WO2015147574A1 - Improvement in survival and proliferation of stem cells by doxycycline - Google Patents
Improvement in survival and proliferation of stem cells by doxycycline Download PDFInfo
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- WO2015147574A1 WO2015147574A1 PCT/KR2015/002992 KR2015002992W WO2015147574A1 WO 2015147574 A1 WO2015147574 A1 WO 2015147574A1 KR 2015002992 W KR2015002992 W KR 2015002992W WO 2015147574 A1 WO2015147574 A1 WO 2015147574A1
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- stem cells
- doxycycline
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Definitions
- the present invention was made by the task number 201201700010001 under the support of the Ministry of Education, Science and Technology of Korea, the research management professional organization of the task is the Korea Research Foundation,
- the present invention was made by the task number 201300000000890 under the support of the Ministry of Science, ICT and Future Planning, the research management organization of the project is the Korea Research Foundation, the research project name is "basic research project / general researcher support project / female scientist” , The research project titled “Cross-differentiation of epithelial cells into dopaminergic neurons through neural stem cells using Sox2 factor", The host institution is Hanyang University Industry-University Cooperation Group, Research period is 2013. 05. 01 ⁇ 2014. 4. 30 . .
- the present invention is the task number under the support of Ministry of Science, ICT and Future Planning
- the host institution is Hanyang University Industry-University Cooperation Group, research period is 2013. 06. 01 ⁇ 2014. 5. 31 .
- This patent application claims priority to Korean Patent Application No. 10-2014-0035610 filed with the Korean Patent Office on March 26, 2014, the disclosure of which is hereby incorporated by reference.
- the present invention relates to a stem cell culture composition and stem cell culture method for increasing the survival and expansion of stem cells.
- hESC human embryonic stem cel l
- iPSCs pluripotent stem eel Is
- hESCs / iPSCs are difficult to cultivate and show slow growth and poor survival, especially in cell separation and dissociation. HESCs are therefore inherently cultured in clusters on the assisting feeder layer 2. If hESC / iPSCs are incubated in Matrigel with chemically defined microtubules (4), feeder-free culture is possible.
- the present inventors have made diligent research efforts to develop a stem cell culture composition for increasing the survival and proliferation of stem cells when the stem cells are cultured. As a result, by using doxycycline to identify the increase in the survival (survival) and proliferation (prol i ferat ion) of stem cells, the present invention was completed.
- an object of the present invention is to provide a composition for stem cell culture.
- Another object of the present invention to provide a stem cell culture method for increasing the survival and proliferation of stem cells.
- Another object of the present invention is to provide a use of the composition for culturing stem cells comprising doxycycline as an active ingredient for increasing stem cell survival and proliferation.
- the present invention provides a composition for culturing stem cells comprising doxycycline as an active ingredient.
- the present inventors made an intensive research effort to develop a stem cell culture composition for increasing the survival and proliferation of stem cells when the stem cells are cultured. As a result, it was found that when doxycycline is used, survival and proliferation of stem cells are increased.
- Doxycycline an active ingredient of the stem cell culture composition of the present invention, is a compound represented by the following Chemical Formula 1 as an antibiotic of the tetracycline series.
- Doxycycline is known to be used for the treatment of lyme disease, chronic prostatitis, sinusitis, pelvic disease, acne, rosacea, rickettsial infections.
- the present inventors have completed the present invention by deriving the conclusion that the use of doxycycline, which is known as above, as a new use for culturing stem cells, is effective in culturing stem cells.
- the stem cell culture composition of the present invention increases the survival (survival) and proliferation (proliferation) of stem cells.
- the composition for stem cell culture should allow the stem cells themselves to survive for a long time without being killed within a short time, and to allow the proliferation of stem cells to be actively performed.
- the present inventors confirmed that stem cell death is suppressed and stem cell proliferation is increased even in a high concentration of insulin environment, which is disadvantageous for stem cell survival and proliferation. One).
- the stem cell of the present invention is totipotent stem cell (pluripotent stem cell), pluripotency.
- stemm cells pluripotent stem cells
- redundant stem cells multipotent stem cells
- unipotent stem cells unipotent stem cells
- the pluripotent stem cells of the above-described embodiment are embryonic stem cells, embryonic germ eel Is or embryonic carcinoma eel Is. More specifically, the omnipotent stem cells are embryonic stem cells.
- the pluripotent stem cell of the above-described embodiment is an induced pluripotent stem cell.
- Reverse differentiated pluripotent stem cells are also described herein by abbreviations such as iPSCs.
- the overlapping stem cells of the above-described embodiment are neural stem cells, hematopoietic] ⁇ ⁇ ] S (hemat opo i et ic stem cells) or mesenchymal stem cells )to be.
- the redundant stem cells are neural stem cells.
- the neural stem cells are neural stem cells derived from viral dedifferentiated stem cells.
- the viral dedifferentiated stem cells are specifically retroviral hiPS-NSCs (Retro human induced Pluri potent stem cell -neural stem cells) or lentiviral hiPS-NSCs (Lent i human induced Plur ipotent stem cell ⁇ neural stem eel Is May be). As shown in FIG.
- the neural stem cells derived from the viral dedifferentiated stem cells can be seen that the cells die while the passage number is increased.
- treatment of doxycycline can solve this problem dramatically (see FIG. 6).
