Method for assessing differentiation state of cells, gelatin nanoparticles and set of gelatin nanoparticles

Abstract

The purpose of the present invention is to provide a method for assessing the differentiation state of cells, said method enabling the assessment of the differentiation state of a great variety of cells, and gelatin nanoparticles and a gelatin nanoparticle set both usable in the method. This purpose can be achieved by a method for assessing the differentiation state of cells, said method comprising a step for monitoring the expression of mRNA encoding peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) or peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) in cells. The aforesaid method can be carried out by using gelatin nanoparticles for assessing the differentiation state of cells, said gelatin nanoparticles carrying a probe capable of detecting mRNA encoding PGC-1α or PGC-1α.

Description

[Technical field]

[0001] The present invention relates to a method for assessing the differentiation state of a cell, gelatin nanoparticles and a set of gelatin nanoparticles. [Background technology]

[0002] Understanding the differentiation state of cells is required in various fields, such as regenerative medicine and disease detection and treatment.

[0003] Conventionally, the differentiation state of cells has been evaluated by immunostaining or quantification of the expression levels of marker genes, but these methods cannot evaluate the differentiation state of cells over time or for individual cells.

[0004] In response to this, Patent Document 1 describes a method for monitoring the differentiation of cardiomyocytes over time by introducing into cardiomyocytes a reporter gene for a luminescent protein that is configured to emit light in response to the expression of a cardiac differentiation marker gene. In Patent Document 1, a vector incorporating a promoter for the marker gene and a gene for a luminescent protein (such as luciferase) located downstream thereof is introduced into cells by electroporation. When a transcription factor is synthesized and the cardiac differentiation marker gene is expressed, the luminescent protein derived from the vector is also expressed and emits light. Patent Document 1 states that the differentiation of cardiomyocytes can be monitored by observing this luminescence. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2015-77122 A Summary of the Invention [Problem to be solved by the invention]

[0006] As described in Patent Document 1, there is a need for the development of a method capable of evaluating cell differentiation over time.

[0007] However, a method using a differentiation marker gene specific to a specific cell, such as the method described in Patent Document 1, can only evaluate the differentiation state of the specific cell. Therefore, when attempting to evaluate the differentiation state of other cells, it is necessary to search for a differentiation marker gene of the other cells that can be used to evaluate the differentiation state.

[0008] The present invention was made based on the above findings, and aims to provide a method for evaluating the differentiation state of a cell that can evaluate the differentiation state of a wide variety of cells, as well as gelatin nanoparticles and a set of gelatin nanoparticles that can be used in the method. [Means for solving the problem]

[0009] The above-mentioned problems are solved by a method for evaluating the differentiation state of a cell, which includes a step of detecting mRNA encoding peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) or peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in the cell.

[0010] The above problem is also solved by gelatin nanoparticles for evaluating the differentiation state of cells, carrying mRNA encoding peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) or a probe capable of detecting peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α).

[0011] The above problem is also solved by a set of gelatin nanoparticles for evaluating the differentiation state of a cell, comprising gelatin nanoparticles carrying mRNA encoding peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) or a probe capable of detecting peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), and gelatin nanoparticles carrying mRNA encoding pyruvate dehydrogenase kinase 1 (Pdk1) or a probe capable of detecting pyruvate dehydrogenase kinase 1 (PDK1). Effect of the Invention

[0012] INDUSTRIAL APPLICABILITY The present invention provides a method for evaluating the differentiation state of a cell, which allows the evaluation of the differentiation state of a wide variety of cells, as well as gelatin nanoparticles and a set of gelatin nanoparticles that can be used in said method. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a flow chart illustrating a method for assessing the differentiation state of a cell according to one embodiment of the present invention. [Diagram 2] FIG. 2A is a graph showing the expression levels of undifferentiation marker mRNA in Test 1, and FIG. 2B is a graph showing the expression levels of early differentiation marker mRNA in Test 1. [Diagram 3] Figure 3A is a graph showing the expression level of mRNA encoding PGC-1α in culture media with LIF addition (w LIF) and without LIF addition (wo LIF) in Test 1, and Figure 3B is a graph showing the expression level of pdk1 in culture media with LIF addition (w LIF) and without LIF addition (woLIF) in Test 1. [Figure 4]FIG. 4A is a fluorescent image (right) of the medium 1 day after the addition of cGNS (PGC-1α MB) under the condition of LIF addition in Test 1, and an image obtained by superimposing a bright field image and a fluorescent image (left). FIG. 4B is a fluorescent image (right) of the medium 2 days after the addition of cGNS (PGC-1α MB) under the condition of LIF addition in Test 1, and an image obtained by superimposing a bright field image and a fluorescent image (left). FIG. 4C is a fluorescent image (right) of the medium 3 days after the addition of cGNS (PGC-1α MB) under the condition of LIF addition in Test 1, and an image obtained by superimposing a bright field image and a fluorescent image (left). [Diagram 5] FIG. 5A is a fluorescent image (right) of the medium 1 day after addition of cGNS (PGC-1α MB) without the addition of LIF in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 5B is a fluorescent image (right) of the medium 2 days after addition of cGNS (PGC-1α MB) without the addition of LIF in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 5C is a fluorescent image (right) of the medium 3 days after addition of cGNS (PGC-1α MB) without the addition of LIF in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. [Figure 6] FIG. 6A is a fluorescent image (right) of the medium 1 day after the addition of cGNS (Pdk1 MB) under the condition of the addition of LIF in Test 1, and an image (left) in which the bright field image and the fluorescent image are superimposed. FIG. 6B is a fluorescent image (right) of the medium 2 days after the addition of cGNS (Pdk1 MB) under the condition of the addition of LIF, and an image (left) in which the bright field image and the fluorescent image are superimposed. FIG. 6C is a fluorescent image (right) of the medium 3 days after the addition of cGNS (Pdk1 MB) under the condition of the addition of LIF, and an image (left) in which the bright field image and the fluorescent image are superimposed. [Figure 7]FIG. 7A is a fluorescent image (right) of the medium 1 day after addition of cGNS (Pdk1 MB) without the addition of LIF in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 7B is a fluorescent image (right) of the medium 2 days after addition of cGNS (Pdk1 MB) without the addition of LIF in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 7C is a fluorescent image (right) of the medium 3 days after addition of cGNS (Pdk1 MB) without the addition of LIF in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. [Figure 8] FIG. 8A is a fluorescent image (right) of the medium 1 day after addition of cGNS (Actb MB) under the condition of LIF addition in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 8B is a fluorescent image (right) of the medium 2 days after addition of cGNS (Actb MB) under the condition of LIF addition in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 8C is a fluorescent image (right) of the medium 3 days after addition of cGNS (Actb MB) under the condition of LIF addition in Test 1, and an image (left) of a bright field image and a fluorescent image superimposed thereon. [Figure 9] FIG. 9A is a fluorescent image (right) of the medium 1 day after addition of cGNS (Actb MB) without the addition of LIF in Test 1, and an image (left) of the bright field image and the fluorescent image superimposed thereon. FIG. 9B is a fluorescent image (right) of the medium 2 days after addition of cGNS (Actb MB) without the addition of LIF in Test 1, and an image (left) of the bright field image and the fluorescent image superimposed thereon. FIG. 9C is a fluorescent image (right) of the medium 3 days after addition of cGNS (Actb MB) without the addition of LIF in Test 1, and an image (left) of the bright field image and the fluorescent image superimposed thereon. [Figure 10]Figure 10A is a graph showing the fluorescence intensity of medium supplemented with cGNS (PGC-1α MB) in Test 1, Figure 10B is a graph showing the fluorescence intensity of medium supplemented with cGNS (Pdk1 MB) in Test 1, and Figure 10C is a graph showing the fluorescence intensity of medium supplemented with cGNS (Actb MB) in Test 1. [Figure 11] Figure 11A shows a fluorescent image (right) of a medium supplemented with cGNS (Pdk1 MB) in Test 1, and an image obtained by superimposing the bright-field image and the fluorescent image (left). Figure 11B shows a fluorescent image (right) of a medium supplemented with a complex of Lipofectamine 2000 and Pdk1 MB in Test 1, and an image obtained by superimposing the bright-field image and the fluorescent image (left). Figure 11C shows a fluorescent image (right) of a medium supplemented with Pdk1 MB alone in Test 1, and an image obtained by superimposing the bright-field image and the fluorescent image (left). [Figure 12] Fig. 12A is a fluorescent image (right) of a medium to which cGNS (Actb MB) was added in Test 1, and an image obtained by superimposing the bright-field image and the fluorescent image (left). Fig. 12B is a fluorescent image (right) of a medium to which a complex of Lipofectamine 2000 and Actb MB was added in Test 1, and an image obtained by superimposing the bright-field image and the fluorescent image (left). Fig. 12C is a fluorescent image (right) of a medium to which Actb MB alone was added in Test 1, and an image obtained by superimposing the bright-field image and the fluorescent image (left). [Figure 13] FIG. 13A is a graph showing the expression level of the mRNA encoding PGC-1α in Test 2, FIG. 13B is a graph showing the expression level of the above mRNA of Pdk1 in Test 2, FIG. 13C is a graph showing the expression level of the above mRNA of Oct-3 / 4 in Test 2, and FIG. 13D is a graph showing the expression level of the above mRNA of Sox2 in Test 2. [Figure 14]FIG. 14A is a graph showing the expression level of the above-mentioned Nanog mRNA in Test 2, FIG. 14B is a graph showing the expression level of the above-mentioned Pax6 mRNA in Test 2, FIG. 14C is a graph showing the expression level of the above-mentioned Nestin mRNA in Test 2, and FIG. 14D is a graph showing the expression level of the above-mentioned Tubb III mRNA in Test 2. [Figure 15] FIG. 15A is a fluorescent image (right) of a medium to which cGNS (PGC-1α MB) was added under the condition of adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 15B is a fluorescent image (right) of a medium 4 days after adding cGNS (PGC-1α MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 15C is a fluorescent image (right) of a medium 7 days after adding cGNS (PGC-1α MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 15D is a fluorescent image (right) of a medium 9 days after adding cGNS (PGC-1α MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. [Figure 16] FIG. 16A is a fluorescent image (right) of a medium to which cGNS (Pdk1 MB) was added under the condition of adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 16B is a fluorescent image (right) of a medium 4 days after adding cGNS (Pdk1 MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 16C is a fluorescent image (right) of a medium 7 days after adding cGNS (Pdk1 MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 16D is a fluorescent image (right) of a medium 9 days after adding cGNS (Pdk1 MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. [Figure 17]FIG. 17A is a fluorescent image (right) of a medium to which cGNS (Actb MB) was added under the condition of adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 17B is a fluorescent image (right) of a medium 4 days after adding cGNS (Actb MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 17C is a fluorescent image (right) of a medium 7 days after adding cGNS (Actb MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 17D is a fluorescent image (right) of a medium 9 days after adding cGNS (Actb MB) under the condition of not adding LIF in Test 2, and an image (left) of a bright field image and a fluorescent image superimposed thereon. [Figure 18] Figure 18A is a graph showing the fluorescence intensity of medium supplemented with cGNS (PGC-1α MB) in Test 2, Figure 18B is a graph showing the fluorescence intensity of medium supplemented with cGNS (Pdk1 MB) in Test 2, and Figure 18C is a graph showing the fluorescence intensity of medium supplemented with cGNS (Actb MB) in Test 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the following embodiments.

