KR101615298B1 - Composition for Inhibiting Teratoma Formation and Growth Comprising Neural Stem Cell Culture Solution - Google Patents

Abstract

The present invention relates to a composition for inhibiting teratoma formation and growth, which comprises a neural stem cell culture liquid as an active ingredient, and more particularly, to a composition for inhibiting the growth of a neural stem cell or embryonic stem of a neural stem cell culture fluid extracted from a ventricular zone of a human brain And to the use of teratoma formation and growth inhibition which are problematic after transplantation of cell-derived neural progenitor cells. The composition for teratoma formation and growth inhibition containing the neural stem cell culture liquid according to the present invention as an active ingredient has no effect on the differentiation ability of neural progenitor cells derived from embryonic stem cells or embryonic stem cells, And can be used as a composition for suppressing growth and growth.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for forming a teratoma and a composition for inhibiting growth, which comprises a neural stem cell culture liquid as an active ingredient.

The present invention relates to a composition for inhibiting teratoma formation and growth, which comprises a neural stem cell culture liquid as an active ingredient, and more particularly, to a composition for inhibiting the growth of a neural stem cell or embryonic stem of a neural stem cell culture fluid extracted from a ventricular zone of a human brain And to the use of teratoma formation and growth inhibition which are problematic after transplantation of cell-derived neural progenitor cells.

Embryonic stem cells are infinitely proliferating by continuous self-renewal and have pluripotency to differentiate into all the cells present in the human body. Therefore, it can be used as a cell therapy agent for intractable diseases such as Parkinson's disease, diabetes and heart disease. However, when embryonic stem cells are not fully differentiated in the differentiation process, it is pointed out as a limit of clinical application that teratoma can be formed in the tissue when transplanted into a patient.

In an effort to suppress teratoma formation by embryonic stem cells, studies to date have been made by selectively removing the remaining cells that can not be differentiated by various methods. A method of separating and removing the undifferentiated pluripotent stem cells into MACS using two types of antibodies specific for pluripotent stem cell surface markers (SSEA-3 and TRA-1-60) (Korean Patent No. 10-1323650) , A method of selectively removing undifferentiated cells by treating small molecules such as quercetin and YM155 that can activate apoptotic factors specifically expressed in undifferentiated cells of embryonic stem cells (Lee et al., PNAS 110: E3281-3290, (Schuldiner et al., Stem Cells 21: 257-265, 1983) and a method of removing teratoma by treating a teratoma formed by embryonic stem cells into which suicide gene (HSV-tk) 2003).

In addition, recent evidence suggests that the conditioned medium (CM) obtained from various kinds of cells can inhibit the proliferation of tumor cells. (U.S. Pat. No. 2009-0186007), the culture medium of adipose stem cells causes death of gallbladder carcinoma and liver cancer cells (Liu et al ., PLoS One 8: e62844, Hou et al . , Tumor Biology , 35: 1239-50).

However, it is not known that the neural stem cell culture fluid inhibits cancer cells, especially teratoma formation and growth.

Accordingly, the present inventors have confirmed the effect of the culture solution of neural stem cells extracted from the ventricular region of the human brain to inhibit the formation and growth of embryonic stem cells or embryonic stem cell-derived neural progenitor cells, and completed the present invention .

It is an object of the present invention to provide a composition for teratoma formation and growth inhibition using a neural stem cell culture solution.

It is another object of the present invention to provide a method for producing a teratoma formation and growth inhibition composition using a culture solution of neural stem cells.

In order to accomplish the above object, the present invention provides a composition for teratoma formation and growth inhibition containing a neural stem cell culture liquid as an active ingredient.

The present invention also provides a method for producing a neural stem cell, comprising the steps of: culturing neural stem cells; And separating and removing the cultured neural stem cells to obtain a neural stem cell culture solution.

The composition for teratoma formation and growth inhibition containing the neural stem cell culture liquid according to the present invention as an active ingredient has no effect on the differentiation ability of neural progenitor cells derived from embryonic stem cells or embryonic stem cells, And can be used as a composition for suppressing growth and growth.

