JP2019033746A - Screening method of staining marker and labeling method of tumorigenic cells - Google Patents

Abstract

To provide screening methods of a label marker for potential tumorigenic cells that may be included in products such as regenerative medicines, and to provide markers that can label potential tumorigenic cells.SOLUTION: To screen a marker capable of specifically labeling pluripotent stem cells and initial differentiated cells having tumorigenicity, a staining marker that matches the purpose is selected by digitizing the staining results obtained by various markers. Tumorigenic cells comprising in a product such as a regenerative medicine can be detected by using SSEA-4 obtained, rBC2 LCN, and alkaline phosphatase as a result thereof.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for screening a marker for staining cells, particularly a specific marker for labeling pluripotent stem cells having tumorigenic potential and pluripotent stem cells (partially differentiated cells) that have started early differentiation. It relates to the screening method.

  Since pluripotent stem cells can be differentiated into almost all cells constituting a living body, application to regenerative medicine research and drug discovery screening is expected. Until now, the development of a technique for inducing differentiation from a pluripotent stem cell to a target cell has progressed.

  Pluripotent stem cells form teratomas when transplanted in vivo, so if cells that have been induced to differentiate from pluripotent stem cells are used for regenerative medicine, etc., the possibility of pluripotent stem cells and tumor formation It is necessary to prevent contamination of certain cells.

  In order to detect a cell having a possibility of tumor formation that can be contained in a pluripotent stem cell-derived cell, a method using a marker specific to the pluripotent stem cell is generally used. However, not only completely undifferentiated pluripotent stem cells but also cells that have induced early differentiation have a risk of tumor formation (Non-patent Document 1), and thus tumor formation with partially advanced differentiation is possible. Sexual cells must also be detected. However, there has been no report so far on a screening method for markers capable of detecting cells having a possibility of tumor formation and markers that can actually be labeled.

Long Z et al. , JOURNAL OF BIOLOGICAL CHEMISTRY, 287, 32338-32345 (2012)

  An object of the present invention is to screen a marker for labeling a cell having a possibility of tumor formation, which can be contained in a cell induced differentiation from a pluripotent stem cell or a product such as regenerative medicine for use in regenerative medicine. And providing a marker capable of labeling cells with a potential for tumor formation.

  In order to solve the above-mentioned problems, the present inventors have intensively studied to arrive at the present invention.

That is, the present invention can be exemplified as follows.
[1]
It is a screening method for markers that can specifically stain pluripotent stem cells, and the results of staining with various markers are quantified and a staining marker that matches the purpose is selected.
[2]
The method according to [1], comprising a marker capable of staining cells partially differentiated from pluripotent stem cells.
[3]
Obtaining initial differentiated cells by partially differentiating pluripotent stem cells;
Staining initial differentiated cells with a staining marker;
The method according to [2], comprising a step of obtaining an image of an early differentiated cell, and a step of detecting staining of the early differentiated cell with a staining marker based on the image.
[4]
The method according to [2] or [3], wherein the partially differentiated cell is one or more selected from the group consisting of an ectoderm cell, a mesoderm cell, and an endoderm cell.
[5]
The method according to [2] or [3], wherein the partially differentiated cells are any two selected from the group consisting of ectoderm cells, mesoderm cells, and endoderm cells. .
[6]
The method according to [2] or [3], wherein the partially differentiated cells are ectoderm cells, mesoderm cells, and endoderm cells.
[7]
The method according to any one of [1] to [6], wherein the staining marker is a fluorescently labeled antibody.
[8]
The method according to any one of [1] to [6], wherein the staining marker is an enzyme.
[9]
The method according to any one of [1] to [6], wherein the staining marker is SSEA-4, rBC2LCN, or alkaline phosphatase.

  Hereinafter, the present invention will be described in detail.

  In the present invention, a pluripotent stem cell refers to a cell that can differentiate into all cell lineages in vivo and in vitro. Specific examples include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

  The partially differentiated cell in the present invention, that is, an early differentiated cell refers to a cell that has been induced to differentiate from a pluripotent stem cell. Specific examples include, but are not limited to, cells that have started to induce differentiation into ectoderm, mesoderm, and endoderm. However, somatic cells that have completely lost the properties of pluripotent stem cells are not included.

