JP2022174608A - Method for evaluating state of differentiation into neuron - Google Patents

Abstract

To evaluate noninvasively whether a pluripotent stem cell has differentiated into a neuron.SOLUTION: The present invention discloses a method for evaluating the state of differentiation of a cultured pluripotent stem cell into a neuron, when performing a plurality of culture stages to induce the pluripotent stem cell into the neuron. The method includes a measurement step of measuring the abundance of an index substance in a conditioned medium in a plurality of culture stages; and a determination step of determining the differentiation state of the cultured cell on the basis of the abundance of the index substance, where the index substance is at least one of 2-amino ethanol, choline, glutamic acid and serine.SELECTED DRAWING: Figure 4

Description

TECHNICAL FIELD The present invention relates to a method for evaluating the state of differentiation into nerve cells.

Conventionally, a method using immunostaining, a method of quantifying the expression level of a marker gene, and the like have been widely used as methods for evaluating the differentiation state of cells. However, since these methods require invasive treatment of cells, the cells subjected to evaluation cannot be used for other purposes, such as cell sources for regenerative medicine. Therefore, in recent years, a method has been proposed in which cells are not invasively treated (see Patent Document 1).

In the method for evaluating the differentiation state of Patent Document 1, pluripotent stem cells with an unknown differentiation state are used as test cells, and pluripotent stem cells with a known differentiation state are used as control cells. kynurenine abundance in culture supernatants of control cells with that in culture supernatants of control cells.

Patent No. 6332440

The evaluation method of Patent Document 1 is effective for evaluating whether pluripotent stem cells are in an undifferentiated state. However, when pluripotent stem cells are induced to differentiate into target cells such as nerve cells, there is a problem that it is not possible to evaluate whether or not they are differentiated into target cells.

An object of the present invention is to provide a method capable of non-invasively evaluating the state of differentiation into nerve cells when pluripotent stem cells are induced to differentiate into nerve cells.

A first aspect of the present invention is a method for evaluating the state of differentiation into nerve cells in cultured cells when performing a plurality of culture steps to induce differentiation of pluripotent stem cells into nerve cells, and a determination step of determining the differentiation state of the cultured cells based on the abundance of the indicator substance, wherein the indicator substance is , 2-aminoethanol, choline, glutamic acid and serine.

According to the first aspect of the present invention, it is possible to non-invasively evaluate whether or not pluripotent stem cells are differentiated into nerve cells.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram illustrating a method for evaluating the state of differentiation into nerve cells according to the first aspect of the present invention; It is an example of components of a medium (StemFit) used in Examples. It is an example of components of a medium (DMEM/F12) used in Examples. 1 is a graph showing changes over time in the abundance of an index substance (2-aminoethanol) determined by LCMS analysis of culture supernatants in Examples. The vertical axis indicates the difference in area ratio of the indicator substance to the standard substance, and the horizontal axis indicates the number of days of culture. 1 is a graph showing changes over time in abundance of an index substance (choline) determined by LCMS analysis of culture supernatants in Examples. 1 is a graph showing changes over time in abundance of an index substance (glutamic acid) determined by LCMS analysis of culture supernatants in Examples. 1 is a graph showing changes over time in abundance of an index substance (serine) determined by LCMS analysis of culture supernatants in Examples. 1 is a graph showing changes over time in abundance of an index substance (ornithine) determined by LCMS analysis of culture supernatants in Reference Example. 2 is a graph showing changes over time in abundance of an index substance (cystine) obtained by LCMS analysis of culture supernatants in Reference Example. 2 is a graph showing changes over time in the abundance of an index substance (riboflavin) determined by LCMS analysis of culture supernatants in Reference Example.

1. First Aspect The first aspect is a method of evaluating the state of differentiation into neurons in cultured cells when performing a plurality of culture stages (culturing steps) to induce differentiation of pluripotent stem cells into neurons. be. That is, according to the first aspect, the differentiation state evaluation method is performed on the cells being cultured when performing a plurality of culture stages.

[Culturing stage]
A first aspect comprises, in order, a preparatory culture stage and a differentiation culture stage.