- This makes it possible to stably culture dedifferentiated stem cells and new pluripotent stem cells, redundant stem cells or unipotent stem cells derived using the same.
- the inventors have identified through several examples that doxycycline has an effect on the culture of embryonic stem cells, pluripotent stem cells, pluripotent stem cells, pluripotent stem cells, and neural stem cells, which are redundant stem cells.
- Stem cells of the present invention are specifically derived from, for example, human, mouse, cow, horse, goat, sheep, dog, cat, rabbit or bird.
- the composition of the present invention comprises doxycycline in the content of 1 n -5000 yM.
- the results of the MTT assay are shown in FIG. 4 of the present specification, and it was observed that the viability of the cultured cells was improved even by the addition of 1 nM of doxycycline.
- FIG. 32 shows the relative viability of the doxycycline untreated control group
- the composition of the present invention comprises doxycycline in an amount of 0.01 ⁇ -5000 ⁇ , even more specifically 0.1 y M-5000 ⁇ , and even more specifically 1 ⁇ -5000 ⁇ . And even more specifically, 10 y M-5000 ⁇ .
- the present invention provides a stem cell culture method for increasing the survival and proliferation of stem cells comprising the following steps:
- step (b) culturing stem cells by treating doxycycline in the medium prepared in step (a).
- the medium for stem cell culture that can be used is not particularly limited, and any medium conventionally used for culturing animal cells can be used. Specifically, for example, Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), a-MEM (Stanner, CP. Et al., Nat. New Biol. 230: 52 (1971) ), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc.Exp.
- Treatment of the doxycycline of the present invention is possible to process once or twice or more during the culturing of stem cells, preferably two or more times, more preferably two or more times at regular time intervals, Specifically, for example, it may be added two or more times at intervals of 24 hours.
- Human embryonic stem cell medium which generally uses doxycycline only once, specifically, for example, cultured by mixing with commercially available Stemcell Technology's mTESR medium and Millipore's Pluristem medium, the medium without doxycycline was considered as a control. At this time, it is possible to culture human embryonic stem cells without changing medium for 6 days more efficiently than the control (see Fig. 19). In particular, when doxycycline is added, the survival rate is higher than that of Millipore's Pluristem medium, which is a product that can be used for one week without changing the medium.
- step (b) of the present invention treats Y-27632 with doxycycline.
- Y- 27632 of the present embodiment corresponds to the compound represented by the following Chemical Formula 2, and specifically has a characteristic of selectively inhibiting pl60ROCK as a ROCK (rh associated protein kinase) inhibitor.
- Y-27632 is known as an additive to help the culture of conventional stem cells.
- Y-27632 there is a problem that deformation occurs in the morphology of cultured cells (see FIG. 16).
- the use of doxycycline does not cause this problem, and the effect of increasing the survival or proliferation of stem cells is more than that of Y-27632.
- Excellent results were obtained (see FIGS. 13, 15 and 17).
- Y-27632 has a problem with the above-described cell morphology when used for a long time, it can be seen that when Y-27632 is used in combination with doxycycline, it is most effective for culturing stem cells (see FIG. 30).
- Y-27632 is treated only the first time of each passage. Specifically, for example, when doxycycline is treated with stem cell culture medium at intervals of 24 hours, Y-27632 is treated together only during the first single treatment of doxycycline. According to this embodiment, there is no fear of occurrence of cell-type modification problems that may occur when Y-27632 is used for a long time, and even more excellent survival and proliferation effect of stem cells is compared with doxycycline or Y-27632 alone. Can be obtained (see Fig. 30). According to another aspect of the present invention, the present invention provides the use of doxycycline for stem cell culture.
- the present invention is to use a stem cell culture composition comprising a doxycycline, which is another aspect of the present invention as an active ingredient, a stem cell culture method for increasing the survival and proliferation of stem cells is another aspect of the present invention.
- a stem cell culture method for increasing the survival and proliferation of stem cells is another aspect of the present invention.
- the duplicated content will be omitted in order to avoid excessive complexity of the description.
- the present invention provides a use for culturing stem cells of a medium composition comprising doxycycline as an active ingredient.
- the present invention is to use a stem cell culture composition comprising a doxycycline as another active aspect of the present invention as an active ingredient, stem cell culture method for increasing the survival and proliferation of stem cells another aspect of the present invention
- stem cell culture method for increasing the survival and proliferation of stem cells another aspect of the present invention
- the duplicated content will be omitted in order to avoid excessive complexity of the description.
- the present invention provides a composition for stem cell culture.
- the present invention provides a stem cell culture method for increasing the survival and proliferation of stem cells.
- the present invention provides a use for stem cell culture of a medium composition comprising doxycycline or doxycycline as an active ingredient.
- stem cell culture composition of the present invention When using the stem cell culture composition of the present invention, it is possible to increase both the survival (survival) and proliferation (prol i ferat ion) of various stem cells.
- composition for stem cell culture of the present invention When using the composition for stem cell culture of the present invention, it is possible to prevent apoptotic cell death and premature aging due to long-term passage of various stem cells derived from viral dedifferentiated stem cells.
- composition for stem cell culture of the present invention When the composition for stem cell culture of the present invention is used, there is an effect of increasing the medium exchange time. In particular, it shows higher efficiency than Pluristem, a commercial medium that increases the medium exchange time of Millipore, and when used in addition to a general commercial medium, the efficiency of the medium is greatly increased.