[0015] FIG. 1 is a flow chart illustrating a method for assessing the differentiation state of a cell according to one embodiment of the present invention.

[0016] In this embodiment, a probe is introduced into a cell (step S110), a signal from the probe is acquired (step S120), and the differentiation state of the cell is evaluated based on the acquired signal (step S130).

[0017] (Introduction of probe (step S110)) First, the probe is introduced into the cell.

[0018] The above-mentioned probe may be any probe capable of detecting mRNA encoding peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) or peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). In this embodiment, the expression of these enzymes or mRNA is detected to detect the metabolic state of the cell, thereby evaluating the differentiation state of the cell.

[0019] The present inventors have found that the expression level of PGC-1α or mRNA encoding PGC-1α is significantly higher in differentiated somatic cells than in undifferentiated cells, and have found that detection of the expression level of PGC-1α or mRNA encoding PGC-1α is extremely useful for determining the differentiation state of cells from an undifferentiated state in which glycolytic metabolism is dominant to a differentiated state in which mitochondrial metabolism is activated, thereby completing the present invention.

[0020] Based on the above new findings, in this process, in order to detect mRNA encoding PGC-1α or the expression level of PGC-1α, a probe capable of detecting mRNA encoding PGC-1α or a probe capable of detecting PGC-1α is introduced into cells.

[0021] The above-mentioned probe may be any compound having a site that directly or indirectly binds to the mRNA encoding PGC-1α or PGC-1α and a site that emits a detectable signal.For example, the above-mentioned probe may be a probe that can specifically bind to the mRNA encoding PGC-1α by a nucleic acid having a sequence complementary to at least a part of the nucleic acid sequence of the mRNA encoding PGC-1α, or a probe that can specifically bind to PGC-1α by an antibody.The above-mentioned probe may also be a probe that contains a fluorophore and emits fluorescence as a signal, or a probe that emits other signals by chemiluminescence or the like.

[0022] The type of the phosphor is not particularly limited, and may be a fluorescent dye or semiconductor nanoparticles.

[0023] Examples of the fluorescent dyes include rhodamine-based dye molecules, squarylium-based dye molecules, fluorescein-based dye molecules, coumarin-based dye molecules, acridine-based dye molecules, pyrene-based dye molecules, erythrosine-based dye molecules, eosine-based dye molecules, cyanine-based dye molecules, aromatic ring-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, and pyrromethene-based dye molecules.

[0024] Examples of the semiconductors constituting the semiconductor nanoparticles include II-VI group compound semiconductors, III-V group compound semiconductors, and IV group semiconductors. Specific examples of the semiconductors constituting the semiconductor nanoparticles include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.

[0025] The probe capable of specifically binding to the above-mentioned PGC-1α-encoding mRNA may be a known probe such as a molecular beacon, Taqman probe, cycling probe, or INAF probe, but a molecular beacon is preferred because it can use a general-purpose fluorescent dye and is easy to detect in various cell types.

[0026] A molecular beacon is a nucleic acid derivative having a stem-loop structure, with a fluorescent dye bound to one of the 5' and 3' ends and a quenching dye bound to the other end. When the molecular beacon forms the stem-loop structure, the fluorescent dye and the quenching dye are in close proximity to each other, so the fluorescence emitted from the fluorescent dye is quenched, but when the molecular beacon approaches a target sequence (mRNA encoding PGC-1α), the loop structure opens and the molecular beacon binds to the mRNA encoding PGC-1α. This causes the fluorescent dye and the quenching dye to separate from each other, allowing the fluorescence emission to be detected.

[0027] The combination of the fluorescent dye and the quenching dye is not particularly limited and may be appropriately selected from the fluorescent dyes described above. The quenching dye may be a molecule that performs quenching by any of fluorescence resonance energy transfer (FRET), contact quenching, and collisional quenching.

[0028] The molecular beacon may have a sequence complementary to at least a part of the nucleic acid sequence of the mRNA encoding PGC-1α. The complementary sequence may be sufficiently complementary to the extent that the molecular beacon can bind to the mRNA encoding PGC-1α, and may have, for example, 80% or more identity to at least a part of the nucleic acid sequence of the mRNA encoding PGC-1α, preferably 90% or more identity, and more preferably 95% or more identity. The sequence complementary to at least a part of the nucleic acid sequence of the mRNA encoding PGC-1α typically constitutes the loop structure of the molecular beacon, and may be, for example, a sequence consisting of 2 to 40 nucleic acids.