(Preparatory culture stage)
In the preparatory culture step, the pluripotent stem cells are cultured in preparation for differentiation culture, depending on purposes such as proliferation and acclimatization. The preparatory culture stage comprises, in order, a maintenance culture stage and an acclimation culture stage.

In the maintenance culture stage (undifferentiated maintenance culture stage), pluripotent stem cells are prepared as cultured cells and grown in an undifferentiated state. Pluripotent stem cells include iPS cells, ES cells and the like. As a medium used for culture, a medium generally used for culturing pluripotent stem cells may be used, and examples thereof include the StemFit (registered trademark) series. FIG. 2 shows an example of the components of StemFit. If desired, the culture vessel may be coated with a cell culture substrate such as iMatrix-511 for efficient expansion of pluripotent stem cells.

In the conditioned culture stage, the expanded pluripotent stem cells are conditioned. Specifically, it facilitates differentiation into nerve cells. The medium includes, for example, the same medium as used in the maintenance culture stage. If necessary, the medium may be supplemented with a receptor inhibitor, such as a TGF-β receptor inhibitor, a BMP inhibitor, a GSK3 inhibitor, etc., for inducing differentiation into nerve cells.

At the preparatory culture stage, either adherent culture or suspension culture may be performed, and the type of pluripotent stem cells or the type of medium is appropriately determined according to a conventional method. In the first aspect, for example, both the maintenance culture stage and the acclimation culture stage may employ adherent culture.

(Differentiation culture stage)
In the differentiation culture stage, pluripotent stem cells are induced to differentiate into nerve cells. The differentiation culture stage includes a first differentiation culture stage and a second differentiation culture stage in order.

In the first differentiation culture stage, the pluripotent stem cells (cultured cells) after the preparatory culture stage are induced to differentiate into neural progenitor cells. Examples of the medium used in the first differentiation culture stage include DMEM/F12, a medium containing DMEM/F12 as a main component (eg, mTeSR1), and the like. FIG. 3 shows an example of the components of DMEM/F12. Additives are added to the medium to induce neural progenitor cells. Additives include saccharides, amino acids, buffers, various growth factors, various inhibitors, various supplements, and the like.

In the second differentiation culture stage, the pluripotent stem cells (cultured cells) after the first differentiation culture stage, ie, neural progenitor cells, are induced to differentiate into cerebral cortical neurons. As the culture used in the second differentiation culture stage, the same culture as used in the first differentiation culture stage can be used. Additives are added to the medium for induction into cerebral cortical neurons. Additives include sugars, amino acids, buffers, various supplements, and the like. If necessary, the culture vessel may be coated with a cell culture substrate such as poly-L-ornithine or laminin to ensure differentiation into neurocortical neurons.

At the stage of differentiation culture, either adherent culture or suspension culture may be performed, and the culture is appropriately determined according to the type of cultured cells or culture medium in accordance with a conventional method. In the first aspect, for example, suspension culture may be adopted in the first differentiation culture stage, and adherent culture may be adopted in the second differentiation culture stage.

In the first aspect, subculturing is carried out at each of the culture stages described above. That is, medium exchange is performed. Medium exchange can be implemented by a well-known method. The medium replacement interval may be, for example, every 1 to 2 days in the preparatory culture stage, and every 2 to 4 days in the differentiation culture stage.

[Evaluation method]
A first aspect includes a measurement step and a determination step in order as an evaluation method.

(measurement step)
In the measurement step, the abundance of the indicator substance in the culture supernatant is measured. That is, every time the medium is replaced, the supernatant of the medium used is obtained, and the abundance of the indicator substance contained in the culture supernatant is measured. Specifically, for both (a) the culture supernatant in the medium before use and (b) the culture supernatant in the medium after the medium has been used in the culture step, the abundance of the indicator substance to measure. Subsequently, the increase/decrease in the index substance before and after use is calculated.

The indicator substance is at least one of 2-aminoethanol, choline, glutamic acid and serine.

At each culture stage, the abundance of the indicator substance may be measured in at least one medium, preferably in half or more of the medium used, more preferably in all of the medium used.

