KR101660450B1 - Composition for differentiating stem cells to neural precursor cells, and method using the same - Google Patents
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Abstract
A composition for the differentiation of stem cells into neural precursor cells containing an inhibitor of histone deacetylase, and a method of using the same. According to this, it is possible to efficiently differentiate into neural progenitor cells in a short time without forming an embryoid body from stem cells.
Description
A composition for the differentiation of stem cells into neural precursor cells, and a method using the same.
Since human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the ability to differentiate into almost all kinds of cells, When a tissue is damaged by disease, it can be used to regenerate the tissue by differentiating the embryonic stem cell into a desired tissue.
When hESCs are differentiated into specific cells or tissues, the hESCs aggregate with one another to form embryoid bodies (EBs) and differentiate into cells or tissues. For example, when hESCs are differentiated into neural precursor cells (NPCs), hESCs are broken up into colonies that are grown to form suspension cells, which form rounded cell masses (EBs) , And the embryoid body is adherent to the bottom of the culture vessel to induce differentiation into neural progenitor cells.
However, because of the process of forming the embryoid body, it takes a lot of time and effort to differentiate the hESC, and it is difficult to differentiate the hESC in large quantities, which makes it difficult to use the hESC clinically.
Therefore, it is necessary to develop a method for inducing the differentiation of hESCs in a quick and simple manner without floating the hESCs to form embryoid bodies.
There is provided a composition for differentiating stem cells into neural precursor cells.
Thereby providing a method of differentiating stem cells into neural progenitor cells.
One aspect provides a composition for the differentiation of stem cells into neural precursor cells (NPCs) comprising an inhibitor of histone deacetylase (HDAC).
Histone deacetylase (HDAC) refers to an enzyme that removes the acetyl group (O = C-CH 3 ) from the N-acetyl lysine amino acid of histone. HDAC is also called lysine deacetylase (KDAC). HDAC may be, for example, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, sirtuin, Sir2, or a combination thereof.
The inhibitor may be an HDAC protein activity inhibitor or an HDAC gene expression inhibitor. The HDAC protein activity inhibitor is also referred to as a histone deacetylase inhibitor (HDAC inhibitor; HDI), and may be, for example, a small molecule compound, an anti-HDAC antibody or an antigen binding fragment thereof. The small molecule compound may be, for example, 4- (dimethylamino) -N- [6- (hydroxyamino) -6-oxohexyl] -benzamide, FK228, MS275, RG2833, CI994, ITF2357, Droxinostat, MC1568, SB939, ACY1215, PCI34051, SB939, JNJ-26481585, JNJ-26481585, or a combination thereof. The HDAC gene expression inhibitor may be, for example, a chemical compound, an HDAC-specific antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, or a combination thereof.
The term "stem cell" refers to a totipotent cell capable of differentiating into all kinds of cells, pluripotent cells, or pluripotent cells capable of differentiating into various types of cells ), And stem cells can be differentiated into cells of a specific tissue as undifferentiated cells. The stem cell may be an embryonic stem cell (ESC), an adult stem cell, or an induced pluripotent stem cell (iPSC). The embryonic stem cells are obtained by extracting the inner cell mass from the blastocyst embryo immediately before embryo implantation into the uterus of the mother and culturing it in vitro. The adult stem cells are undifferentiated cells in which only a small amount exists in each tissue of the body, and refer to dead cells or cells replacing damaged tissues. Induced pluripotent stem cell (iPSC) refers to a cell that induces pluripotency, such as embryonic stem cells, by injecting a cell differentiation-related gene into a differentiated somatic cell and returning it to a pre-differentiation cell stage. The inducible pluripotent stem cells may be, for example, human feeder free-iPSC (hFF-iPSC) or urine-induced pluripotent stem cells (urine-iPSC).
The neural progenitor cell is a cell that has markers of neuronal progenitor cells and can make various neural cells after differentiation.
The term " differentiation " refers to a phenomenon in which the structure or function of a cell is specialized while the cell is proliferating and growing. Whole, pluripotent, or multipotent stem cells can be differentiated into specific types of progenitor cells (e.g., neural progenitor cells) and then differentiated into specific cells (e.g., neurons, glial cells, etc.) .