  In the present invention, the partial differentiation induction method is a method in which undifferentiated pluripotent stem cells are subjected to a specific treatment and cultured for a specific period, but the treatment method and the period are not limited. For example, early differentiated cells can be obtained by using a Human Pluripotent Stem Cell Functional Identification Kit (manufactured by R & D Systems). Moreover, initial differentiation can be induced by the addition of Y-27632, which is a ROCK inhibitor (Maldonado M et al., Stem Cell Research, 17, 222-227, 2016).

  For comparison of marker selection, a negative control cell is required. Although the kind of cell used as a negative control is not limited, it needs to be a somatic cell that is more differentiated than pluripotent stem cells and early differentiated cells. Moreover, it is desirable that the cells are normal cells, not cancer cells or immortalized cells. Examples include mesenchymal stem cells, fibroblast cell lines, and other normal cells. Furthermore, somatic cells that have been induced to differentiate from pluripotent stem cells can also be used as negative controls.

  For the labeling of cells with the potential for tumor formation, a reagent that specifically stains pluripotent stem cells and does not stain differentiated somatic cells is used. For example, antibodies against cell surface antigens or intracellular antigens, proteins that bind to cell surface sugar chains, or substrates for enzymes specific for pluripotent stem cells are used. Specifically, cell surface antigens (TRA-1-60, TRA-1-81, SSEA-3, SSEA-4), intracellular antigens (POU5F1, NANOG), cell surface sugar chain binding protein (lectin) ( rBC2LCN) and enzyme (alkaline phosphatase) substrates are preferred, but not limited thereto. In addition, antibodies and cell surface sugar chain binding proteins are labeled using a fluorescent labeling substance (such as an antibody) that binds a fluorescent dye directly or binds to a staining reagent, but the method is not limited. A known method or a commercially available staining reagent can be used to detect the enzyme. For example, Stemgent Alkaline Phosphatase Staining Kit II (manufactured by Reprocell) can be mentioned.

  The labeled cells are imaged under a microscope, and the staining intensity is digitized using image analysis software. The image analysis software to be used is not limited and can be converted into a numerical value using Image J or the like. In order to quantify the staining intensity of each cell, for example, there is a method of setting a Region of Interest (ROI) using a nuclear staining image of each cell as a mask. Although the reagent used for nuclear staining is not limited, for example, a reagent that emits fluorescence, such as 2- (4-amidophenyl) -1H-indole-6-carboxamide (DAPI), Hoechst 33342, and Propidium Iodide, is preferable. However, when a fluorescent dye is used as a reagent for staining pluripotent stem cells, the wavelength of fluorescence needs to be different from that of the nuclear staining reagent. For quantification of nuclear antigens (POU5F1, NANOG), the mask of the nuclear staining image is used as ROI. For cell surface antigens, the range of ROI is expanded centering on the nucleus, and ROI is further applied to the region not containing the nucleus. It can be quantified by setting.

  By comparing the staining intensity of individual cells, an optimal marker for screening for tumorigenic cells can be selected. For example, a histogram is created using software such as Microsoft EXCEL, and an optimal marker is selected from parameters such as peak spread and number. As an index for selection, (I) that there is one peak, (II) that the spread of the peak is small and uniform, and (III) the peak of undifferentiated cells and the peak of early differentiated cells are not too far apart. And (IV) that it can be distinguished from a negative control (somatic cell etc.) used as a comparison.

  In addition to the method of setting the ROI using the nucleus as a mask, the staining intensity can be compared with a simple method by digitizing the staining intensity of the entire region where cells exist and obtaining an average value. For example, using software such as Image J, the staining intensity of randomly selected cell regions can be quantified. Quantitative comparison is possible by using the intensity of the area where no cells exist as a blank. When using a simple method, as an index for selecting a marker for staining cells with a potential for tumor formation, (i) the numbers of undifferentiated pluripotent stem cells and early differentiated cells are relatively equivalent, (Ii) It can be distinguished from negative controls (such as somatic cells) used for comparison.