- FIG. 1 shows that apoptosis-induced apoptosis induced by high levels of insulin in hNSC culture can be inhibited by doxycycline addition.
- 4 and 5 show a comparison of the cells, survival effects of doxycycline, analogs thereof (minocycline, tetracycline) and other antibiotics.
- 6 and 7 show that doxycycline supplementation prevents early aging and apoptotic cell death in viral hiPS-derived NSCs during cell passage.
- FIG. 7 shows representative ⁇ -galactosidase (gastric, cell aging) and activation casing at the last day of passage 4 in retro-2-hiPS-NSC cultures treated with doxycycline (right) and untreated (left) Phase 3 (bottom apoptosis)-shows stained cells.
- FIG. 10 shows expression arrays for anti- / pro-apoptotic genes modified by doxycycline treatment.
- FIG. 11 shows cell growth curves in long-term (l ong-term) culture for hESC cell expansion.
- Figure 13 compares the effects of doxycycline and Y-27632 on hESC survival and maintenance, showing cell growth for 5 days.
- Figure 14 shows the number of colonies formed as a comparison of the effects of doxycycline and Y-27632 on hESC survival and maintenance.
- Figure 15 shows the size of colonies formed as a comparison of the effects of doxycycline and Y-27632 on hESC survival and maintenance.
- FIG. 16 shows phase contrast images of representative colonies from untreated, Y-27632-treated, and doxycycline-treated cultures.
- Figure 18 shows the expression level of genes encoding human stem cell transcription factors in H9 cells treated with doxycycline compared to the expression level in untreated culture.
- Figure 23 shows the estimation of the intracellular signal activated by doxycycline.
- FIG. 25-27 show that the doxycycline-induced effect on the number (FIG. 25), size (FIG. 26), and percentage ratio (FIG. 27) of AP + colonies was blocked by blockers specific for PI3K-Akt signal.
- 28 shows a general radiographic based PI3K activity assay. 29 shows that the effect of doxycycline on PI3K activity chukjeong using the PI3 kinase act ivi ty / inhi bi ter assay ki t R.
- Figure 30 shows the result of increased survival and proliferation of stem cells when Y-27632 was treated on the first day of each passage with doxycycline.
- Figure 31 shows that even when cultured up to 20 passages with doxycycline does not change the properties of the basic human embryonic stem cells.
- Fig. 32 shows the relative viability of cultured stem cells in the doxycycline treated example to the doxycycline untreated control.
- bFGF basic fibroblast growth factor
- hESC / iPSC colonies were isolated into 1-5 cells using TrypLE (Invitrogen) or collagenase IV (0.1 mg / mL; STEMCELL technology) and without feeder layers, TESR medium ( Cultured on Matrigel TM using STEMCELL technology). Medium replacement and bFGF complement were performed daily without special mention.
- hNSC colonies were derived by in vitro differentiation of previously known hESCs and hiPSCs (16, 18).
- hNSCs were expanded bFGF (20 ng / mL) in ITSA medium (13, 18) and weekly passaged for long term expansion.
- hNSCs Final differentiation of hNSCs is free of bFGF, but brain-derived neurotrophic factor (20 ng / mL) (R & D Systems), and glial cell line derived neurotrophic factor (20 ng / mL) (R & D Systems), or dibutyryl cAMP (0.5 ⁇ L ol / L) (Sigma).
- doxycycline (Sigma), minocycline (Sigma), tetracycline (Sigma), kanamycin (Sigma), blasticidine (Invitrogen), chloramphenicol (Sigma), Y-27632 (Calniochem), LY294002 (LY294002) Calbiochem), Wortmannin (Mi 1 lipore), PD98059 (Calbiochem). Effect analysis of doxycycline alone
- Cultured cells were fixed in 4% paraformaldehyde in PBS and incubated at 4 ° C. with the primary antibodies described in Table 3. Appropriate fluorescently-labeled secondary antibodies (Jackson Immunoresearch Laboratories) were used for visualization. Stained samples were mounted in VECTASHIELD using DAPI mount solution (Vector Laboratories).
- FACS (Canto TM II) (Chemomet ec) and Nuc 1 eoCounter ® (NC-3000 TM) (Chemomet ec) were further counted for FACS analysis to determine the cell distribution accumulated on each cell cycle step.
- Aging-Galactosidase staining kit (Senescence ⁇ -Galactosidase Staining Kit (Cell Signaling) was used to determine cell aging according to the manufacturer's instructions Alkaline phosphatase activity assay
- Alkali phosphatase substrate kit 1 (vector) was used to analyze alkaline phosphatase activity in colonies according to the manufacturer's instructions. Measurement of starch capacity
- PI3 Kinase Activity Assay (31) General radiographic-based PI3K activity assays were performed by known methods. In short, PI3K was immunoprecipitated with anti-PI3K antibody (anti P 85) from total cell extracts from H9 hESCs cultured with or without doxycycline. The enzymatic reaction was initiated by adding [ ⁇ -32 ⁇ ] ⁇ and L-al7 phosphatidylinosi (substrate) to the precipitate, and the radiolabeled material by the reaction was detected using radiographs.
- PI3K activator / inhibitor activity was measured according to the slightly modified manufacturer's instructions. Effect analysis of combination with doxycycline and Y-27632
- hNSCs Human NSCs
- hESC / iPSC-derived NSCs hES / iPS-NSCs
- nestin + hNSCs are bFGF (basic f ibrobl ast growth factor) and differentiate into neuronal cells in the absence of bFGF and in the presence of binding neurotrophic factors (13, 14).