[0029] Moreover, the molecular beacon has mutually complementary sequences at both the 5'-end and 3'-end of the sequence complementary to at least a part of the nucleic acid sequence of PGC-1α. The mutually complementary sequences bind to each other to form a stem region of a stem-loop structure. The mutually complementary sequences may be, for example, sequences consisting of 5 to 10 nucleic acids. From the viewpoint of increasing the stability of the molecular beacon, it is preferable that the mutually complementary sequences have a total amount of cytosine (C) and thymine (T) of 50% or more of the total amount of adenine (A), cytosine (C), thymine (T) and guanine (G) of 50% or more.

[0030] The probe capable of specifically binding to PGC-1α by the antibody is preferably a phosphor integrated dot (PID). The PID is a nano-sized particle that is based on an organic or inorganic particle and contains multiple phosphors. The PID directly or indirectly binds to an antibody that specifically binds to PGC-1α to label PGC-1α. The multiple phosphors may be present within the particle or on the surface of the particle. The phosphor integrated particle can emit fluorescence of sufficient intensity to show each molecule of the target substance as a bright spot.

[0031] Examples of organic materials for the matrix include thermosetting resins such as melamine resin, urea resin, aniline resin, guanamine resin, phenol resin, xylene resin, and furan resin, thermoplastic resins including styrene resin, acrylic resin, acrylonitrile resin, AS resin (acrylonitrile-styrene copolymer), and ASA resin (acrylonitrile-styrene-methyl acrylate copolymer), other resins such as polylactic acid, and polysaccharides. Examples of inorganic materials for the matrix include silica and glass. It is preferable that the matrix and the fluorescent substance have substituents or sites with opposite charges and are electrostatically interacting with each other.

[0032] The average particle size of the phosphor-accumulated particles is not particularly limited, but taking into consideration the ease of detection as bright spots, it is preferably 10 nm or more and 500 nm or less, and more preferably 50 nm or more and 200 nm or less.

[0033] The particle size of the phosphor-aggregated particles can be measured by measuring the projected area of ​​the phosphor-aggregated particles using a scanning electron microscope (SEM) and converting it into a circle-equivalent diameter. The average particle size and coefficient of variation of a group consisting of multiple phosphor-aggregated particles are calculated using the particle sizes (circle-equivalent diameters) calculated for a sufficient number of phosphor-aggregated particles (e.g., 1,000 particles).

[0034] Although there is no particular limitation on the method for introducing the probe into the cell, in this embodiment, it is preferable to introduce the probe into the cell by using gelatin nanoparticles carrying the probe.

[0035] Gelatin nanoparticles are taken up into cells by the cell's own activity. Therefore, compared with other methods such as electroporation, gelatin nanoparticles allow the above-mentioned probe to be easily introduced into cells while having less influence on the activity of living cells. In addition, since gelatin particles can carry a large amount of the above-mentioned probe, it is possible to introduce a large amount of the probe into cells at once. Furthermore, gelatin nanoparticles release the probe over a long period of time after being taken up into cells, making it possible to detect mRNA encoding PGC-1α or the expression of PGC-1α over time.

[0036] Furthermore, the probe capable of specifically binding to the mRNA encoding PGC-1α is composed of a negatively charged nucleic acid, and therefore is difficult to enter the negatively charged cell membrane as it is. In contrast, the probe is supported on gelatin nanoparticles, and the gelatin nanoparticles are taken up into cells, so that the probe can be more easily introduced into cells.

[0037] The gelatin nanoparticles may be any known gelatin nanoparticles obtained by denaturing collagen derived from cow bone, cowhide, pig skin, pig tendon, fish scales, fish meat, etc. Gelatin has been used for food and medical purposes for a long time, and is less harmful to the human body when ingested. In addition, gelatin has the advantage that it does not need to be removed from the body because it disperses and disappears in the body.

[0038] The weight-average molecular weight of the gelatin constituting the gelatin nanoparticles is preferably from 1000 to 100000. The weight-average molecular weight can be a value measured, for example, according to the PAGI method, 10th edition (2006).

[0039] The gelatin constituting the gelatin nanoparticles may be crosslinked. The crosslinking may be performed by a crosslinking agent or may be self-crosslinking without using a crosslinking agent.

[0040] From the viewpoint of making it easier to carry the probe capable of detecting PGC-1α, the gelatin nanoparticles are preferably cationized by introducing a primary amino group, a secondary amino group, a tertiary amino group, or a quaternary ammonium group, etc. Nucleic acids have a negative charge, and therefore can electrostatically interact with cationized gelatin to bind more strongly.

[0041] Gelatin nanoparticles can be cationized by a known method of introducing a functional group that cationizes under physiological conditions during production. For example, alkyl diamines including ethylenediamine and N,N-dimethyl-1,3-diaminopropane, trimethylammonium acetohydrazide, spermine, spermidine, and diethylamide chloride can be reacted with condensing agents including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, cyanuric chloride, N,N'-carbodiimidazole, cyanogen bromide, diepoxy compounds, tosyl chloride, diethyltriamine-N,N,N',N'',N''-pentanoic acid dianhydride, and trisyl chloride to introduce the amino groups into hydroxyl or carboxyl groups of gelatin.

[0042] The gelatin nanoparticle carries the probe. For example, when the probe is a molecular beacon, the gelatin nanoparticle carries the molecular beacon. When the probe is a PID, the gelatin nanoparticle carries the PID, an antibody that specifically binds to PGC-1α, and a medium molecule that binds the antibody to the PID.

[0043] The term "gelatin nanoparticles carrying a probe" means that the probe is immobilized on the surface of the gelatin nanoparticles or incorporated inside the gelatin nanoparticles.

[0044] In addition, it is preferable that the amount of probe in the gelatin nanoparticle is greater than the amount of probe in the surface layer. By reducing the amount of probe in the surface layer of the gelatin nanoparticle, the amount of probe exposed on the surface of the gelatin nanoparticle can be reduced. This makes it difficult for the gelatin nanoparticle to be recognized as a foreign body by cells, and makes it easier to be taken up into the cell by activities such as endocytosis. The surface layer refers to a region up to a depth of 1% of the average particle diameter of the gelatin nanoparticle.

[0045] The average particle size of the gelatin nanoparticles is preferably 100 nm or more and 1000 nm or less. Although the gelatin nanoparticles carry a probe, they do not substantially have a probe on their surface, so that even if the average particle size is 1000 nm, they are easily taken up into cells by the activity of the cells themselves. In order to take up many gelatin nanoparticles into cells in a shorter time, the average particle size of the gelatin nanoparticles is more preferably 800 nm or less. On the other hand, gelatin nanoparticles having an average particle size of 100 nm or more can easily carry a probe within the particle, and the probe capacity can be increased. From the above viewpoint, the average particle size of the gelatin nanoparticles is preferably 200 nm or more, more preferably 300 nm or more.

[0046] The average particle size of the gelatin nanoparticles can be the apparent particle size of the gelatin nanoparticles measured by dynamic light scattering. Alternatively, the average particle size of the gelatin nanoparticles can be the average of the major axis and minor axis. The minor axis and major axis of the gelatin nanoparticles can be values ​​obtained by analyzing images of gelatin nanoparticles in a dry state after standing in the air at 80°C for 24 hours, which are captured by a scanning electron microscope (SEM). Since gelatin nanoparticles are usually aggregates of multiple gelatin nanoparticles, the major axis, minor axis, and particle size of the gelatin nanoparticles can be the average of the major axis, minor axis, and particle size of multiple gelatin nanoparticles (for example, 20 gelatin nanoparticles) arbitrarily selected from the aggregate. When there is a difference between the average particle sizes measured by these methods, the average particle size measured by dynamic light scattering can be used.