The composition may further comprise insulin, transferrin, sodium selenite, or a combination thereof. Insulin is a peptide hormone secreted from beta cells of the islet of Langerhans. Transferrin is a type of β globulin that binds to two molecules of trivalent iron ions absorbed in the serum to transport iron required for cell proliferation and hemoglobin production into iron-transporting proteins via the transferrin receptor. Sodium selenite is an inorganic compound having the formula Na 2 SeO 3 .
The composition may be a medium. The composition may further comprise, for example, DMEM (Dulbecco's Modified Eagle Medium) medium, Ham's F12 medium, glutamine, or a combination thereof.
Another aspect provides a step of culturing stem cells in the presence of an HDAC inhibitor to differentiate stem cells into neural progenitor cells.
The stem cells, HDAC, HDAC inhibitors, neural precursor cells, and differentiation are as described above.
The stem cells can be cultured in the presence of an inhibitor at a final concentration of 0.01 nM to 3 μM, 0.1 nM to 2 μM, or 0.2 to 1 μM.
The above method can be carried out by an attachment culture in which stem cells are adhered to the bottom of a culture dish and cultured. In the suspension culture of stem cells, an embryoid body, which is a mass of cells in which the stem cells are assembled in the early stage of cell division, may be formed. However, according to one aspect, So that an embryoid body may not be formed.
The method may be used to culture stem cells in a culture dish to which the polypeptide is immobilized, in order to adhere and culture the stem cells to the culture dish. The polypeptide may be a single protein or protein complex. The single protein may be, for example, vitronectin. The protein complex may be MATRIGEL (TM) (BD Biosciences).
The method can be used to culture stem cells in the presence of insulin, transferrin, sodium selenite, or a combination thereof. For example, insulin, transferrin, sodium selenite, or a combination thereof may be included in the stem cell culture medium.
In this method, the culturing time of the stem cells may vary depending on the culture conditions. For example, stem cells can be cultured for, for example, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days.
The method may further comprise differentiating the differentiated neural progenitor cells into neural cells. The step of differentiating neural progenitor cells into neural cells may be carried out by methods known in the art. For example, differentiated neural progenitor cells can be cultured in the presence of basic fibroblast growth factor (bFGF) to differentiate neural progenitor cells into neural progenitor cells. The neural cell may be a nerve cell or a neuroglia cell. Neurons, also called neurons, are cells that transmit the stimulation and excitement of the nervous system. Neurons are cells that provide the necessary substances for neurons and create a chemical environment suitable for the activity of neurons. The glial cells may be, for example, an astrocyte, an oligodendrocyte, a microglia, an ependymal cell, or a Schwann cell.
According to one aspect, the composition for differentiating stem cells into neural precursor cells and the neural progenitor cells, neurons, or glial cells differentiated according to the method of using the composition can be used as a therapeutic composition for brain or nervous system diseases. The composition may be a cell therapy agent. Wherein said brain or nervous system disease is selected from the group consisting of Parkinson's disease, neuralgia, arthritis, headache, schizophrenia, epilepsy, stroke, insomnia, dementia, depression, dyskinesia, Alzheimer's disease, lewy body dementia, Lou Gehrig's disease, multiple sclerosis Bipolar disorder, autism, attention deficit hyperactivity disorder), multiple system atrophy, Huntington's disease, Tourette's syndrome, anxiety, learning and memory impairment, various degenerative neurological disorders, bipolar disorder, ; ADHD), which is caused by a decrease or malfunction of the nervous system.
According to one aspect of the present invention, there is provided a composition for differentiating stem cells into neural progenitor cells containing the HDAC inhibitor, and a method of using the same, wherein the stem cells can be efficiently differentiated into neural progenitor cells in a short period of time without forming an embryoid body.
FIG. 1 is a schematic diagram for inducing differentiation into neural progenitor cells by adhering hPSCs in the presence of an inhibitor for each HDAC isotype.
FIG. 2 is a graph showing flow cytometry results showing the percentage (%) of cells expressing Sox1 as neural progenitor cells when hESCs were differentiated in the presence of an inhibitor for each HDAC isotype.
FIG. 3A is a schematic diagram showing the induction of differentiation into neural progenitor cells by adhesion culturing of hPSC in the presence of HDAC isotype-specific siRNA. FIG. 3B is a graph showing the effect of HDAC mRNA on siRNA when hESC was cultured in the presence of HDAC- A graph showing relative amounts.