- doxycycline complement shows the effect of doxycycline complement on neurons derived from culture of hNSCs and hESC / iPSCs.
- Doxycycline prevents apoptosis cell death caused by high levels of insulin (see FIG. 1) or low cell concentrations (see FIG. 2) in hNSC cultures.
- H9 hESC-derived NSCs plated with either (a) 1.4 ⁇ IO 5 / cm 2 or (b) 3.4 ⁇ IO 4 / cm 2 were incubated with or without supplementation of doxycycline complement (: g / mL).
- Apoptotic cell death was measured by viable cell counts and cells positive for activated (cut) caspase 3.
- representative phase differences and truncated Casphase 3 stained images are shown in the left panel. Scale bars represent 50 ⁇ .
- FIG. 3 shows the effect of doxycycline on neuronal survival.
- Subtypes of neurons were induced by differentiation of H9 hESC-NSCs for 9 days. Neuronal cultures were maintained for 5 days with or without supplementation of doxycycline. On the last day of culture, immunostaining was performed to distinguish dopamine activity (TH), serotoni n and GABA activity (GABA) neurons. Scale bar represents 50 ⁇ .
- NSCs derived from retroviral (Retro-2, middle degree), lentiviral hiPSC strain (l ineKLent i— 1, right side) and H9 hESCs (control, left side) were found every 7 hours in the presence or absence of doxycycline strata. Expanded by subculture every day. The cell growth curves shown in FIG. 6 are indicated by counting the total number of viable cells at the end of each passage. In doxycycline-layered cultures at p ⁇ 0.01, there was a significant increase in viable cell numbers.
- FIG. 7 shows representative ⁇ -galactosidase (gastric, cell aging) and activation casing at the last day of passage 4 in retro-2-hiPS-NSC cultures treated with doxycycline (right) and untreated (left) Phase 3 (bottom apoptosis)-shows stained cells. Scale bar represents 50 ⁇ .
- Doxycycline complement layer enhanced cell survival and maintenance of undifferentiated hESC / iPSCs in culture.
- FIG. 10 shows expression arrays for anti- / pro-apoptotic genes modified by doxycycline treatment.
- FIG. 11 shows cell growth curves in long-term culture for hESC cell expansion.
- H9 hESCs were incubated in 8 passages for 56 days on MEF feeder in bFGF-supplemented KSR medium with or without doxycycline (1 y g / ml).
- the curve is shown by counting the total viable cell number on the last day of each passage. Significantly different cell numbers were observed with treatment of doxycycline in the same cell passage at p ⁇ 0.001.
- Figures 13-18 show doxycycline and Y- in hESC survival and maintenance
- Dissociated H9 cells were plated at low cell concentrations (500 cells / well, 96 well plates) and in metrigel-mTeSR, with or without doxycycline (1 ug / ml) or Y-27632 10 uM). Incubated for days. Cell growth for 5 days is shown in FIG. 13. In the case of treatment with doxycycline, a clear increase in the number of cells was observed as well as in the untreated control as compared to Y-27632. The number of colonies formed (see FIG. 14) and the size (see FIG. 15) were compared. The number of clones is related to cell survival and the size of the colon is related to cell proliferation.
- 16 shows phase contrast images of representative colonies from untreated, Y-27632-treated, and doxycycline-treated cultures.
- High performance image of the designated cells, scale bar represents 50 ⁇ .
- ⁇ -27632 was treated, the morphology of the cells was observed to change, which means that the cells had an adverse effect.
- this type of change was not observed when treating doxycycline, which means that the treatment of doxycycline may solve the problems caused by Y-27632 treatment.
- Figure 18 shows the expression level of genes encoding human stem cell transcription factors in H9 cells treated with doxycycline compared to the expression level in untreated culture.
- the dichroic straight line represents a 2-fold change in the gene expression threshold. Expression of genes associated with pluripotency (red dot) was selected and the fold change in doxycycline treated culture relative to the control is shown in the graph to the right.
- Doxycycline enables cell survival and maintenance of undifferentiated hESCs through PI3K-Akt signal activation.
- 20-22 show that ectomycin overactivation of cell dissociation-induced was prevented by Y-27632 but not by doxycycline treatment.
- H9 hESCs were incubated for 1 hour prior to cell dissociation with or without doxycycline or Y-27632 (control). Cells were harvested after 0, 10 and 30 minutes of cell dissociation and Western blot was performed to detect proteins associated with cell-dissociation-induced apoptosis (see FIG. 20).
- FIG. 21 and 22 show representative pMLC-immunostained images and phase contrast images of dissociated hESCs in Y-27632 or doxycycline treated cultures.
- Cells pretreated with Y-27632 or doxycycline were dissociated and plated directly on metrigel with mTESR and incubated in the continued presence of the chemical. The images were collected at designated times after smearing (dissociation). The scale bar is 10 u m.
- ⁇ -27632 pretreatment ⁇ -cadherin does not decrease and pMLC decreases
- doxycycline pretreatment as in the untreated control E-cad Herrin decreases and pMLC increases. This shows that doxycycline is not a ROCK inhibitor like Y-27632.
- Figure 23 shows the estimation of the intracellular signal activated by doxycycline.