  By using the above method, a marker capable of detecting cells having a possibility of tumor formation can be selected by comparing the staining intensity of cells labeled with the respective markers. From the results of examining the above-mentioned eight types of markers, three types of SSEA-4, rBC2LCN, and alkaline phosphatase are excellent as markers that can label pluripotent stem cells and early differentiated cells.

  In the present invention, three types of SSEA-4, rBC2LCN, and alkaline phosphatase are proposed as markers capable of labeling tumorigenic cells. However, when a novel labeling marker is identified using the above screening method, Not limited to species. Further, when screening is performed, it is not limited whether the cells are in an adherent state or are dispersed as single cells. In the case of screening in an adhered state, it is preferable from the viewpoint of obtaining the expression location of the marker, position information in the colony of the cell expressing the marker, and the like. On the other hand, when using a cell suspension dispersed as a single cell by enzyme treatment or the like, the cell suspension is introduced by introducing the cell suspension onto a substrate having a holding part capable of holding one cell. Staining with a specific marker is preferable because the target cells expressing the marker can be easily separated and collected. What is necessary is just to select the screening form according to the objective suitably.

  In addition, there is no particular limitation on the method for holding the cells on the substrate having the holding unit described above, and the cell suspension may be simply introduced into the holding unit, or a centrifugal force may be used after introducing a liquid containing cells. Then, the cells may be forcibly introduced into the holding part. In particular, after introducing the cell suspension, it is preferable to introduce the cells into the holding part using the dielectrophoretic force because the undifferentiated cells can be efficiently held in the holding part.

  According to the present invention, it is possible to select a specific marker capable of labeling a cell having a possibility of forming a tumor and to evaluate the quality of a product such as regenerative medicine by detecting a cell having a possibility of forming a tumor using the marker. .

It is a figure which shows the cell-staining result obtained in Example 2 and 3. It is a figure which shows the numerical result of the dyeing | staining intensity | strength of the fluorescence image obtained in Example 4. FIG. It is a figure which shows the numerical result of the dyeing | staining intensity | strength of the alkaline phosphatase obtained in Example 4. FIG. It is the figure (exploded view) which showed the board | substrate used in Example 5. FIG. It is a front view of the board | substrate shown in FIG. It is the figure which showed an example of the detection method of an undifferentiated cell using the board | substrate shown in FIG.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to the said example.

Example 1 Culture of human iPS cells and somatic cells and induction of early differentiated cells (1) 201B7 strain (purchased from iPS Academia Japan), a human iPS cell line, is on a polystyrene plate coated with iMatrix (Nippi), StemFit
Culturing was performed using AK02N medium (Takara Bio Inc.).

  (2) As a somatic cell used as a negative control, human mesenchymal stem cells (hMSC: manufactured by LONZA) and MRC-5 cells (distributed from JCRB cell bank) were used. Human mesenchymal stem cells are cultured using MSCGM medium (manufactured by LONZA), and MRC-5 cells are treated with Dulbecco's Modified Eagle Medium (DMEM: Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum. Culture. At the time of staining, the cells were cultured on a polystyrene plate coated with iMatrix (Nippi) using StemFit AK02N medium (Takara Bio).

  (3) Early differentiated cells (ectodermal, mesoderm, endoderm) are started using Human Pluripotent Stem Cell Functional Identification Kit (manufactured by R & D Systems) from the day after 201B7 cells are seeded on iMatrix. Differentiation induction was performed. The method followed the protocol attached to the kit. In addition, cells cultured by adding 10 μM ROCK inhibitor (Y-27632: manufactured by Wako Pure Chemical Industries, Ltd.) were also used for comparison.

Example 2 Staining of cells using antibody and lectin (1) Cells cultured according to Example 1 were washed once with phosphate buffered saline (PBS), and then a pathological tissue fixing agent (mildform 10N: Jun Wako) (Manufactured by Yakuhin Co., Ltd.) was added, and the cells were fixed by allowing to stand at room temperature for 10 minutes.