[0047] The amount of probe carried by the gelatin nanoparticles, the average concentration of the probe in the surface layer of the gelatin nanoparticles, and the average concentration of the probe in the inside can be determined by XPS depth profile measurement. In the XPS depth profile measurement, the surface composition analysis can be performed sequentially while exposing the inside of the sample by using X-ray photoelectron spectroscopy (XPS) measurement and rare gas ion sputtering such as argon. The distribution curve obtained by such a measurement can be created, for example, with the atomic ratio of each element (unit: at%) on the vertical axis and the etching time (sputtering time) on the horizontal axis. In addition, in such an element distribution curve with the etching time on the horizontal axis, the etching time is roughly correlated with the distance from the surface. Therefore, elemental analysis is performed from the surface to the center of the gelatin nanoparticle to obtain an elemental distribution curve of the gelatin nanoparticle, and the amount of probe in the surface layer is obtained from the elemental distribution from the measurement start point to the etching time corresponding to 0.01X (X is the average particle diameter), and further the amount of probe inside is obtained from the elemental distribution from the etching time corresponding to 0.01X to the etching time corresponding to the particle center.

[0048] The amount of probe is measured at multiple randomly selected locations (e.g., 10 locations) using the above method, the average value (mass) of the probe contained in each of the surface layer and the interior is calculated, and the concentration relative to the total mass of the gelatin particle (i.e., the total mass of gelatin and probe) is calculated to obtain the average concentration of each. Since gelatin nanoparticles are usually an aggregate of multiple particles, the average concentration of the probe can be calculated by averaging the average concentrations of multiple gelatin particles (e.g., 20 gelatin particles) randomly selected from the aggregate.

[0049] When the gelatin nanoparticles carrying the above-mentioned probes are brought into contact with cells, the gelatin nanoparticles are taken up into the cells by the activity of the cells themselves.

[0050] The above-mentioned cells may be any cells whose differentiation state is to be evaluated, and are preferably cells whose differentiation or dedifferentiation switches between a state in which glycolysis is dominant and a state in which mitochondrial metabolism is activated, and are particularly preferably cells whose glycolysis is dominant in the undifferentiated state and whose mitochondrial metabolism is activated in the differentiated state. Examples of the above-mentioned cells include stem cells, including embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), nerve cells, and cancer cells.

[0051] For example, when cells or tissues induced to differentiate from pluripotent stem cells are transplanted into living organisms, there is a risk of tumor formation if undifferentiated pluripotent stem cells remain. Therefore, it is expected that the safety of regenerative medicine such as transplantation will be improved by introducing the above-mentioned probe into pluripotent stem cells and evaluating their differentiation state.

[0052] The above cells may not be undifferentiated cells, but may be differentiated somatic cells derived from biological samples or specimens extracted from various organs. By introducing the above probe into these cells and observing whether the expression of mRNA encoding PGC-1α or PGC-1α is reduced, it is also possible to evaluate the canceration of these cells or the acquisition of pluripotency through dedifferentiation.

[0053] These cells are collected from a living body and the above-mentioned probe is introduced by a known method. The above-mentioned introduction may be performed by a known method such as electroporation or microinjection, but from the viewpoint of suppressing the decrease in the activity of the cells, a method in which gelatin nanoparticles carrying the above-mentioned probe are mixed with the cells in a liquid and cultured is preferred.

[0054] In this step, a probe capable of detecting mRNA encoding pyruvate dehydrogenase kinase 1 (Pdk1) or pyruvate dehydrogenase kinase 1 (PDK1) may be introduced.

[0055] Cellular metabolism includes glycolysis, which takes place in the cytoplasm, and the TCA cycle and oxidative phosphorylation, which take place in mitochondria. In undifferentiated cells, glycolysis is predominant, but in differentiated somatic cells, it is known that mitochondrial metabolism (TCA cycle and oxidative phosphorylation) is also activated.

[0056] Pyruvate, the final product of glycolysis, is oxidatively decarboxylated by a complex consisting of pyruvate dehydrogenase (PDH), dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase (pyruvate dehydrogenase complex (PDC)), converted to acetyl-CoA, and sent to the TCA cycle. PDH is phosphorylated by four PDH kinases, PDK1, PDK2, PDK3, and PDK4, to inhibit its activity, and dephosphorylated by two PDH phosphatases, pyruvate dehydrogenase phosphatase 1 (PDP1) and PDP2, to activate it.

[0057] The conversion of pyruvate to acetyl-CoA involves many of the enzymes and their coenzymes mentioned above, but it was unclear whether the expression of these enzymes or coenzymes was useful as a marker for cell differentiation, and if so, which enzymes or coenzymes could serve as a marker.

[0058] In contrast, the present inventors found that the expression levels of pdk1 or PDK1 are significantly higher in undifferentiated cells than in differentiated somatic cells, and found that detection of the expression levels of pdk1 or PDK1 is extremely useful for determining the differentiation state of cells from an undifferentiated state in which glycolytic metabolism is dominant to a differentiated state in which mitochondrial metabolism is activated.

[0059] The above-mentioned probe may be any compound having a site that directly or indirectly binds to pdk1 or PDK1 and a site that emits a detectable signal. For example, the above-mentioned probe may be a probe that can specifically bind to pdk1 via a nucleic acid having a sequence complementary to at least a portion of the nucleic acid sequence of pdk1, or a probe that can specifically bind to PDK1-1α via an antibody. The above-mentioned probe may also be a probe that contains a fluorophore and emits fluorescence as a signal, or a probe that emits other signals such as chemiluminescence.

[0060] The configuration of these probes can be similar to that of the above-mentioned probe capable of detecting mRNA encoding PGC-1α and probe capable of detecting PGC-1α.

[0061] In this case, the gelatin nanoparticles may be a set of gelatin nanoparticles including gelatin nanoparticles carrying mRNA encoding PGC-1α or a probe capable of detecting PGC-1α, and gelatin nanoparticles carrying a probe capable of detecting Pdk1 or PDK1.

[0062] In addition, at this time, a probe capable of detecting an mRNA (e.g., Actb) or a protein (e.g., β-actin (ACTB)) whose expression level does not change depending on the differentiation state of the cells may be introduced as a control. The configuration of this probe may be the same as that of the above-mentioned probe capable of detecting the mRNA encoding PGC-1α and the probe capable of detecting PGC-1α.

[0063] In this case, the gelatin nanoparticles may be a set of gelatin nanoparticles including gelatin nanoparticles carrying mRNA encoding PGC-1α or a probe capable of detecting PGC-1α, gelatin nanoparticles carrying a probe capable of detecting Pdk1 or PDK1, and gelatin nanoparticles carrying a probe capable of detecting mRNA or protein whose expression level does not change depending on the differentiation state of the cell.

[0064] (Acquisition of signal from probe (step S120)) Next, a signal derived from the probe and emitted from the cells into which the probe has been introduced is obtained, thereby making it possible to detect the expression of PDK1 or Pdk1 in the cells.

[0065] The signal may be acquired by a method according to the type of signal emitted from the probe. For example, when the probe contains a fluorescent substance, a fluorescent image may be obtained by capturing the fluorescence emitted from the cell using a fluorescent microscope or the like.

[0066] The signal may be obtained by a method that can confirm the presence or absence of the signal, or by a method that can quantitatively measure the amount of the signal. The signal may be obtained by a qualitative method or a quantitative method.

[0067] The signal may be acquired immediately after the introduction of the probe, or after a predetermined time has elapsed. The signal may be acquired only once, or over time (continuously, or multiple times at intervals). When it is desired to determine the current state of the cell, the signal may be acquired immediately after the introduction of the probe. When it is desired to observe the timing at which the cell differentiates, the signal may be acquired over time after the introduction of the probe.

[0068] In particular, if the probe is introduced into cells using gelatin nanoparticles carrying the probe, the gelatin nanoparticles gradually release the probe, making it easy to obtain the signal over time.