- H9 hESCs may be combined with doxycycline (1 wg / ml) prior to acquisition or The cells were incubated for 30 min without doxycycline (control) and used for immunoblot analysis using human phosphokinase blot array R designed to detect 46 phosphorylated intracellular proteins. The array analysis was performed three times. Representative pairs of blots of untreated control and doxycycline treated cells are shown. In the blot, the dots in the doxycycline-treated group whose intensity is 1.5 times or more compared to the control were indicated by dotted circles. Reference points are underlined.
- the graph on the right shows the numerically analyzed values of the intensity of the points shown from the three blots.
- the results of the pai red t-test at ⁇ .001 ** and P ⁇ 0.05 * were significantly different from the control.
- the signal activation after 0, 10 and 30 minutes of doxycycline treatment was further measured by Western blot analysis (see Figure 24).
- FIG. 20 an increase in pAKT signal during doxycycline treatment was observed in FIG. 23.
- the activity of the protein was screened using human phospho-kinase assay (Proteome Prof. TM, R & D Systems) (see FIG. 23).
- FIG. 23 human phospho-kinase assay
- Doxycycline (1 g / ml) or vehicle (with or without PI3K-Akt blocker LY29002 (20 ⁇ ) or wortmannin (wortmanninX LOO ⁇ ) or Raf-Erk blocker PD98059 (10 ⁇ )
- Colony formation assays were performed by culturing dissociated H9 cells for 5 days in the presence of DMS0).
- n 3 independent experiments, Tucky In one-way AN0VA using post-hawk analysis, it was significantly different from control cultures treated with vehicle at * p ⁇ 0.001 and cultures treated with doxycycline alone at #p ⁇ 0.005.
- 28 and 29 show PI3K enzyme activity enhanced by doxycycline.
- 28 shows a general radiographic based PI3K activity assay.
- PI3K activity was simultaneously determined in control cell lines (human breast cancer cell MDA-MB-231) with or without a known PI3K inhibitor (Accol) (31) (left diagram).
- 29 shows that the effect of doxycycline on PI3K activity as measured with a PI3 kinase act ivi ty / inhibi ter assay ki t R.
- the assay was also performed in the presence of other antibiotics or known PI3k inhibitors (control): doxycycline (Dox); Tetracycline (Tet); Minocycline (Mino); Kanamycin (Kan); Chlorampheni col (Chi or);
- Blastcidine (Bl as).
- the graph shows the calculated PI3K enzyme activity after excluding the activity without inhibitor / activator. Doxycycline dose-dependent PI3K activity curves are also indicated.
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Abstract
The present invention relates to a composition for culturing stem cells, containing doxycycline as an active ingredient, and a method for culturing stem cells to increase the survival and proliferation of stem cells. When the stem cells are cultured, the survival and proliferation of stem cells can be increased by using the composition and culturing method of the present invention.
Description
【명세서】 【Specification】
【발명의 명칭】 [Name of invention]
독시사이클린에 의한 줄기세포의 생존 및 증식 향상 【기술분야】 Survival and Proliferation of Stem Cells by Doxycycline [Technical Field]
본 발명은 대한민국 교육과학기술부의 지원 하에서 과제번호 201201700010001 에 의해 이루어진 것으로서， 상기 과제의 연구관리전문기관은 재단법인 한국연구재단, 연구사업명은 The present invention was made by the task number 201201700010001 under the support of the Ministry of Education, Science and Technology of Korea, the research management professional organization of the task is the Korea Research Foundation,
"기초연구사업 /선도연구센터육성지원사업 /기초의과학분야 (MRC) " ， "Basic Research Project / Leading Research Center Development Support Project / Basic Medical Science (MRC)",
연구과제명은 "줄기세포로부터 중뇌 도파민 신경세포로의 분화 및 이식연구" , 주관기관은 한양대학교 산학협력단, 연구기간은 2012. 09. 01 ~ 2013. 08. 31 이다. The research project titled "Differentiation and Transplantation Study of Stem Cells into Midbrain Dopamine Neurons", and the host institution is Hanyang University Industry-Academic Cooperation Group.
또한 본 발명은 대한민국 미래창조과학부의 지원 하에서 과제번호 201300000000890 에 의해 이루어진 것으로서， 상기 과제의 연구관리전문기관은 재단법인 한국연구재단， 연구사업명은 "이공분야 기초연구사업 /일반연구자지원사업 /여성과학자" , 연구과제명은 "Sox2 인자를 이용한 상피세포의 신경줄기세포를 통한 도파민성 신경세포로의 교차분화 연구" , 주관기관은 한양대학교 산학협력단, 연구기간은 2013. 05. 01 ~ 2014. 4. 30이다. . In addition, the present invention was made by the task number 201300000000890 under the support of the Ministry of Science, ICT and Future Planning, the research management organization of the project is the Korea Research Foundation, the research project name is "basic research project / general researcher support project / female scientist" , The research project titled "Cross-differentiation of epithelial cells into dopaminergic neurons through neural stem cells using Sox2 factor", The host institution is Hanyang University Industry-University Cooperation Group, Research period is 2013. 05. 01 ~ 2014. 4. 30 . .
