST OPHTHALMOL VIS SCI., vol. 55, 2014, pages 8309 - 8318
LANE APHILIP LRRUBAN L ET AL.: "Engineering efficient retinal pigment epithelium differentiation from human pluripotent stem cells", STEM CELLS TRANSL MED, vol. 3, 2014, pages 1295 - 1304, XP055303892, DOI: 10.5966/sctm.2014-0094
SUEMORI H. ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 345, 2006, pages 926 - 932
TAKAHASHI K. ET AL., CELL, vol. 131, 2007, pages 861 - 872
TAKAHASHI K.YAMANAKA D., CELL, vol. 126, 2006, pages 663 - 676
VUGLER ACARR AJLAWRENCE J ET AL.: "Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation", EXP NEUROL, vol. 214, 2008, pages 347 - 361, XP025695837, DOI: 10.1016/j.expneurol.2008.09.007

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