4 is a graph showing the results of flow cytometry showing the percentage (%) of cells expressing Sox1 in hPSC cultured in the presence of siRNA that inhibits the expression of HDAC1 or HDAC4. Randomized siRNAs were used as negative controls.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are for illustrative purposes only, and the scope of the present invention is not limited to these embodiments.
Example 1. Promotes differentiation of human pluripotent stem cells into neural progenitor cells under adherent culture conditions Histone Deacetylase Identification of inhibitory compounds
1.1. Promoting differentiation of human pluripotent stem cells into neural progenitor cells under adherent culture conditions Histone Deacetylase Screening of inhibitory compounds
An isotype-dependent inhibitor of histone deacetylase (HDAC) is used to express human pluripotent stem cells (hPSC) into neural precursor cells (NPCs) under adherent culture conditions HPSCs were cultured in the presence of an isotype-specific inhibitor of HDAC (Fig. 1).
Specifically, colonies obtained by culturing in H9 hESC (WiCell Research Institute, Inc. Madison, WI, USA) were divided into about 20 cell clusters, and each cell mass was incubated with 12-well plates coated with vitronectin Well plate. To the inoculated cells, DMEM / F12 (Life Technologies), 1x GlutaMAX ™ (Life Technologies), and 10 μg / ml insulin (Sigma-Aldrich), 9 μg / ml transferrin (Sigma-Aldrich), and 14 ng / And a medium (Sigma-Aldrich) medium (ITS medium) was added.
The hESCs were incubated at 37 ° C. and 5% CO 2 for 5 days at 37 ° C. and 5% CO 2 by adding HDAC isotype-specific inhibitors (Selleck Chemicals, Huston, TX, USA) dissolved in DMSO (Sigma-Aldrich) Lt; / RTI > The dosage at least IC 50 within the range of HDAC isotype-specific cytotoxicity does not appear to be inhibited . For example, CI994 was administered at 1 μM with 1 μM of HDAC1 and 2, while CI994 was administered at 1 μM to reduce the effect of HDAC3 with an IC 50 of 1.2 μM. In addition, Droxinostat was treated with 2 μM to inhibit HDAC8, but treated with 2 μM Droxinostat to reduce HDAC6.
HDAC2: 0.9 [mu] M
HDAC3: 1.2 [mu] M
HDAC8: 1.46 [mu] M
1.2. HDAC Isotype Cultured in the presence of a star inhibitor hESC Cell Flow cell analysis
Flow cytometry was performed in Example 1.1 to confirm whether the cells cultured in the presence of the inhibitor for each HDAC isotype were neural progenitor cells.
For flow cytometry, differentiated cells in the presence of HDAC isotype-specific inhibitor in Example 1.1 were added to one well of a 12 well plate and accutase (Life Technologies) was added. (V / v) paraformaldehyde (Sigma-Aldrich) was added to the cells and incubated at room temperature for 10 min at room temperature. The cells were incubated for 5 min. Lt; RTI ID = 0.0 > min. ≪ / RTI > (V / v) normal serum / 1 x PBS (Vector Laboratories, Inc., Burlingame, Calif.) Containing 0.1% (v / v) TRITON ™ X-100 (Sigma-Aldrich) Incubation was carried out for 30 minutes to block the fixation. In order to analyze the ratio of cells (Sox1 + cells) expressing Sox1 protein which is a marker of neuroectodermal cells as neural progenitor cell markers, phycoerythrin (PE) -labeled anti-Sox1 monoclonal antibody (1:20 dilution ) (BD Biosciences). Immunostained cells were analyzed by flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences, Sparks, MD, USA).
As a result of flow cytometry, the percentage (%) of cells expressing Sox1 in the cells cultured in the presence of the HDAC isotype-specific inhibitor is shown in Fig. As shown in FIG. 2, it was confirmed that CI994, an inhibitor of
Example 2. Histone Deacetylase About siRNA Determination of whether human pluripotent stem cells induce differentiation into neural progenitor cells under adherent culture conditions
When small interfering RNA (siRNA), which inhibits the expression of HDAC, was administered, it was confirmed whether hESC induced differentiation into neural progenitor cells under adherent culture conditions.
Specifically, siRNA was administered to hESC to inhibit the expression of HDAC1 or HDAC4. A random polynucleotide sequence was used as a negative control.