또한 본 발명은 대한민국 미래창조과학부의 지원 하에서 과제번호 In addition, the present invention is the task number under the support of Ministry of Science, ICT and Future Planning
201300000260001 에 의해 이루어진 것으로서, 상기 과제의 연구관리전문기관은 재단법인 한국연구재단， 연구사업명은201300000260001, the research management specialized organization for the above task is the Korea Research Foundation,
"원천기술개발사업 /바이오의료기술개발사업 /줄기세포연구사업" , "Source Technology Development Project / Biomedical Technology Development Project / Stem Cell Research Project",
연구과제명은 "중뇌도파민신경세포 분화， 생존， 기능획득기전 이해를 통한 파킨슨 증후군 줄기세포 치료기술 개발" ， 주관기관은 한양대학교 산학협력단， 연구기간은 2013. 06. 01 ~ 2014. 5. 31이다. 본 특허출원은 2014 년 3 월 26 일에 대한민국 특허청에 제출된 대한민국 특허출원 제 10-2014-0035610 호에 대하여 우선권을 주장하몌 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다.
본 발명은 줄기세포의 생존 및 증삭을 증대시키는 줄기세포 배양용 조성물 및 줄기세포 배양 방법에 관한 것이다. The research project titled "Development of Parkinson's syndrome stem cell treatment technology through understanding of mesenchymal dopaminergic neural cell differentiation, survival, and function acquisition", The host institution is Hanyang University Industry-University Cooperation Group, research period is 2013. 06. 01 ~ 2014. 5. 31 . This patent application claims priority to Korean Patent Application No. 10-2014-0035610 filed with the Korean Patent Office on March 26, 2014, the disclosure of which is hereby incorporated by reference. The present invention relates to a stem cell culture composition and stem cell culture method for increasing the survival and expansion of stem cells.
【배경기술】 Background Art
줄기세포 특히 hESC( Human embryoni c stem cel l ) 및 iPSCs( induces plur i potent stem eel I s)는 발생 생물학， 질병 모델링 및 재생 의약에 대한 중요한 도약의 발판올 제공한다 ( 1， 2 , 3 , 4) . 그러나 기술적으로 hESC/ iPSCs 는 배양하기 어렵고， 특히 세포 분리 및 해리에 있어서， 느린 성장 및 불량한 생존율을 보인다. 그러므로， hESCs 는 보조하는 피더 층 (2) 상의 클러스터 내에서 고유하게 배양된다. hESC/ iPSCs 가 화학적으로 정의된ᅳ배지 (4)를 갖는 매트리겔 내에서 배양된다면, 피더 -프리 배양은 가능하다. 게다가 현탁액 (5) 내에서， 마이크로 어레이 (4 , 6)를 가지고 또는 합성 폴리머 (7) 상에서 , hESC/iPSCs 를 배양하는 것과 같은 방법이 소개되었다. 그러나 상기의 새로운 기술들은 비용이 많이 들고， 제한된 확장성을 가지며 , 높은 뱃치 (batch) 간의 변동성을 갖는다. ROCK 억제제인 Y-27632 가 세포 해리 후의 세포 아팝토시스를 방지하고ᅳ 플레이트에 도말 후의 세포 생존성을 향상 (8 , 9)시키기 위해 사용되지만, 상기 화학물질의 이점은 세포 해리 후의 짧은 주기로 제한되며, 세포 생존 및 증식에 대한 그 지속적인 효과가 의심된다 ( 10) . 그러므로 낮은 비용으로 가능하고， 강력하며， 확장가능하고, 사용이 쉬우며， 효과가 지속적인 배양 방법들이， 기초 연구 및 임상 웅용에서의 hESC/ iPSCs 의 광범위한 적용을 가능케하기 위하여 더욱 발전되어야 한다. 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다. Stem cells, in particular human embryonic stem cel l (hESC) and induces pluripotent stem eel Is (iPSCs), provide a springboard for important leaps in developmental biology, disease modeling and regenerative medicine (1, 2, 3, 4). ). Technically, however, hESCs / iPSCs are difficult to cultivate and show slow growth and poor survival, especially in cell separation and dissociation. HESCs are therefore inherently cultured in clusters on the assisting feeder layer 2. If hESC / iPSCs are incubated in Matrigel with chemically defined microtubules (4), feeder-free culture is possible. In addition, methods such as culturing hESC / iPSCs in suspension (5) with microarrays (4, 6) or on synthetic polymer (7) have been introduced. However, these new technologies are expensive, have limited scalability, and have high batch-to-batch variability. Although the ROCK inhibitor Y-27632 is used to prevent cell apoptosis after cell dissociation and to improve cell viability after smearing on plates (8, 9), the benefits of this chemical are limited to a short cycle after cell dissociation. (10) Sustained effects on cell survival and proliferation are suspected. Therefore, low cost, viable, powerful, scalable, easy to use and lasting effective culture methods should be further developed to enable widespread application of hESCs / iPSCs in basic research and clinical use. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
[발명의 내용】 [Contents of the Invention]
【해결하고자 하는 과제】
본 발명자들은 줄기세포를 배양시 줄기세포의 생존 (survival ) 및 증식 (prol i ferat i on)을 증대시키기 위한 줄기세포 배양용 조성물을 개발하고자 예의 연구 노력하였다. 그 결과 독시사이클린을 이용하는 경우 줄기세포의 생존 (survival ) 및 증식 (prol i ferat ion)이 증대되는 것을 규명함으로써, 본 발명을 완성하게 되었다. Problem to be solved The present inventors have made diligent research efforts to develop a stem cell culture composition for increasing the survival and proliferation of stem cells when the stem cells are cultured. As a result, by using doxycycline to identify the increase in the survival (survival) and proliferation (prol i ferat ion) of stem cells, the present invention was completed.