[0069] The cells are maintained in a viable state until the signal is acquired. At this time, the cells may be cultured in a medium or may be returned to a living body. In addition, at this time, the differentiation or dedifferentiation of the cells may be promoted or inhibited.

[0070] (Evaluation of cell differentiation state (step S130)) Based on the above obtained signals, the differentiation state of the cells can be evaluated.

[0071] According to the new findings of the present inventors, the expression level of PGC-1α changes with the differentiation of cells. When cells are undifferentiated and the metabolism by glycolysis is dominant, the expression level of mRNA encoding PGC-1α or PGC-1α is lower. Conversely, when cells are differentiated and the metabolism in mitochondria is activated, the expression level of mRNA encoding PGC-1α or PGC-1α is higher. Therefore, when the expression level of mRNA encoding PGC-1α or PGC-1α is lower, it can be determined that the cells are undifferentiated, and when the expression level of mRNA encoding PGC-1α or PGC-1α is higher, it can be determined that the cells are differentiated. In addition, by observing the cells into which the above-mentioned probe is introduced over time, it can be determined that the cells have differentiated when the expression level of mRNA encoding PGC-1α or PGC-1α is higher, and it can be determined that the cells have dedifferentiated when the expression level of mRNA encoding PGC-1α or PGC-1α is lower.

[0072] In addition, according to the new findings of the present inventors, the expression level of PDK1 also changes with cell differentiation. When cells are undifferentiated and glycolytic metabolism is dominant, the expression level of Pdk1 or PDK1 is higher. Conversely, when cells are differentiated and metabolism in mitochondria is activated, the expression level of Pdk1 or PDK1 is lower. Therefore, when the expression level of Pdk1 or PDK1 is higher, the cell can be judged to be undifferentiated, and when the expression level of Pdk1 or PDK1 is lower, the cell can be judged to be differentiated. In addition, by observing the cells into which the above-mentioned probe is introduced over time, when the expression level of Pdk1 or PDK1 is lower, the cell can be judged to be differentiated, and when the expression level of Pdk1 or PDK1 is higher, the cell can be judged to be dedifferentiated.

[0073] Furthermore, by observing the expression levels of the mRNAs encoding these two proteins or the proteins themselves over time, the differentiation state of cells can also be observed in a superimposed manner. EXAMPLES

[0074] Specific examples of the present invention will be described below together with comparative examples, but the present invention is not limited to these.

[0075] In the figures related to the following explanation, "*" indicates a significant difference (a p-value of less than 0.05 is considered statistically significant), and "ns" indicates no significant difference.

[0076] The following experiment was carried out using a molecular beacon capable of detecting mRNA encoding PGC-1α, a molecular beacon capable of detecting Pdk1, and, as a control, a molecular beacon capable of detecting mRNA for β-actin (Actb), which is expressed at a constant level regardless of the differentiation state of cells.

[0077] 1. Probe The following probes were used:

[0078] PGC-1α MB: A probe modified at the 5' end with TYE563 and at the 3' end with IBRQ (lowa black RQ) of SEQ ID NO: 1. SEQ ID NO: 1 is a molecular beacon in which positions 1 to 7 and positions 31 to 37 are complementary sequences that constitute a stem region, and positions 8 to 30 are a sequence that constitutes a loop structure. Pdk1 MB: A probe modified at the 5' end with AlexaFlour488 and at the 3' end with IBFQ (lowa black FQ) of SEQ ID NO: 1. SEQ ID NO: 1 is a molecular beacon in which positions 1 to 7 and positions 31 to 37 are complementary sequences that constitute a stem region, and positions 8 to 30 are a sequence that constitutes a loop structure. Actb MB: A probe modified at the 5' end with TYE665 and at the 3' end with IBRQ (lowa black RQ) of SEQ ID NO: 2. SEQ ID NO: 2 is a molecular beacon in which positions 1 to 6 and positions 24 to 30 are complementary sequences that constitute a stem region, and positions 7 to 23 are a sequence that constitutes a loop structure.

[0079] It was previously confirmed that the fluorescence intensity from these molecular beacons only emitted fluorescence when they reacted with mRNA encoding PGC-1α, pdk1, and actb, respectively, and that the fluorescence intensity increased depending on the amount of each mRNA.

[0080] 2. Gelatin nanoparticles carrying probes 2-1. Preparation of gelatin nanoparticles Gelatin (G-2613P, manufactured by Nitta Gelatin Co., Ltd.) was dissolved in 24 ml of 0.1 M phosphate buffer solution (pH 5.0) at 37 ° C. An appropriate amount of ethylenediamine was added to this solution. Furthermore, an aqueous hydrochloric acid solution was added to adjust the pH of the solution to 5.0. Furthermore, an appropriate amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the concentration of gelatin was adjusted to 2 mass% by adding an aqueous 0.1 M phosphate buffer solution. This solution was stirred at 37 ° C. for 4 hours to introduce ethylenediamine into the carboxyl group of the gelatin. The reaction product was then dialyzed against double distilled water for 3 days to obtain a slurry of cationized gelatin. Then, acetone was added as a phase separation inducer, and the mixture was mixed at 50 ° C. to collect particles precipitated in the slurry, which were then washed with pure water to obtain cationized gelatin nanoparticles. These cationized gelatin nanoparticles are called cGNS.

[0081] The apparent average particle size of cGNS was determined to be 168.0 nm by dynamic light scattering at 37°C using DLS-7000, manufactured by Otsuka Electronics Co., Ltd. The zeta potential of cGNS was determined to be 8.41 mV by electrophoretic light scattering using DLS-8000, manufactured by Otsuka Electronics Co., Ltd.

[0082] 2-2. Molecular beacon support by gelatin nanoparticles cGNS and PGC-1α MB were mixed at room temperature for 15 minutes, then centrifuged and washed with water to obtain gelatin nanoparticles carrying the above probe. These gelatin nanoparticles were designated as cGNS (PGC-1α MB).

[0083] cGNS and Pdk1 MB were mixed at room temperature for 15 minutes, then centrifuged and washed with water to obtain gelatin nanoparticles carrying the above-mentioned probe. These gelatin nanoparticles are referred to as cGNS (Pdk1 MB).

[0084] cGNS and Actb MB were mixed at room temperature for 15 minutes, then centrifuged and washed with water to obtain gelatin nanoparticles carrying the above-mentioned probe. These gelatin nanoparticles are referred to as cGNS(Actb MB).

[0085] The amount of probe carried by cGNS (PGC-1α MB), cGNS (Pdk1 MB) and cGNS (Actb MB) was determined by a conventional method. The apparent average particle size and zeta potential of cGNS (PGC-1α MB), cGNS (Pdk1 MB) and cGNS (Actb MB) were also determined in the same manner as for cGNS. The results are shown in Table 1. The values ​​in Table 1 indicate the mean ± standard deviation.

[0086] [Table 1]

[0087] As is clear from Table 1, no significant changes were observed between the physical properties of cGNS(PGC-1α MB), cGNS(Pdk1 MB), and cGNS(Actb MB) depending on the type (sequence) of the probe carried.

[0088] It was also confirmed in advance that these gelatin nanoparticles were taken up to the same extent by the cells used in the following test, and that the uptake of these gelatin nanoparticles did not cause any change in the expression level and staining level of the target mRNA and various marker genes mRNA. It was also confirmed in advance that the amount of these gelatin nanoparticles introduced into the inside of the above cells was proportional to both the contact time between the gelatin nanoparticles and the cells and the concentration of the gelatin nanoparticles, and tended to increase according to the number of cells. The following experiment was also performed under conditions that confirmed that the uptake of gelatin nanoparticles into the inside of the cells did not significantly reduce the viability of the cells, and that the fluorescence from the probes carried by the gelatin nanoparticles could be sufficiently detected.