1 is a schematic diagram of treatment of a neural stem cell culture solution.
FIG. 2A shows the morphological change of embryonic stem cells by the treatment of neural stem cell culture medium, and FIG. 2B shows the result of increasing the division time.
FIG. 3 shows the results of the total cell number reduction of embryonic stem cells according to the treatment time of the neural stem cell culture medium.
FIGS. 4A and 4B show the reduction of embryonic stem cells expressing the cell proliferation marker Ki67 by treatment with a neural stem cell culture medium.
5A and 5B show the increase of embryonic stem cells expressing the activated form of Caspase 3, a cell death marker by treatment with neural stem cell culture medium.
FIGS. 6A and 6B are graphs showing that the expression of the cyclin D1 gene, which is a major cell cycle factor, was reduced in the embryonic stem cells after treatment with neural stem cells.
FIG. 7A and FIG. 7B are the results of analysis of changes in the expression of allelic marker and differentiation marker genes in embryonic stem cells after treatment with neural stem cell culture medium.
FIG. 8 shows the result of confirming the difference in cleavage time of mouse fibroblasts into which an allosteric marker gene was introduced using retrovirus.
Fig. 9 is a schematic diagram of the differentiation from embryonic stem cells into neural precursor cells.
FIG. 10A is a graph showing the change in shape of neural progenitor cells by treatment with neural stem cell culture medium, and FIG. 10B is a graph showing a decrease in total cell number of neural progenitor cells according to treatment time of neural stem cell culture medium.
FIG. 11A and FIG. 11B show the decrease in neural progenitor cells expressing the cell proliferation marker Ki67 by treatment with neural stem cell culture medium.
12A and 12B are graphs showing that the expression of the cyclin D1 gene, which is a major cell cycle factor, was decreased after treatment of neural progenitor cells with neural progenitor cells.
13 is a schematic diagram for analyzing a change in the differentiation potential of neural progenitor cells after treatment with a neural stem cell culture medium.
FIGS. 14A, 14B, and 14C are the results of analysis of genes and protein expression of neuro progenitor cell markers (Sox1, Nestin) and differentiated neuronal cell markers (Tuj1) after treatment of neural precursor cells with neural progenitor cells.
15A, 15B, and 15C show the results of analysis of gene and protein expression of neuro progenitor cell markers (Sox1, Nestin) and differentiated neuronal cell markers (Tuj1) by treating neural progenitor cells after 7 days of differentiation to be.
FIG. 16 is a schematic diagram of analysis of inhibition of teratoma formation by embryonic stem cells in the treatment of neural stem cell culture medium. FIG.
FIG. 17A is a result of comparing differences in teratoma sizes by embryonic stem cells in the treatment of neural stem cell cultures, and FIG. 17B is a result of examining embryonic stem cell-derived teratoma 3 germ layers.
18 is a schematic diagram of analysis of inhibition of teratoma growth by embryonic stem cells in the treatment of neural stem cell culture medium.
FIG. 19A is a result of comparing differences in the sizes of teratomas and sizes of the neural stem cell cultures after the formation of teratomas by embryonic stem cells, and FIG. 19B is a result of examining embryonic stem cell- .

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a composition for teratoma formation and growth inhibition containing a neural stem cell culture liquid as an effective ingredient.

The neural stem cells of the present invention are not limited in their kind but are preferably adult stem cells derived from a fetus and more preferably neural stem cells extracted from the ventricular zone of the fetal brain do.

In the present invention, "Neural Stem Cell Culture Solution" is a substance containing constituents contained in a medium obtained by subculturing neural stem cells, which is a type of ectodermal stem cells.

The neural stem cell culture solution of the present invention includes a variety of growth factors and cytokines to inhibit the proliferation of embryonic stem cells and neural progenitor cells, and it is also known that cyclin D1 To inhibit the formation and growth of embryonic stem cell-derived teratoma.

The neural stem cell culture solution of the present invention has at least one of the following characteristics.

1) inhibition of Ki-67 expression;

2) inhibiting the expression of cyclin-D1 (cyclin-D1);

3) promoting caspase-3 activation; And

4) Inhibition of Oct4 and Sox2 expression.

The neural stem cell culture solution of the present invention is characterized by inhibiting the proliferation of embryonic stem cells or neural progenitor cells derived from embryonic stem cells, and not affecting the differentiation ability.

In the present invention, "composition for inhibiting teratoma formation and growth (inhibitor)" is an inhibitor comprising the above-described neural stem cell culture solution, and the formulation can be in any form.