따라서， 본 발명의 목적은 줄기세포 배양용 조성물을 제공하는데 있다. Accordingly, an object of the present invention is to provide a composition for stem cell culture.
본 발명의 다른 목적은 줄기세포의 생존 및 증식을 증대시키는 줄기세포 배양 방법을 제공하는데 있다. Another object of the present invention to provide a stem cell culture method for increasing the survival and proliferation of stem cells.
본 발명의 다른 목적은 줄기세포 생존 및 증식을 증대시키기 위한 독시사이클린을 유효성분으로 포함하는 줄기세포 배양용 조성물의 용도를 제공하는데 있다. 본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다. Another object of the present invention is to provide a use of the composition for culturing stem cells comprising doxycycline as an active ingredient for increasing stem cell survival and proliferation. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
【과제 해결 수단】 [Task solution]
본 발명의 일 양태에 따르면, 본 발명은 독시사이클린을 유효성분으로 포함하는 줄기세포 배양용 조성물을 제공한다. 본 발명자들은 줄기세포를 배양시 줄기세포의 생존 (survival ) 및 증식 (prol i ferat ion)을 증대시키기 위한 줄기세포 배양용 조성물을 개발하고자 예의 연구 노력하였다. 그 결과 독시사이클린을 이용하는 경우 줄기세포의 생존 (survival ) 및 증식 (pro l i ferat ion)이 증대되는 것을 규명하였다. According to one aspect of the present invention, the present invention provides a composition for culturing stem cells comprising doxycycline as an active ingredient. The present inventors made an intensive research effort to develop a stem cell culture composition for increasing the survival and proliferation of stem cells when the stem cells are cultured. As a result, it was found that when doxycycline is used, survival and proliferation of stem cells are increased.
본 발명의 줄기세포 배양용 조성물의 유효성분인 독시사이클린은 테트라사이클린 계열의 항생제로서 하기 화학식 1로 나타낸 화합물이다. Doxycycline, an active ingredient of the stem cell culture composition of the present invention, is a compound represented by the following Chemical Formula 1 as an antibiotic of the tetracycline series.
[화학식 1]
[Formula 1]
독시사이클린은 라임병 (lyme disease), 만성 전립성염， 부비강염， 골반 연증성 질환, 여드름, 주사비 (rosacea), 리케치아 (rickettsial ) 감염의 치료에 사용되는 것으로 알려져 있다. 본 발명자들은 상기와 같은 용도가 공지되어 있는 독시사이클린을 줄기세포의 배양을 위한 새로운 용도로서 이용하는 경우， 줄기세포의 배양에 효과적이라는 결론을 도출하여 본 발명을 완성하였다. Doxycycline is known to be used for the treatment of lyme disease, chronic prostatitis, sinusitis, pelvic disease, acne, rosacea, rickettsial infections. The present inventors have completed the present invention by deriving the conclusion that the use of doxycycline, which is known as above, as a new use for culturing stem cells, is effective in culturing stem cells.
본 발명의 일 구현예에 의하면， 본 발명인 줄기세포 배양용 조성물은 줄기세포의 생존 (survival) 및 증식 (proliferation)을 증대시킨다. 줄기세포 배양을 위한 조성물은 줄기세포 자체가 단시간 내에 사멸하지 않고 장기간 생존할 수 있도록 해야 하며， 줄기세포의 증식이 활발하게 이루어지도록 해야 한다. 본 발명자들은 줄기세포 배양용 배지에 독시사이클린을 처리하는 경우, 줄기세포가 생존 및 증식하는데 불리한 고농도 인슐린 환경 하에서도 줄기세포의 사멸이 억제되고， 줄기세포의 증식이 증가되는 것을 확인하였다 (참조: 도 1). 또한 줄기세포가 저농도로 존재하는 '환경이 줄기세포 증식에 불리함에도 불구하고, 독시사이클린을 처리하는 경우에는 저농도의 줄기세포의 생존 및 증식도 증대되는 것을 확인하였다 (참조: 도 2). 이는 다른 계열의 항생제에서는 나타나지 않는 효과이며, 같은 테트라사이클린 계열의 항생제 중에서도 뛰어난 효과이다 (참조: 도 4). 또한， 이를 통하여 독시사이클린-매개의 세포 생존 증대 효과가 그것의 항생제 효능과 연관되어 있는 것은 아님을 본 발명의 일 구현예에 의하면， 본 발명의 줄기세포는 전능성 줄기세포 (totipotent stem cell), 다능성 줄기세포 (plur ipotent stem cell), 중복성 줄기세포 (Multipotent stem cell) 또는 단분화성 줄기세포 (Unipotent stem cell)이다.