  (2) After the fixed cells were washed once with PBS, a PBS solution containing 0.2% (v / v) Triton X-100 (trade name) was added, and the mixture was allowed to stand at room temperature for 2 minutes. Cell membrane permeabilization was performed.

  (3) After washing 3 times with PBS, a PBS solution (BSA / PBS solution) containing 1% (w / v) bovine serum albumin (BSA) was added and left undisturbed at room temperature for 1 hour. The suppression reaction was performed.

  (4) After removing the BSA / PBS solution, the primary antibody listed in Table 1 or a lectin diluted with the BSA / PBS solution was added, and allowed to stand at room temperature for 2 hours for labeling.

  (5) For antibodies that were not directly labeled (POU5F1 (OCT3 / 4) and NANOG), after washing 3 times with PBS, a fluorescently labeled antibody (secondary antibody) was further added and allowed to react at room temperature for 2 hours. .

  (6) In the process of (4) or (5) above, 2- (4-amidophenyl) -1H-indole-6-carboxamide (DAPI) is added to stain the nucleus simultaneously with the treatment with the fluorescence-modified antibody. And reacted.

  (7) After washing three times with PBS, fluorescence images of the nucleus and each marker were taken using a fluorescence microscope and a CCD camera.

  The result dye | stained by said process is shown in FIG. It was revealed that the staining properties of undifferentiated human iPS cells and early differentiated cells differ depending on the type of marker. Moreover, it was shown that hMSC and MRC-5 cells, which are negative controls, are not stained with any of the markers.

Example 3 Alkaline phosphatase staining (1) Cells cultured according to Example 1 were stained using Stemgent Alkaline Phosphatase Staining Kit II (manufactured by Reprocell). The method was performed according to the protocol of the kit.

  (2) A bright field image of the stained cells was taken under a microscope.

  The result dye | stained by said process is shown in FIG. HMSC and MRC-5 cells used as negative controls were shown not to be stained.

Example 4 Screening of markers capable of staining early differentiated cells by digitizing staining intensity (part 1)
From the stained image obtained in Example 2 above, the staining intensity was digitized by the following steps.

  (1) From the fluorescence-stained image obtained in Example 2, a mask for the nucleus region was created from the DAPI-stained image using Image J, which is image analysis software. Because it is difficult to select all cell nuclei at once due to aggregation or uneven staining, the masks created by changing the threshold value several times are integrated, and finally the integrated mask It was created.

  (2) Region of Interest (ROI) region was set using Image J software from the integrated mask created in the step (1).

  (3) For images labeled with antibodies against nucleoprotein (POU5F1 (OCT3 / 4) and NANOG), the ROI of the above step (2) is used as it is, and the staining intensity of the labeled image is digitized using Image J software. did.

  (4) For markers (TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, rBC2LCN) present in the cell membrane, the ROI (ROI-1) created in the step (2) is expanded. A new ROI (ROI-2) was created, and an area surrounded by ROI-1 and ROI-2 was set as a new ROI (ROI-3). Using the set ROI-3, the staining intensity of the labeled image was quantified using Image J software.

  (5) A histogram was prepared using the numerical values of the staining intensity obtained in the steps (3) and (4).

The results of the histogram obtained by the above process are shown in FIG. From the histogram results,
Screening for markers suitable for detection of cells with potential for tumor formation was performed. For TRA-1-60, expression was enhanced in endodermal and mesodermal early differentiated cells, and in TRA-1-81, expression was enhanced in endodermal early differentiated cells. For this reason, the distribution of histograms was extremely different, and it was considered unsuitable for detection of cells with the potential for tumor formation. For SSEA-3 and POU5F1 (OCT3 / 4), since there are multiple peaks even in cells in the same state, there is a problem in the uniformity of staining and it is judged that they are not suitable as markers for detecting tumorigenic cells. did. In addition, since NANOG expression was lost in the outer pulmonary lobe and mesodermal early differentiated cells, it was judged that detection of tumorigenic cells was impossible. On the other hand, for SSEA-4 and rBC2LCN, (I) there is one peak in cells in all states, (II) there is little spread of peaks, and (III) the peak of undifferentiated cells and early differentiated cells This is a marker that is suitable for detection of cells that are likely to form tumors because it is not separated from the peak of (IV) and can be distinguished from the negative control (somatic cells) used as a comparison. It has been shown.