In another aspect, the present invention provides a method for producing a neural stem cell comprising: (a) culturing a neural stem cell; And (b) separating and removing the cultured neural stem cells to obtain a neural stem cell culture solution. The preparation of the teratoma formation inhibitor may be prepared by any conventional method, and the process of isolating, culturing and isolating the neural stem cells is not limited to the method of the present invention, but may be carried out by a method commonly practiced in the art It is possible.

In the present invention, the step (a) is a step of culturing a human neurosphere culture medium containing N-2 additive, heparin, fibroblast growth factor (bFGF), epidermal growth factor (EGF), and leukemia inhibitory factor media, and then the immortalized cells are cultured in non-inoculated medium containing DMEM (Dulbecco's Moeifiee Eagle's Medium), 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin, It is not.

The medium for culturing neural stem cells of the present invention can be used without limitation in a basic medium known in the art. The basic medium may be prepared by artificial synthesis, or a commercially prepared medium may be used. Examples of commercially produced media include Dulbecco's Modified Eagle's Medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and Isocove's Modified Dulbecco's Medium, but the present invention is not limited thereto and may be a DMEM medium. In addition, the basic medium preferably contains 0.01 to 15% (v / v) FBS. In a specific example of the present invention, the medium is cultured in DMEM medium, and 10% FBS is used to inhibit teratoma formation Respectively.

The ectodermal-derived neural stem cell culture medium of the present invention can be useful as a raw material for the development of drugs capable of preventing and treating teratoma formation and growth during stem cell transplantation therapy. Although the above description has been made with reference to the teratoma formation inhibitor during the stem cell transplantation therapy, the present invention can be utilized as a pharmaceutical and quasi-drug composition having a teratoma formation inhibitory effect comprising an ectodermal-derived neural stem cell culture liquid as an effective ingredient It will be apparent to those skilled in the art to which the present invention pertains.

[Example]

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.

Example 1: Production of neural stem cell culture fluid

Cells were obtained by immortalizing neural stem cells (NSCs) isolated from the ventricular zone of the fetal brain. 14-week fetal nerve cell tissue was treated with 0.1% collagenase and 0.1% hyaluronidase solution at 37 ° C for 1 hour, and then treated with 0.05% Trypsin-EDTA for 2-3 minutes to separate into single cells. The markers (CD45- / CD133 + CD34-). ≪ / RTI > The cells were cultured for 10-14 days in human neurosphere culture media supplemented with N-2 supplements, 0.2 mg / ml heparin, 20 ng / ml basic fibroblast growth factors (bFGF), 20 ng / ml EGF (Epidermal Growth Factor) After that, the formed neurospheres were treated with collagenase and separated into single cells. Cells obtained by transduction and selection of the v-myc gene using a retroviral vector were cultured in DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum), and 1 % penicillin / streptomycin (Flax et al., Nature Biotechnology vol.16, 1998; Lim et al., Neuroscience Letters 435: 175-180, 2008). The cell 5X10 ⁵ was dispensed into a 150 mm culture dish, 15 ml of a culture medium was added, and a culture solution was obtained at 80% confluence in a 5% CO ₂ incubator at 37 ° C. At this time, non-cultured medium containing DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum) and 1% penicillin streptomycin was used, and non-adherent cells were removed after culturing. In the course of subculturing neural stem cells (NSCs) cultured in the above procedure, a culture solution was obtained, and the supernatant was filtered by centrifugation. The neural stem cell extract was washed twice with PBS, and then subjected to Trypsin-EDTA The obtained cells were homogenized with a homogenizer at 6,000 RPM for 5 minutes by adding 100 ml of tertiary distilled water and centrifuged (1500 rpm, 5 minutes) to obtain a supernatant.

Example 2: Confirmation of embryonic stem cell proliferation inhibition of neural stem cell cultures

Adult embryonic stem cells of J1 and B6 were inoculated into a 6-well plate at a rate of 1 × 10 ⁵ cells per well. The next day, the non-adherent cells were removed and the neural stem cell culture medium was cultured for 1 day, 2 days Day, and 3 days, respectively. Then, the cells were rinsed with phosphate buffered saline (PBS), and the cell doubling time was measured with trypsin.