[0089] 3. Test 1: Early differentiation of ES cells 3-1. Observation of changes in mRNA expression levels depending on the differentiation state of cells (qRT-PCR) Mouse ES cells (EB5, 2 × 10 5 Cells (cells / well) were seeded in 6-well plates and cultured for 48 h in the presence of leukemia inhibitory factor (LIF) to maintain the undifferentiated state. The medium was then replaced with OptiMEM, and the cells were further cultured with or without LIF. After 1, 2, and 3 days of culture, the cells were harvested from each medium, RNA was extracted, and cDNA was synthesized by reverse transcription. In addition, qRT-PCR was used to amplify the undifferentiated markers Oct-3 / 4, Sox2, and Nanog, as well as the early differentiation markers Gata4, Gata6, and Sox17 (definitive endoderm markers), T and GSC (embryonic mesoderm markers), Pax6 and Nestin (embryonic ectoderm markers), Eomes and Cdx2 (embryonic trophectoderm markers), as well as mRNAs encoding PGC-1α and pdk1. Using the ΔΔCt method, the expression levels of these markers were first normalized using Actb as an internal standard, and then the expression levels of these mRNAs in the presence of LIF were normalized to the expression levels in the absence of LIF.

[0090] FIG. 2A is a graph showing the expression levels of the above-mentioned mRNAs of undifferentiation markers, and FIG. 2B is a graph showing the expression levels of the above-mentioned mRNAs of early differentiation markers.

[0091] As is clear from Figures 2A and 2B, when cell differentiation was induced without the addition of LIF, the expression levels of undifferentiated markers significantly decreased over time, while the expression levels of early differentiation markers significantly increased.

[0092] Figure 3A is a graph showing the expression level of mRNA encoding PGC-1α in culture media with (w LIF) and without (wo LIF), and Figure 3B is a graph showing the expression level of pdk1 in culture media with (w LIF) and without (wo LIF).

[0093] As shown in Figure 3A, when differentiation of cells was induced without the addition of LIF, the expression level of the mRNA encoding PGC-1α was significantly increased, and as shown in Figure 3B, when differentiation of cells was induced without the addition of LIF, the expression level of pdk1 was significantly decreased.

[0094] 3-2. Observation of changes in mRNA expression levels depending on the differentiation state of cells (gelatin nanoparticles) Mouse ES cells (EB5, 2 × 10 5 cells / well) were seeded in 6-well plates and cultured for 48 hours in the presence of leukemia inhibitory factor (LIF) to maintain the undifferentiated state. The medium was then replaced with OptiMEM, and the cells were further cultured with or without LIF. After 1, 2, and 3 days of culture, cGNS (PGC-1α MB) was added at 10 μg / mL, and the cells were co-cultured for 1 hr and observed under a fluorescent microscope.

[0095] Similarly, at the time of changing the medium to OptiMEM, cGNS (Pdk1 MB) was added at 10 μg / mL after 1, 2, and 3 days of culture, with and without LIF, and the cells were co-cultured for 1 hr and then observed under a fluorescent microscope.

[0096] Similarly, at the time of changing the medium to OptiMEM, cGNS (Actb MB) was added at 10 μg / mL after 1, 2, and 3 days of culture, with and without LIF addition, and the cells were co-cultured for 1 hour and then observed under a fluorescent microscope.

[0097] Figure 4A shows a fluorescent image (right) of the medium 1 day after addition of cGNS (PGC-1α MB) under the condition of LIF addition, and an image obtained by superimposing a bright field image and a fluorescent image (left). Figure 4B shows a fluorescent image (right) of the medium 2 days after addition of cGNS (PGC-1α MB) under the condition of LIF addition, and an image obtained by superimposing a bright field image and a fluorescent image (left). Figure 4C shows a fluorescent image (right) of the medium 3 days after addition of cGNS (PGC-1α MB) under the condition of LIF addition, and an image obtained by superimposing a bright field image and a fluorescent image (left).

[0098] FIG. 5A shows a fluorescent image (right) of the medium 1 day after addition of cGNS (PGC-1α MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon. FIG. 5B shows a fluorescent image (right) of the medium 2 days after addition of cGNS (PGC-1α MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon. FIG. 5C shows a fluorescent image (right) of the medium 3 days after addition of cGNS (PGC-1α MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon.

[0099] FIG. 6A shows a fluorescent image (right) of the medium one day after the addition of cGNS (Pdk1 MB) under the condition of the addition of LIF, and an image obtained by superimposing the bright field image and the fluorescent image (left). FIG. 6B shows a fluorescent image (right) of the medium two days after the addition of cGNS (Pdk1 MB) under the condition of the addition of LIF, and an image obtained by superimposing the bright field image and the fluorescent image (left). FIG. 6C shows a fluorescent image (right) of the medium three days after the addition of cGNS (Pdk1 MB) under the condition of the addition of LIF, and an image obtained by superimposing the bright field image and the fluorescent image (left).

[0100] Figure 7A shows a fluorescent image (right) of the medium 1 day after addition of cGNS (Pdk1 MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon. Figure 7B shows a fluorescent image (right) of the medium 2 days after addition of cGNS (Pdk1 MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon. Figure 7C shows a fluorescent image (right) of the medium 3 days after addition of cGNS (Pdk1 MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon.

[0101] FIG. 8A is a fluorescent image (right) of the medium one day after the addition of cGNS (Actb MB) under the condition of the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon. FIG. 8B is a fluorescent image (right) of the medium two days after the addition of cGNS (Actb MB) under the condition of the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon. FIG. 8C is a fluorescent image (right) of the medium three days after the addition of cGNS (Actb MB) under the condition of the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon.

[0102] Figure 9A shows a fluorescent image (right) of the medium 1 day after addition of cGNS (Actb MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon. Figure 9B shows a fluorescent image (right) of the medium 2 days after addition of cGNS (Actb MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon. Figure 9C shows a fluorescent image (right) of the medium 3 days after addition of cGNS (Actb MB) without the addition of LIF, and an image (left) of the bright field image and the fluorescent image superimposed thereon.

[0103] As is clear from Figures 4A to 4C and Figures 5A to 5C, when LIF was added and the cells were maintained in an undifferentiated state, the fluorescence derived from PGC-1α MB did not increase significantly in almost all cells, but when differentiation of the cells was induced without the addition of LIF, the fluorescence derived from PGC-1α MB increased significantly.

[0104] As is clear from Figures 6A to 6C and Figures 7A to 7C, when LIF was added and the cells were maintained in an undifferentiated state, the fluorescence derived from Pdk1 MB increased in almost all cells, but when differentiation of the cells was induced without the addition of LIF, the number of cells in which the fluorescence derived from Pdk1 MB was quenched increased.

[0105] On the other hand, as is clear from FIGS. 8A to 8C and 9A to 9C, no change was observed in the expression of fluorescence derived from Actb MB due to changes in the differentiation state of the cells caused by the presence or absence of addition of LIF.

[0106] In addition, the brightness of six randomly selected fields of view was measured from the fluorescent images captured by the fluorescent microscope for each medium, and the average brightness was taken as the fluorescence intensity of the fluorescent image.

[0107] Figure 10A is a graph showing the fluorescence intensity of medium supplemented with cGNS (PGC-1α MB), Figure 10B is a graph showing the fluorescence intensity of medium supplemented with cGNS (Pdk1 MB), and Figure 10C is a graph showing the fluorescence intensity of medium supplemented with cGNS (Actb MB).

[0108] As is clear from FIG. 10A, the fluorescence intensity of the cells induced to differentiate without adding LIF when cGNS (PGC-1α MB) was introduced was greater over time than that of the cells maintained in an undifferentiated state by adding LIF. Also, as is clear from FIG. 10B, the fluorescence intensity of the cells induced to differentiate without adding LIF when cGNS (Pdk1 MB) was introduced was smaller over time than that of the cells maintained in an undifferentiated state by adding LIF. In contrast, as is clear from FIG. 10C, the fluorescence intensity of the cells induced to differentiate without adding LIF when cGNS (Actb MB) was introduced was not different from that of the cells maintained in an undifferentiated state by adding LIF.