Example 5 Screening of markers capable of staining early differentiated cells by digitizing staining intensity (part 2)
The alkaline phosphatase image obtained in Example 3 was digitized by the following steps.
(1) The intensity of a random area was digitized as a blank, and the intensity of a random area where cells exist was digitized.
(2) An average value was calculated from the staining intensity of a plurality of regions and graphed.

  The results obtained by the above steps are shown in FIG. Both undifferentiated iPS cells and early differentiated cells (ectodermal, mesoderm, endoderm) have higher staining intensity than negative control hMSC and MRC-5 cells, and can detect cells with potential for tumor formation. It was shown to be suitable.

Example 6
In the above-described examples, the cell population was screened for markers. However, it was examined whether screening could be performed in the same manner even when dispersed as single cells. Note that iPS cells were used as undifferentiated cells, MRC-5 cells were used as differentiated cells, and SSEA-4 was used as a marker. Further, as a means for holding the cells in the dispersion, the substrate 100 shown in FIGS. 4 and 5 was used.

The substrate 100 shown in FIG. 4 and FIG.
It comprises a flat light shielding member 11 having a through hole 11a, a flat insulator 12 having a through hole 12a, and a flat spacer 13 having an inlet 13a, a discharge port 13b, and a through part 13c. Cell introduction holding means 10;
Electrodes 21 and 22 provided so as to sandwich the cell introduction holding means 10 in the vertical direction;
A conductive wire 30 connecting the electrodes 21 and 22;
A signal generator 40 for applying signals to the electrodes 21 and 22;
It has.

  The through hole 11a included in the light shielding member 11 and the through hole 12a included in the insulator 12 have the same size and shape, and the light shielding member 11 and the insulator 12 are provided so that the positions of the respective through holes coincide with each other. . When the holding portion 50 is configured by the electrode substrate 21 provided in close contact with the lower portion of the through-hole 11a, the through-hole 12a, and the light shielding member 11, when a liquid containing undifferentiated cells 210 and differentiated cells 220 is introduced from the introduction port 13a, the through-hole is formed. Cells are introduced into the holding part 50 through the part 13c. The electrode 22 is provided in close contact with the upper portion of the spacer 13 and prevents scattering and evaporation of the liquid containing cells introduced from the introduction port 13a.

(1) Preparation of human iPS cells (1-1) After washing iPS cells cultured for 7 days with PBS, the cells are mixed with TrypLE select (manufactured by Thermo Fisher) and Versene Solution (manufactured by Thermo Fisher). It peeled.
(1-2) The detached cells were collected, washed with PBS, and fixed in a 4% (w / v) paraformaldehyde solution for 40 minutes at room temperature.
(1-3) After fixation, washed with PBS and stored.

(2) Preparation of MRC-5 cells (2-1) MRC-5 cells cultured for 7 days were washed with PBS, and then the cells were detached using 0.05% Trypsin EDTA (manufactured by Thermo Fisher).
(2-2) The detached cells were collected, washed with PBS, and then immersed in a 4% (w / v) paraformaldehyde solution at room temperature for 40 minutes to fix the cells.
(2-3) After fixation, washed with PBS and stored.

(3) Preparation of solution for dielectrophoresis (3-1) Polyethylene glycol (mPEG-NHS) having a molecular weight of 5000, one end of which is a methoxy group and the other end is an N-hydroxysuccinimide ester group, and BSA (300 mg, 0.3 mmol) was dissolved in sodium bicarbonate buffer (0.1 M, 15 mL), and the solution was stirred at room temperature for 3 hours to prepare BSA (PEG-BSA) bound with polyethylene glycol. did. In the preparation, the molar ratio of mPEG-NHS to BSA (mPEG-NHS / BSA) was set to 2. After the preparation, the solution was replaced with pure water for 3 days using a dialysis membrane having a molecular weight cut off of 10,000.
(3-2) A solution containing PEG-BSA (1% (w / v) as BSA) and 300 mM mannitol prepared in (3-1) was prepared and used as a solution for dielectrophoresis.