As a result, when the neural stem cell culture medium was not treated, the normal division time was about 20 hours, whereas when the neural stem cell culture medium was treated, the disruption time was increased (FIG. 2B). In addition, the proliferation inhibition effect of immunofluorescence (IF) decreases the expression of Ki67, which is a cell proliferation marker, and the expression of Caspase 3 activated form, which is a cell death marker, is increased (Fig. 4 and Fig. 5).

When the embryonic stem cells were treated with the neural stem cell cultures at 8 hour intervals for 2 days, the total number of cells was significantly reduced between 16 and 24 hours (FIG. 3), and expression of the cell cycle related genes The amount of cyclin D1 was detected by RT-PCR, and the amount of cyclin D1 expression, which is a major factor of the cell cycle, was found to differ (FIG. 6). Thus, it was confirmed that the neural stem cell culture solution according to the present invention inhibited the proliferation of embryonic stem cells through inhibition of cyclin D1 expression.

Example 3: Effect of neural stem cell culture on embryonic stem cell potential and differentiation

The morphology of the cells was observed when the neural stem cells were treated with embryonic stem cells (Fig. 2A), and the effect of embryonic stem cells on the competence and differentiation of embryonic stem cells was confirmed by RT-PCR.

As a result, it was confirmed that the omnipotent markers Oct4 and Sox2 decreased but the differentiation markers did not change significantly (Fig. 7).

Although Oct4 is well known as a competent marker, it is also known as a regulator of the cell cycle of embryonic stem cells (Lee et al., Biochemical Journal 426: 171-181, 2010). To confirm this, retrovirus GFP, Oct4, and Sox2 were injected and the changes of cell proliferation level were confirmed. To construct a retrovirus capable of transferring the Oct4 gene, pMXs vector plasmid from Addgene was used. 293GPG cells for retrovirus production were cultured in a 10 cm cell culture dish to grow at a density of about 80%, and Lipofectamin 2000 was used to introduce the corresponding plasmid into 293GPG cells. After 48 hours, 5 ml of retrovirus secreted as a supernatant of the cell culture was collected for 2 weeks and stored at -80 ° C. The retroviruses were mixed with a medium containing 2-4 μg / ml of polybrene and cultured with the cells for 4 to 5 hours to infect the cells and deliver the genes into the cells.

As a result, it was confirmed that the proliferation of Oct4-injected cells occurred more rapidly than the control group in which GFP was injected (FIG. 8). Therefore, it can be seen that the effect of the neural stem cell culture medium of the present invention, which is confirmed in Example 2, on the proliferation of embryonic stem cells is due to the decrease of expression of cyclin D1 and Oct4.

Example 4: Confirmation of inhibition of proliferation of neural progenitor cells in neural stem cells

In order to examine the effect of neural stem cell culture media on neural progenitor cells, embryonic stem cells were differentiated into neural progenitor cells. Embryonic stem cells were treated with trypsin to separate into single cells and induced embryonic body (EB) formation. In this process, a method of culturing in a floating state in a culture condition without LIF, which inhibits the differentiation of embryonic stem cells, was used. Specifically, the embryonic stem cells in culture were washed with PBS, treated with 0.05% Trypsin-EDTA for 2-3 min, and separated into single cells. Then, 2 mM L-glutamine, 10 mM beta-mercapto The cells were incubated for 4 days in a floating state with a cell culture-free culture dish with ethanol, 0.1 mM MEM NEAA, 10% FBS, and Pen / strep, an antibiotic, to obtain suspended EBs. The obtained EBs were transferred to culture dishes for cell culture and then cultured for 10 days in a medium supplemented with Insulin-Transferrin-Selenium-A (ITSA) in DMEM-F12 of GIBCO to induce neural progenitor cells. Then, the neural progenitor cells were transferred to a new cell culture culture dish, and neuronal progenitor cells were secured by adding N2 and 10 ng / ml bFGF to DMEM-F12 (FIG. 9).

The obtained neural progenitor cells were inoculated into a 6-well plate at 4 × 10 ⁵ cells per well. The next day, the non-adherent cells were removed and the neural stem cell culture medium was treated.

As a result, it was confirmed that the total number of cells treated with the neural stem cell culture solution was small (Fig. 10). In addition, the expression level of Ki67 and cyclin D1 decreased in the same manner as in Example 2, and the effect was highest when the neural stem cell culture medium was treated for 2 days (FIGS. 11 and 12).