Methods for measuring the abundance of indicator substances in culture supernatants include, for example, quantitative analysis by mass spectrometry, particularly liquid chromatograph-mass spectrometer (LC-MS) and gas chromatograph-mass spectrometer (GC-MS). The quantitative analysis used can be suitably used. In addition, for example, the components eluted by injecting the culture supernatant together with the eluent into a liquid chromatography device are detected with a spectroscopic detector (e.g., ultraviolet-visible spectroscopic detector, infrared spectroscopic detector). Method: A method of adding a reagent that specifically causes the indicator substance to emit light to the culture supernatant and detecting the luminescence intensity can also be mentioned.

Moreover, the abundance of the indicator substance may be determined either as an absolute amount or as a relative amount. For example, when measuring the relative amount by mass spectrometry, a predetermined amount of standard substance (e.g., isopropyl malic acid) is added to a sample containing the indicator substance, mass spectrometry is performed, and the measurement result of the indicator substance ( The area of the mass chromatogram) is normalized with the measurement result of the standard substance to calculate the relative value. Then, the determination step may determine the relative value as the abundance of the index substance.

(judgment step)
In the determination step, the differentiation state of the cultured cells is determined based on the abundance of the indicator substance. Specifically, changes over time in the abundance of the indicator substance (more specifically, increase or decrease in the indicator substance) measured at each culture stage are confirmed. In particular, in the two stages, the preparatory culture stage and the differentiation culture stage, the tendency of increase and decrease of the indicator substance changes before and after the medium is used.

For example, when the indicator substance is 2-aminoethanol, the abundance of 2-aminoethanol tends to decrease as a whole in the preliminary culture stage. That is, it shows a tendency to consume 2-aminoethanol in the medium. On the other hand, at the stage of differentiation culture, the amount of 2-aminoethanol present tends to be almost constant throughout.

When the indicator substance is choline, the abundance of choline tends to decrease as a whole in the preliminary culture stage. On the other hand, at the stage of differentiation culture, choline tends to increase overall. That is, it shows a tendency to produce choline.

When the indicator substance is glutamic acid, the abundance of glutamic acid tends to increase overall in the preliminary culture stage. On the other hand, at the differentiation culture stage, the abundance of glutamic acid tends to decrease overall.

When the indicator substance is seric acid, the abundance of glutamic acid tends to decrease as a whole in the preliminary culture stage. On the other hand, at the differentiation culture stage, the abundance of glutamic acid tends to increase overall.

In addition, each stage may be compared in two stages, a preparatory culture stage and a differentiation culture stage, and a maintenance culture stage, an acclimation culture stage, a first differentiation culture stage, and a second differentiation culture stage. can be compared in four stages.

The increase/decrease tendency of the indicator substance may be determined by the average value for each stage, or may be determined by the larger number of increases/decreases for each stage. For example, when ten culture media are used in the preparatory culture stage, the trend of increase or decrease may be determined by the average value of the increase or decrease of the ten media. Alternatively, the 10-point culture medium may be divided into "increase," "constant," and "decrease," respectively, and the largest number of divisions may be adopted to determine the trend of increase or decrease.

For each classification of increase, constant, and decrease, for example, when the range of increase/decrease is within a predetermined threshold, the amount is constant, and when the threshold is exceeded, the amount is increased or decreased. The threshold may be appropriately set from the viewpoint of the degree of increase or decrease of the indicator substance before and after use, the amount of the standard substance added, and the like.

Measurement of each culture supernatant may be performed immediately after collecting the culture supernatant, or the collected culture supernatant is temporarily frozen and stored, and all the culture supernatant is collected. Measurements may be taken at a later time.

The first aspect of the present invention enables non-invasive evaluation of whether or not pluripotent stem cells are differentiated into nerve cells (in particular, neural progenitor cells or cerebral cortical neurons). In other words, since the culture supernatant is measured rather than the cultured cells (pluripotent stem cells or neurons induced to differentiate from them), there is no need to invasively treat the cultured cells, and the cells are separated after culture. It can be used for various purposes. In addition, the state of differentiation into nerve cells can be determined simply by measuring the amount of 2-aminoethanol, choline, glutamic acid, or serine in the culture supernatant, so the evaluation method is easy. In addition, in the first aspect, both adherent culture and suspension culture can be measured, so the differentiation state can be evaluated without being limited to the culture form.