[0109] These results indicate that the expression levels of PGC-1α-encoding mRNA and Pdk1 in ES cells change depending on the differentiation state of the cells, and therefore it is possible to determine the differentiation state of ES cells by observing the expression levels of PGC-1α-encoding mRNA or Pdk1.

[0110] In addition, 125 The amount of gelatin particles taken up into the cells was measured based on the radioactivity of I. There was no difference in the amount of taken up into the cells among cGNS(PGC-1α MB), cGNS(Pdk1 MB), and cGNS(Actb MB).

[0111] 3-3. Evaluation of differences in signal sensitivity due to probe introduction method Mouse ES cells (EB5, 2 × 10 5 The cells (1000 x 1000 cells / well) were seeded in a 6-well plate and cultured for 48 hours in the presence of leukemia inhibitory factor (LIF) added to maintain the undifferentiated state. The medium was then replaced with OptiMEM, and cGNS (Pdk1 MB) was added and co-cultured for 1 hour. In addition, instead of cGNS (Pdk1 MB), a complex of Lipofectamine 2000, a gene transfer reagent consisting of cationic lipids (liposomes), and Pdk1 MB, or Pdk1 MB alone were added and co-cultured for 1 hour in the same manner. The cells were then washed with PBS and cultured for an additional 6 hours before observation under a fluorescent microscope.

[0112] Similarly, after changing the medium to OptiMEM, cGNS (Actb MB), a complex of Lipofectamine 2000 and Actb MB, or Actb MB alone was added and co-cultured for 1 hour. The cells were then washed with PBS and further cultured for 6 hours before being observed under a fluorescent microscope.

[0113] Figure 11A shows a fluorescent image (right) of a medium containing cGNS (Pdk1 MB) and an overlay image (left) of the bright-field image and the fluorescent image. Figure 11B shows a fluorescent image (right) of a medium containing a complex of Lipofectamine 2000 and Pdk1 MB and an overlay image (left) of the bright-field image and the fluorescent image. Figure 11C shows a fluorescent image (right) of a medium containing Pdk1 MB alone and an overlay image (left) of the bright-field image and the fluorescent image.

[0114] Figure 12A shows a fluorescent image (right) of a medium containing cGNS (Actb MB) and an image obtained by superimposing a bright-field image and a fluorescent image (left). Figure 12B shows a fluorescent image (right) of a medium containing a complex of Lipofectamine 2000 and Actb MB and an image obtained by superimposing a bright-field image and a fluorescent image (left). Figure 12C shows a fluorescent image (right) of a medium containing ActbMB alone and an image obtained by superimposing a bright-field image and a fluorescent image (left).

[0115] As is clear from Figures 11A to 11C and Figures 12A to 12C, when the probe was loaded onto the gelatin nanoparticles, a stronger signal (fluorescence) was observed than when a gene transfer reagent (Lipofectamine 2000) was used or when the probe was added alone.

[0116] When the gene transfer reagent (Lipofectamine 2000) was used, many dead cells were observed, but when gelatin nanoparticles were used, almost no dead cells were observed.

[0117] These results indicate that by loading a probe onto gelatin nanoparticles, a larger amount of the probe can be delivered into cells more safely.

[0118] 4. Test 2: Differentiation into neural cells 4-1. Observation of changes in mRNA expression levels depending on the differentiation state of cells (qRT-PCR) Mouse ES cells (EB5, 2 × 10 5Cells (cells / well) were seeded in 6-well plates and cultured for 48 h in the presence of leukemia inhibitory factor (LIF) to maintain the undifferentiated state. The medium was then replaced with neuronal differentiation medium (NDiff227) and further cultured with or without LIF. Cells were harvested from each medium on days 4, 7, and 9 of culture, and RNA was extracted and cDNA was synthesized by reverse transcription. Furthermore, mRNAs encoding PGC-1α and pdk1, as well as the undifferentiation markers Oct-3 / 4, Sox2, and Nanog, the neural progenitor markers Pax6 and Nestin, and the neuronal marker Tubb III were amplified by qRT-PCR. The expression levels of the mRNAs of these markers were first normalized using Actb as an internal standard, and then normalized to the expression levels of these mRNAs in the presence of LIF against the expression levels of these mRNAs in the absence of LIF, using the ΔΔCt method.

[0119] Figure 13A is a graph showing the expression level of the mRNA encoding PGC-1α, Figure 13B is a graph showing the expression level of the above mRNA for Pdk1, Figure 13C is a graph showing the expression level of the above mRNA for Oct-3 / 4, and Figure 13D is a graph showing the expression level of the above mRNA for Sox2.

[0120] Figure 14A is a graph showing the expression level of the above-mentioned Nanog mRNA, Figure 14B is a graph showing the expression level of the above-mentioned Pax6 mRNA, Figure 14C is a graph showing the expression level of the above-mentioned Nestin mRNA, and Figure 14D is a graph showing the expression level of the above-mentioned Tubb III mRNA.

[0121] As is clear from Figures 13A to 13D and Figures 14A to 14D, when differentiation into neural cells was induced, the expression level of undifferentiated markers significantly decreased over time, while the expression levels of neural progenitor differentiation markers and neuronal markers significantly increased, and simultaneously, the expression level of mRNA encoding PGC-1α increased over time, and the expression level of Pdk1 decreased over time.

[0122] These results indicate that the expression levels of mRNA encoding PGC-1α and Pdk1 in nerve cells change with cell differentiation.

[0123] 4-2. Observation of changes in mRNA expression levels depending on the differentiation state of cells (gelatin nanoparticles) Mouse ES cells (EB5, 2 × 10 5 cells / well) were seeded in 6-well plates and cultured for 48 hours in the presence of leukemia inhibitory factor (LIF) to maintain the undifferentiated state. The medium was then replaced with neural differentiation medium (NDiff227) and the cells were further cultured with or without LIF. The cells were further cultured with or without LIF. After 4, 7, and 9 days of culture, cGNS (PGC-1α MB) was added at 10 μg / mL, and the cells were co-cultured for 1 hr and observed under a fluorescent microscope.

[0124] Similarly, at the time of changing the medium to neural differentiation medium (NDiff227), cGNS (Actb MB) was added at 10 μg / mL on the 4th, 7th, and 9th days of culture, with and without LIF, and the cells were co-cultured for 1 hour and then observed under a fluorescent microscope.

[0125] Similarly, at the time of changing the medium to neural differentiation medium (NDiff227), cGNS (Actb MB) was added at 10 μg / mL on the 4th, 7th, and 9th days of culture, with and without LIF, and the cells were co-cultured for 1 hour and then observed under a fluorescent microscope.

[0126] FIG. 15A is a fluorescent image (right) of a medium to which cGNS (PGC-1α MB) was added under the condition of adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 15B is a fluorescent image (right) of a medium 4 days after adding cGNS (PGC-1α MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 15C is a fluorescent image (right) of a medium 7 days after adding cGNS (PGC-1α MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 15D is a fluorescent image (right) of a medium 9 days after adding cGNS (PGC-1α MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon.

[0127] FIG. 16A is a fluorescent image (right) of a medium to which cGNS (Pdk1 MB) was added under the condition of adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 16B is a fluorescent image (right) of a medium 4 days after adding cGNS (Pdk1 MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 16C is a fluorescent image (right) of a medium 7 days after adding cGNS (Pdk1 MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 16D is a fluorescent image (right) of a medium 9 days after adding cGNS (Pdk1 MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon.

[0128] FIG. 17A is a fluorescent image (right) of a medium to which cGNS (Actb MB) was added under the condition of adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 17B is a fluorescent image (right) of a medium 4 days after adding cGNS (Actb MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 17C is a fluorescent image (right) of a medium 7 days after adding cGNS (Actb MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon. FIG. 17D is a fluorescent image (right) of a medium 9 days after adding cGNS (Actb MB) under the condition of not adding LIF, and an image (left) of a bright field image and a fluorescent image superimposed thereon.