Negative control: sense strand 5'-agcguguagcuagcagaggtt-3 '(SEQ ID NO: 1)
The antisense strand 5'-ccucugcuagcuacacgcutt-3 '(SEQ ID NO: 2)
HDAC1 siRNA: sense strand 5'-gcuucaaucuaacuaucaatt-3 '(SEQ ID NO: 3)
The antisense strand 5'-uugauaguuagauugaagctt-3 '(SEQ ID NO: 4)
HDAC4 siRNA: sense strand 5'-agcguguagcuagcagaggtt-3 '(SEQ ID NO: 5)
The antisense strand 5'-uugauaguuagauugaagctt-3 '(SEQ ID NO: 6)
The siRNA and the Visufect sample that transferred them into the cell were prepared by Seolin Bioscience, Seongnam, Korea.
hESC are the hESC were cultured for 5 days at a temperature and conditions of 5% CO 2 in the preparation, and 37 ℃ as described in Example 1.1. As shown in Fig. 3A, siRNA prepared above was added to hESCs once every 24 hours six times (arrow; addition of siRNA) to the culture solution while hESC was being cultured.
To confirm whether the siRNA reduced the level of the target mRNA, the amount of target mRNA was confirmed by quantitative RT-PCR (qRT-PCR) method.
HESC cultured in the presence of each siRNA was obtained and total RNA was extracted from hESC using NucleoSpin RNA II kit (MACHEREY-NAGEL GmbH, Duren, Germany). 1 ㎍ of total RNA was converted into cDNA using ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan).
For quantitative real-time PCR, the primers of the polynucleotide sequences of Table 2 below were used.
(SEQ ID NO: 7)
(SEQ ID NO: 8)
(SEQ ID NO: 9)
(SEQ ID NO: 10)
(SEQ ID NO: 11)
(SEQ ID NO: 12)
Using a StepOnePlus ™ Real-Time PCR System (Applied Biosystems, Life Technologies) for quantitative real-time PCR, 40 cycles of 95 ° C. for 40 seconds, 55 ° C. to 63 ° C. for 30 seconds, The cycle was repeated and incubated at 72 占 폚 for 10 minutes. Normalization was performed using GAPDH to compare the amount of mRNA between samples. The ratio of the amount of reduced HDAC1 mRNA and HDAC4 mRNA when treated with HDAC1 siRNA or HDAC4 siRNA was calculated by comparing the amount of HDAC1 mRNA and HDAC4 mRNA of the cells obtained from the negative control, and the result is shown in Fig. 3B. As shown in Figure 3B, each siRNA was found to reduce the amount of target mRNA by about 80%.
On the other hand, in order to confirm whether the hESC cultured in the presence of siRNA was differentiated into neural progenitor cells, Sox1-expressing cells were subjected to flow cytometry as described in Example 1.2, and the results are shown in Fig. As shown in Fig. 4, about 88.2% of the cells expressed Sox1 when treated with siRNA inhibiting the expression of HDAC1. When siRNAs that inhibited the expression of HDAC4 were treated, the proportion of cells expressing Sox1 was similar to that of the negative control. Therefore, in the case of inhibiting the expression of HDAC1, it was confirmed that hESC was induced to differentiate into neural precursor cells.
<110> College of Medicine Pochon CHA University Industry-Academic Cooperation <120> Composition for differentiating stem cells to neural precursor cells, and method using the same <130> PN106852 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA sense strand <400> 1 agcguguagc uagcagaggt t 21 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA anstisnese strand <400> 2 ccucugcuag cuacacgcut t 21 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA sense strand specific to HDAC1 <400> 3 gcuucaaucu aacuaucaat t 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA antisense strand specific to HDAC1 <400> 4 uugauaguua gauugaagct t 21 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA sense strand specific to HDAC4 <400> 5 agcguguagc uagcagaggt t 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA antisense strand specific to HDAC4 <400> 6 uugauaguua gauugaagct t 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for HDAC1 <400> 7 tgatattcac catggtgacg gc 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for HDAC1 <400> 8 gcctcatagg actcgtcatc aa 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for HDAC4 <400> 9 gcagcaactg cagatgaaca ag 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for HDAC4 <400> 10 ctcttcctca tcgctctcaa tg 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 11 gtggacctga cctgccgtct 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 12 ggaggagtgg gtgtcgctgt 20
Claims (15)
Wherein said HDAC inhibitor is selected from the group consisting of CI994 and HDAC1 siRNA.
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