2. Second Aspect In the first aspect, the preparatory culture step includes a maintenance culture step and an acclimation culture step. For example, in the second aspect, the acclimation culture step may not be provided. . That is, in the second aspect, the culture stage has, in order, a maintenance culture stage, a first differentiation culture stage, and a second differentiation culture stage. From the viewpoint of reliably inducing differentiation into nerve cells, the first aspect is preferable.

3. Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are specific examples of the following aspects.

(Section 1) A method for evaluating the state of differentiation into nerve cells in cultured cells when performing a plurality of culture steps to induce differentiation of pluripotent stem cells into nerve cells, and a determination step of determining the differentiation state of the cultured cells based on the abundance of the indicator substance, wherein the indicator substance is 2-amino At least one of ethanol, choline, glutamic acid and serine may be used.

(Section 2) In the evaluation method described in Section 1, the determination step may determine the state of differentiation of the cultured cells by confirming a change over time in the abundance of the indicator substance.

(Section 3) In the evaluation method according to Section 1 or 2, the plurality of culture steps include a differentiation culture step of inducing differentiation of the pluripotent stem cells into nerve cells, and the differentiation culture step prior to the differentiation culture step. A preparatory culture step for culturing pluripotent stem cells is provided in order, and the abundance of the index substance measured in the preparatory culture step is compared with the abundance of the index substance measured in the differentiation culture step, A differentiation state of the cultured cells may be determined.

(Section 4) In the evaluation method according to Section 3, the preparatory culture step includes a maintenance culture step in which the pluripotent stem cells are grown in an undifferentiated state, and an conditioned culture step in which the pluripotent stem cells are conditioned. and a culturing step may be provided in order.

(Section 5) In the evaluation method according to Section 3 or 4, the differentiation culture stage includes a first differentiation culture stage for inducing neural progenitor cells and a second differentiation culture stage for inducing cerebral cortical neurons. A differentiation culture step may be provided in order.

(Item 6) In the evaluation method according to any one of items 1 to 5, the indicator substance may be 2-aminoethanol.

(Item 7) In the evaluation method according to any one of items 1 to 5, the indicator substance may be at least one of choline and serine.

(Item 8) In the evaluation method according to any one of items 1 to 5, the indicator substance may be glutamic acid.

EXAMPLES Next, the present invention will be described in detail with reference to Examples and Comparative Examples, but the scope of the present invention is not limited by these.

<Culturing stage>
Using iPS cells, induction of differentiation into cerebral cortical neurons was performed as shown in the following stages (see FIG. 1). For the sake of convenience, the day on which the maintenance culture was performed was defined as "minus day 14", and the day on which the first differentiation culture was performed was defined as "day 0".

(Maintenance culture stage)
Human peripheral blood mononuclear cells were transfected with pCE-HoCT3/4, pCE-mp53DD, pCE-hSK, pCE-hUL, and pCXB-EBNA1 using episomal vectors to establish iPS cells.

StemFit AK02N medium was then added to culture vessels coated with iMatrix-511 (cell culture substrate). Using this culture vessel, the established iPS cells were subcultured.

(Acclimatization culture stage)
StemFit AK02N medium was then supplemented with SB431532 (TGF-β inhibitor), LDN193189 (BMP inhibitor) and CHIR99021 (GSK3 inhibitor). Using this medium, the iPS cells were subcultured.

(First differentiation culture stage)
Then, DMEM / F12 medium was added to the low-adhesion culture vessel, followed by sugars, amino acids, buffers, FGF2 (fibroblast growth factor), LIF (leukemia inhibitory factor), SB431542, IWP2 (Wnt signal inhibition agent), N2 supplement and B27 supplement were added. Using this culture vessel, the iPS cells were cultured by suspension culture. As a result, neurospheres, which are neural progenitor cells, were obtained.