(4) Preparation of cell dispersion (4-1) iPS cells preserved in (1) and MRC-5 cells preserved in (2) were prepared with anti-SSEA4 antibody and final concentration 2 μg / mL DAPI (3) The cells were stained by suspending in the dielectrophoresis solution and reacting at room temperature for 60 minutes.
(4-2) After washing twice with the dielectrophoresis solution prepared in (3), the number of iPS cells and the number of MRC-5 cells were counted.
(4-3) One million, 5 or 10 MPS-5 cells were spiked with iPS cells, and the total volume was adjusted to 850 μL to obtain a cell dispersion.

(5) Retaining the cell in the holding part The cell dispersion prepared in (4) is introduced from the inlet 13a provided in the substrate 100 shown in FIG. 4, and an AC voltage 40 having a frequency of 1 MHz is applied to the electrodes 21 and 22. The cells were held on the holding unit 50 of the substrate 100 using the dielectrophoretic force 60 by applying for a minute (FIG. 6a).

(6) Detection of cells The fluorescence of the cells held in the holding unit 50 is observed by photographing with a CMOS camera using the fluorescence microscope 400, and the number of cells recognized as DAPI positive is the total number of cells, DAPI positive. The number of SSEA4-positive cells was determined as the number of iPS cells (undifferentiated cells) (FIG. 6b).

  Table 2 shows the number of iPS cells detected. The average value of the actual number of detected cells was 0.83 (when the number of spikes was 1), 4.67 (when the number of spikes was 5) and 9 (when the number of spikes was 10), iPS cell spikes The result was almost equivalent to the number (theoretical value). From this, it was found that undifferentiated cells and differentiated cells can also be distinguished from the cell dispersion, and the screening method of the present invention can be carried out.

  In order to realize regenerative medicine using pluripotent stem cells, it is indispensable to evaluate the quality of products such as regenerative medicine produced by inducing differentiation from pluripotent stem cells. In addition, as one of the important items for quality evaluation, there is to confirm the presence or absence of cells with the possibility of tumor formation. Therefore, by using the marker selected by the screening method of the present invention, it is possible to contribute to the realization of industrial use of a product such as regenerative medicine by detecting cells with the possibility of tumor formation mixed in the product such as regenerative medicine. .

DESCRIPTION OF SYMBOLS 100: Board | substrate 10: Cell introduction | transduction holding means 11: Light-shielding member 12: Insulator 11a, 12a: Through-hole 13: Spacer 13a: Inlet 13b: Outlet 13c: Through-hole 21/22: Electrode substrate 30: Conductor 40: Signal Generator (AC voltage)
50: Holding unit 60: Dielectrophoretic force 200: Cell 210: Undifferentiated cell 220: Differentiated cell 300: Labeled protein 400: Detection unit (fluorescence microscope)

Claims (9)

It is a screening method for markers that can specifically stain pluripotent stem cells, and the results of staining with various markers are quantified and a staining marker that matches the purpose is selected. The method according to claim 1, comprising a marker capable of staining cells partially differentiated from pluripotent stem cells. Obtaining initial differentiated cells by partially differentiating pluripotent stem cells;
Staining initial differentiated cells with a staining marker;
The method according to claim 2, comprising a step of obtaining an image of an early differentiated cell, and a step of detecting staining of the early differentiated cell with a staining marker based on the image. The method according to claim 2 or 3, wherein the partially differentiated cells are one or more selected from the group consisting of ectoderm cells, mesoderm cells, and endoderm cells. The method according to claim 2 or 3, wherein the partially differentiated cells are any two selected from the group consisting of ectoderm cells, mesoderm cells, and endoderm cells. The method according to claim 2 or 3, wherein the partially differentiated cells are ectoderm cells, mesoderm cells, and endoderm cells. The method according to any one of claims 1 to 6, wherein the staining marker is a fluorescently labeled antibody. The method according to claim 1, wherein the staining marker is an enzyme. The method according to any one of claims 1 to 6, wherein the staining marker is SSEA-4, rBC2LCN, or alkaline phosphatase.

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