[0129] As is clear from Figures 15A to 15D, when LIF was added and the cells were maintained in an undifferentiated state, the fluorescence derived from PGC-1α MB did not increase significantly in almost all cells, but when differentiation of the cells was induced without the addition of LIF, the number of cells with increased fluorescence derived from PGC-1α MB significantly increased.

[0130] Furthermore, as is clear from Figures 16A to 16D, when LIF was added and the cells were maintained in an undifferentiated state, fluorescence derived from Pdk1 MB was expressed in almost all cells, but when differentiation of the cells was induced without the addition of LIF, the number of cells in which the fluorescence derived from Pdk1 MB was quenched increased over time.

[0131] On the other hand, as is clear from FIGS. 17A to 17D, no change was observed in the expression of fluorescence derived from Actb MB due to changes in the differentiation state of the cells caused by the presence or absence of addition of LIF.

[0132] In addition, the brightness of six randomly selected fields of view was measured from the fluorescent images captured by the fluorescent microscope for each medium, and the average brightness was taken as the fluorescence intensity of the fluorescent image.

[0133] Fig. 18A is a graph showing the fluorescence intensity of the medium to which cGNS (PGC-1α MB) was added, Fig. 18B is a graph showing the fluorescence intensity of the medium to which cGNS (Pdk1 MB) was added, and Fig. 18C is a graph showing the fluorescence intensity of the medium to which cGNS (Actb MB) was added. In Fig. 18A to Fig. 18C, "Ctrl" is the intensity from the medium to which LIF was added to maintain the cells in an undifferentiated state, and "day 4", "day 7", and "day 9" are the intensities from the medium after the respective days had elapsed after differentiation was induced without the addition of LIF.

[0134] As is clear from FIG. 18A, the fluorescence intensity of the cells induced to differentiate without adding LIF when cGNS (PGC-1α MB) was introduced was greater over time than that of the cells maintained in an undifferentiated state by adding LIF. Also, as is clear from FIG. 18B, the fluorescence intensity of the cells induced to differentiate without adding LIF when cGNS (Pdk1 MB) was introduced was smaller than that of the cells maintained in an undifferentiated state by adding LIF. In contrast, as is clear from FIG. 18C, the fluorescence intensity of the cells induced to differentiate without adding LIF when cGNS (ActbMB) was introduced was not different from that of the cells maintained in an undifferentiated state by adding LIF.

[0135] These results indicate that the expression levels of PGC-1α-encoding mRNA and Pdk1 in ES cells change depending on the differentiation state of the cells, and therefore it is possible to determine the differentiation state of ES cells by observing the expression levels of PGC-1α-encoding mRNA or Pdk1.

[0136] The above-mentioned test results show that the differentiation state of a wide variety of cell types can be determined by observing the expression levels of PGC-1α-encoding mRNA or Pdk1. These results also show that the differentiation state of a wide variety of cell types can be determined by observing the expression levels of PGC-1α or PDK1. [Industrial Applicability]

[0137] According to the present invention, the differentiation state of cells can be observed more easily. Therefore, the present invention is applicable to a wide variety of applications including regenerative medicine, and the detection and treatment of diseases, and is expected to contribute to the development of these fields.

Claims

1. detecting mRNA encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) or peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) continuously over time in the cells; and evaluating the differentiation state of the cell based on the result of detection of the mRNA encoding PGC-1α or PGC-1α; The step of evaluating includes a step of determining whether the cell is in a differentiation state in which glycolytic metabolism is predominant or in which mitochondrial metabolism is activated; In the determining step, when the expression level of the RNA encoding PGC-1α or PGC-1α is low, it is determined that the differentiation state is one in which metabolism via glycolysis is predominant, and when the expression level of the RNA encoding PGC-1α or PGC-1α is high, it is determined that the differentiation state is one in which metabolism in mitochondria is activated; In the detecting step, the cells are maintained in a viable state. A method for assessing the differentiation state of a cell.

2. The method for evaluating the differentiation state of a cell according to claim 1, wherein the step of detecting is a step of detecting mRNA encoding PGC-1α or PGC-1α over time.

3. The method for evaluating the differentiation state of a cell according to claim 1 or 2, wherein the detecting step is a step of simultaneously detecting mRNA encoding PGC-1α or PGC-1α and mRNA encoding pyruvate dehydrogenase kinase 1 (Pdk1) or pyruvate dehydrogenase kinase 1 (PDK1).

4. The detecting step includes: introducing into said cells mRNA encoding PGC-1α or a probe capable of detecting PGC-1α; and acquiring a signal from the introduced probe. A method for evaluating the differentiation state of a cell according to any one of claims 1 to 3.

5. 5. The method for evaluating a differentiation state of a cell according to claim 4, wherein the probe has a sequence complementary to at least a part of a nucleic acid sequence of an mRNA encoding PGC-1α.

6. The method for evaluating the differentiation state of a cell according to claim 4 or 5, wherein the probe is a molecular beacon.

7. The method for evaluating the differentiation state of a cell according to any one of claims 4 to 6, wherein the step of introducing the probe is a step of contacting gelatin nanoparticles carrying the probe with the cell.

8. The method for evaluating the differentiation state of a cell according to any one of claims 1 to 7, wherein the cell is a cell that, upon differentiation or dedifferentiation, switches between a state in which glycolytic metabolism is dominant and a state in which mitochondrial metabolism is activated.

9. The method for evaluating the differentiation state of a cell according to any one of claims 1 to 8, wherein the cell is a stem cell.

10. The method for evaluating the differentiation state of a cell according to any one of claims 1 to 8, wherein the cell is an immune cell.

11. The method for evaluating the differentiation state of a cell according to any one of claims 1 to 8, wherein the cell is a cancer cell.

12. A gelatin nanoparticle for assessing the differentiation state of a cell, comprising: carrying an mRNA encoding peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) or a probe capable of detecting peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α); The differentiation state to be evaluated is whether the cell is in a differentiation state in which the metabolism by glycolysis is predominant or in which the metabolism in mitochondria is activated; In the evaluation, when the expression level of the RNA encoding PGC-1α or PGC-1α is low, it is determined that the differentiation state is one in which metabolism via glycolysis is predominant, and when the expression level of the RNA encoding PGC-1α or PGC-1α is high, it is determined that the differentiation state is one in which metabolism in mitochondria is activated, capable of maintaining the cell in a viable state after being taken up by the cell until the time of detection; Gelatin nanoparticles.

13. The gelatin nanoparticle according to claim 12, wherein the probe has a sequence complementary to at least a portion of the mRNA encoding the PGC-1α.

14. The gelatin nanoparticle of claim 12 or 13, wherein the probe is a molecular beacon.

15. A set of gelatin nanoparticles for assessing the differentiation state of a cell, comprising: A gelatin nanoparticle carrying at least one of a probe capable of detecting mRNA encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) and a probe capable of detecting peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha); and gelatin nanoparticles carrying at least one of a probe capable of detecting mRNA encoding pyruvate dehydrogenase kinase 1 (Pdk1) and a probe capable of detecting pyruvate dehydrogenase kinase 1 (PDK1); The differentiation state to be evaluated is whether the cell is in a differentiation state in which the metabolism by glycolysis is predominant or in which the metabolism in mitochondria is activated; In the evaluation, when the expression level of the RNA encoding PGC-1α or PGC-1α is low, it is determined that the differentiation state is one in which metabolism via glycolysis is predominant, and when the expression level of the RNA encoding PGC-1α or PGC-1α is high, it is determined that the differentiation state is one in which metabolism in mitochondria is activated, Any of the gelatin nanoparticles are capable of being taken up by the cells and maintaining the cells viable until detection by the probe. A set of gelatin nanoparticles.

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