(Second differentiation culture stage)
DMEM/F12 medium, sugars, amino acids, buffers, N2 supplement and B27 supplement were then added to culture vessels coated with cell culture substrate containing poly-L-ornithine and laminin. Using this culture vessel, the neurospheres were subcultured and induced to differentiate into cerebral cortical neurons.

In maintenance culture and acclimation culture, the medium is changed every 1 to 2 days until the cell coverage area reaches 70 to 80% of the culture container (subconfluent), and the cells are subcultured. (these two operations are collectively referred to as passaging) were carried out. In the primary differentiation culture, the medium was changed every 2-4 days and passaged to the secondary differentiation induction on the 12th day. In the second induction of differentiation, the medium was changed every 2 to 3 days and the cells were cultured until the 26th day. For passage, a cell detachment solution containing 1 mM ethylenediaminetetraacetic acid (trade name: TrypLE Select, manufactured by Thermo Fisher Scientific) was used.

<Evaluation step>
The difference in the area ratios of the mass chromatograms was measured according to the following LC-MS analysis method as the increase or decrease in the amount of the indicator substance in the culture supernatant used at each medium exchange.

(Example 1: LC-MS analysis of 2-aminoethanol)
1. The supernatant of the medium before use for culture was collected and used as a sample. To 100 μL of the sample, 20 μL of 0.5 mM isopropylmalic acid aqueous solution as an internal standard was added and mixed, and then 200 μL of acetonitrile was added to deproteinize. After that, the sample was centrifuged (15000 rpm, room temperature, 15 minutes) to collect the supernatant, and diluted 10-fold with ultrapure water (Milli-Q (registered trademark) water, Merck Co.) for LC-MS analysis. provided. In the LC-MS analysis, the analysis conditions included in "LC/MS/MS Method Package Cell Culture Profiling" (hereinafter abbreviated as "MP") manufactured by Shimadzu Corporation were followed. MP is a collection of analysis condition parameters for analyzing compounds contained in the medium and metabolites secreted from cells by LC-MS. Compound identification is based on the fact that the difference between the retention time of the standard registered in MP and the retention time of the compound in the sample is within ±0.3 minutes, and that both peaks of quantitation ions and confirmation ions are detected. It was carried out on the basis that the strength value was 1000 or more. In addition, quantification of compounds was carried out by a method of calculating the peak area of the mass chromatogram for ions characteristic of each compound in the sample (quantitation ions).

In the mass chromatogram obtained by the above measurement, the peak area (A) corresponding to 2-aminoethanol is divided by the peak area (B) corresponding to isopropylmalic acid (A / B), 2-amino It was calculated as the amount present (C) before the use of ethanol.

2. The supernatant of the culture medium after use was collected and used as a sample. This is described in 1. above. The abundance (D) after the use of 2-aminoethanol was calculated in the same manner.

3. The amount of 2-aminoethanol present after use (D) was subtracted by the amount of 2-aminoethanol present before use (C) to calculate the increase or decrease in the amount present before and after use (DC). The results are shown in FIG.

(Examples 2-4: LC-MS analysis of choline, glutamic acid and serine)
For choline, glutamic acid, and serine, the increase/decrease in abundance before and after use was calculated in the same manner as in Example 1. The results are shown in FIG. 5 (Example 2: Choline), FIG. 6 (Example 3: Glutamic acid), and FIG. 7 (Example 4: Serine).

(Reference Example 1: LC-MS analysis of substances other than indicator substances)
As examples other than the indicator substances described above, changes in the amount of ornithine, cystine and riboflavin before and after use were calculated in the same manner as in Example 1. The results are shown in Figure 8 (ornithine), Figure 9 (cystine) and Figure 10 (riboflavin).

<Confirmation of differentiation induction>
At each medium change, a portion of the cells used for culture was harvested. mRNA is extracted from the sorted cells, cDNA synthesis is performed with reverse transcriptase, and using a real-time PCR device, FOXG1 (genetic marker expressed in telencephalic progenitor cells), MAP2 (expressed gene marker) and CTIP2 (gene marker expressed in the cerebral cortex) were measured.