Example 5: Effect of neural stem cell culture on neuronal progenitor cell potential and differentiation

In order to confirm that the neural stem cell culture medium does not decrease the differentiation ability of neural progenitor cells, the neural progenitor cells obtained in Example 4 were treated with a neural stem cell culture medium, and neural progenitor cells were differentiated. In this procedure, culturing was carried out for 7 days in the absence of bFGF, which inhibits differentiation of neural progenitor cells (Fig. 13). Immunofluorescence (IF) and RT-PCR were performed to analyze the expression levels of Sox1, Nestin, and Tuj1, a marker of differentiated neuronal cells, in neuronal progenitor cells.

As a result, it was confirmed that there was no significant difference in the amount of expression of Sox1, Nestin, and Tuj1 between the neural progenitor cells (FIG. 14) and the differentiated neurons (FIG. 15) Therefore, it can be understood that the neural stem cell culture does not affect the differentiation ability of neural progenitor cells.

Example 6: Confirmation of teratoma formation and growth inhibitory effect of embryonic stem cells in neural stem cell culture

Five-week-old BALB / C-nu female mice were subjected to adaptation at 24 ± 2 ° C for 7 days before subcutaneous injection of embryonic stem cells to form teratomas.

To investigate the effect of neural stem cell culture on teratoma formation and growth, embryonic stem cells were injected into the group treated with a neural stem cell culture medium (Fig. 16) from the day following injection, and neuroblastoma cell cultures were treated Group (Fig. 18). As a control, complete culture media (DMEM + 10% FBS) was used, and the neural stem cell culture solution of the present invention was treated once a day. After 21 days of embryonic stem cell injection, the mice were sacrificed, the teratomas were separated, fixed on 4% formaldehyde, the tissue paraffin was cut out, and the tissue cut to 3 thickness was attached to the slide, and paraffin removal and rehydration (deparaffinize and rehydrate). After that, Haematoxylin & Eosin (H & E) staining revealed that it was a teratoma formed in embryonic stem cells having all three germ layers (FIGS. 17B and 19B).

As a result of examining the size of the teratoma, when 6 neurons were added to the neural stem cell culture medium after the embryonic stem cell injection, and 4 neurons were cultured in the neuronal stem cell culture medium, It was confirmed that the teratoma size was smaller (Figs. 17A and 19A). In particular, it was confirmed that no malformed species was formed when the neural stem cell culture medium was treated with two electron groups. Thus, it can be seen that the neural stem cell culture liquid inhibits the teratoma formation and growth by embryonic stem cells.

Claims (9)

A pharmaceutical composition for inhibiting teratoma formation and growth by transplantation of embryonic stem cells or embryonic stem cell-derived neural progenitor cells containing neural stem cell culture medium as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the neural stem cells are extracted from the ventricular zone of the brain. 2. The pharmaceutical composition according to claim 1, wherein the neural stem cells are extracted from the ventricular zone of the brain.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the neural stem cell culture fluid has at least one of the following characteristics: (a) a neoplastic progenitor cell derived from embryonic stem cells or embryonic stem cells;
1) inhibition of Ki-67 expression;
2) inhibiting the expression of cyclin-D1 (cyclin-D1);
3) promoting caspase-3 activation; And
4) Inhibition of Oct4 and Sox2 expression.
[Claim 2] The method according to claim 1, wherein the neural stem cell culture solution inhibits the proliferation of embryonic stem cells or neural progenitor cells derived from embryonic stem cells. A pharmaceutical composition for inhibiting growth.
The method according to claim 1, wherein the culture medium of the neural stem cells does not affect the pluripotency of neural progenitor cells derived from embryonic stem cells or embryonic stem cells. And inhibiting growth.
delete (a) Culturing the neural stem cells; And
(b) separating and removing the cultured neural stem cells to obtain a neural stem cell culture solution.
The method according to claim 7, wherein the neural stem cells of step (a) are extracted from the ventricular zone of the brain.
The method of claim 7, wherein the step (a) comprises culturing a human neural tube culture medium containing N-2 additive, heparin, fibroblast growth factor (bFGF), epidermal growth factor (EGF), and leukemia inhibitory factor neurosphere culture media) and then the immortalized cells are cultured in a non-inductive medium containing DMEM (Dulbecco's Moeifiee Eagle's Medium), 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin A method for producing a composition for teratoma formation and growth inhibition.

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