In addition, the immunofluorescence staining method targeting the nerve cell marker NeuN and the cerebral lipid marker Tbr1 was performed on the cells after differentiation-inducing culture.

From these, it was confirmed that differentiation was not performed at the maintenance culture stage and the acclimatization culture stage, differentiation was induced to neural progenitor cells at the first differentiation stage, and differentiation was induced to neurocortical neurons at the second differentiation stage. confirmed.

<Discussion>
From FIG. 4, 2-aminoethanol tended to decrease (consumed) in the culture supernatant before day 0 (ie, preparatory culture stage), but after day 0 (ie, differentiation culture stage). In the culture supernatant of , the amount of increase or decrease was within 0.05 and was constant. From this, it is found that 2-aminoethanol is effective as an index substance for nerve cells because the tendency of increase and decrease changes when the induction of differentiation into nerve cells begins.

5 and 7, choline and serine tended to decrease in the culture supernatant before day 0, but tended to increase (produce) in the culture supernatant after day 0. . This shows that choline and serine are effective as indicator substances for nerve cells because their increasing and decreasing tendencies change when differentiation induction into nerve cells begins.

From FIG. 6, glutamic acid tended to increase in the culture supernatant before day 0, but tended to decrease in the culture supernatant after day 0. This shows that glutamic acid is effective as an index substance for nerve cells because the tendency of increase and decrease changes when the induction of differentiation into nerve cells begins.

From FIG. 8, ornithine tended to increase in the culture supernatant before day 0, and also tended to increase in the culture supernatant after day 0. From this, it can be seen that ornithine is not effective as an index substance for nerve cells, because once the induction of differentiation into nerve cells begins, the increasing or decreasing tendency does not change.

From FIG. 9, cystine tends to decrease in the culture supernatant before day 0, and also in the culture supernatant after day 0. This shows that cystine is not effective as an index substance for nerve cells because once the induction of differentiation into nerve cells begins, the increasing and decreasing tendency does not change.

From FIG. 10, riboflavin showed a constant tendency in the culture supernatant before day 0, and also showed a constant tendency in the culture supernatant after day 0. This shows that riboflavin is not effective as an index substance for nerve cells because once the induction of differentiation into nerve cells begins, the tendency of increase and decrease does not change.

Claims (8)

A method for evaluating the state of differentiation into nerve cells in cultured cells when performing a plurality of culture steps to induce differentiation of pluripotent stem cells into nerve cells, comprising:
a measuring step of measuring the abundance of the indicator substance in the culture supernatant in the plurality of culture stages;
a determination step of determining the differentiation state of the cultured cells based on the abundance of the indicator substance;
A method for evaluating the state of differentiation into nerve cells, wherein the indicator substance is at least one of 2-aminoethanol, choline, glutamic acid and serine. 2. The evaluation method according to claim 1, wherein in the determination step, the differentiated state of the cultured cells is determined by confirming the time-dependent change in the abundance of the index substance. The multiple culture stages are
a differentiation culture step of inducing differentiation of the pluripotent stem cells into nerve cells;
a preliminary culturing step of culturing the pluripotent stem cells prior to the differentiation culturing step;
3. The differentiated state of the cultured cells is determined by comparing the abundance of the indicator substance measured in the preparatory culture stage and the abundance of the indicator substance measured in the differentiation culture stage. The evaluation method described in . The preliminary culturing step includes:
a maintenance culture step of growing the pluripotent stem cells in an undifferentiated state;
The evaluation method according to claim 3, further comprising: an conditioned culture step of conditioned culturing the pluripotent stem cells. The differentiation culture step is
a first differentiation culture stage for induction into neural progenitor cells;
5. The evaluation method according to claim 3 or 4, further comprising a second differentiation culture step for induction into cerebral cortical neurons. The evaluation method according to any one of claims 1 to 5, wherein the indicator substance is 2-aminoethanol. The evaluation method according to any one of claims 1 to 5, wherein the indicator substance is at least one of choline and serine. The evaluation method according to any one of claims 1 to 5, wherein the indicator substance is glutamic acid.

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