본 발명의 일 구체예에 의하면， 전술한 일 구현예의 전능성 줄기세포는 배아줄기세포 (embryonic stem cell), 배아생식세포 (embryonic germ eel Is) 또는 배아종양세포 (embryonic carcinoma eel Is)이다. 더욱 구체적으로 상기 전능성 줄기세포는 배아줄기세포이다. According to one embodiment of the invention, the stem cell culture composition of the present invention increases the survival (survival) and proliferation (proliferation) of stem cells. The composition for stem cell culture should allow the stem cells themselves to survive for a long time without being killed within a short time, and to allow the proliferation of stem cells to be actively performed. When the doxycycline is treated in the stem cell culture medium, the present inventors confirmed that stem cell death is suppressed and stem cell proliferation is increased even in a high concentration of insulin environment, which is disadvantageous for stem cell survival and proliferation. One). In addition, even though the "environment in which stem cells are present in a low concentration disadvantages of stem cell proliferation and, when processing a doxycycline has also been confirmed that the increasing survival and proliferation of stem cells at a low concentration (Fig. 2). This is an effect that does not appear in other antibiotics, and is excellent among antibiotics of the same tetracycline series (see Fig. 4). In addition, according to one embodiment of the present invention, the stem cell of the present invention is totipotent stem cell (pluripotent stem cell), pluripotency. Stem cells (plur ipotent stem cells), redundant stem cells (Multipotent stem cells) or unipotent stem cells (Unipotent stem cells). According to one embodiment of the present invention, the pluripotent stem cells of the above-described embodiment are embryonic stem cells, embryonic germ eel Is or embryonic carcinoma eel Is. More specifically, the omnipotent stem cells are embryonic stem cells.
본 발명의 다른 구체예에 의하면 전술한 일 구현예의 다능성 줄기세포는 역분화 다능성 줄기세포 (induced Pluri otent stem cell)이다. 역분화 다능성 줄기세포는 본 명세서 상에서 iPSCs 와 같은 약칭으로도 기재된다. According to another embodiment of the present invention, the pluripotent stem cell of the above-described embodiment is an induced pluripotent stem cell. Reverse differentiated pluripotent stem cells are also described herein by abbreviations such as iPSCs.
본 발명의 다른 구체예에 의하면， 전술한 일 구현예의 중복성 줄기세포는 신경 줄기세포 (neural stem cell), 조혈 ] λ^] S (hemat opo i et i c stem cell) 또는 간엽 줄기세포 (mesenchymal stem cell)이다. 더욱 구체적으로 상기 중복성 줄기세포는 신경 줄기세포이다. 더욱 더 구체적으로 상기 신경 줄기세포는 바이러스성 역분화 줄기세포 유래의 신경 줄기세포이다. 상기 바이러스성 역분화 줄기세포는 구체적으로 레트로 바이러스성 hiPS-NSCs (Retro human induced Pluri potent stem cell -neural stem cells) 또는 렌티 바이러스성 hiPS-NSCs(Lent i human induced Plur ipotent stem cellᅳ neural stem eel Is)일 수 있다. 상기 바이러스성 역분화 줄기세포 유래의 신경 줄기세포는 도 6 에 나타낸 바와 같이 계대 배양을 진행하는 동안 계대 수가 증가하면서, 세포가 사멸함을 알 수 있다. 그러나 독시사이클린을 처리하는 경우 이러한 문제를 극적으로 해결할 수 있음을 확인하였다 (참조: 도 6). 이는 역분화 줄기세포 및 이를 이용하여 유도한 새로운 다능성 줄기세포, 중복성 줄기세포 또는 단분화성 줄기세포를 안정적으로 배양할 수 있도록 한다. 본 발명자들은 대표적인 전능성 줄기세포인 배아줄기세포ᅳ 다능성 줄기세포인 역분화 다능성 줄기세포, 중복성 줄기세포인 신경 줄기세포의 배양에 독시사이클린이 효과가 있음을 여러 실시예를 통하여 규명하였다. 각기 다른 발생의 진행 정도를 보이는 여러 줄기세포의 배양에 대하여 예외 없이 독시사이클린이 줄기세포의 생존 및 증식을 증대시키는 효과를 보임을 알 수 있으며， 이를 통해 다양한 줄기세포에 대하여 동일 또는 이에 준하는 생존 및 증식 증대 효과를 보일 것을 쉽게 유추할 수 있다.
본 발명의 줄기세포는 구체적으로 예를 들면， 인간， 마우스 , 소， 말， 염소， 양, 개， 고양이, 토끼 또는 조류로부터 유래된 것이다. According to another embodiment of the present invention, the overlapping stem cells of the above-described embodiment are neural stem cells, hematopoietic] λ ^] S (hemat opo i et ic stem cells) or mesenchymal stem cells )to be. More specifically, the redundant stem cells are neural stem cells. More specifically, the neural stem cells are neural stem cells derived from viral dedifferentiated stem cells. The viral dedifferentiated stem cells are specifically retroviral hiPS-NSCs (Retro human induced Pluri potent stem cell -neural stem cells) or lentiviral hiPS-NSCs (Lent i human induced Plur ipotent stem cell ᅳ neural stem eel Is May be). As shown in FIG. 6, the neural stem cells derived from the viral dedifferentiated stem cells can be seen that the cells die while the passage number is increased. However, it was confirmed that treatment of doxycycline can solve this problem dramatically (see FIG. 6). This makes it possible to stably culture dedifferentiated stem cells and new pluripotent stem cells, redundant stem cells or unipotent stem cells derived using the same. The inventors have identified through several examples that doxycycline has an effect on the culture of embryonic stem cells, pluripotent stem cells, pluripotent stem cells, pluripotent stem cells, and neural stem cells, which are redundant stem cells. It can be seen that doxycycline has the effect of increasing the survival and proliferation of stem cells in the culture of several stem cells showing different progression of development, and thus the same or equivalent survival and proliferation of various stem cells. It can be easily inferred to show augmentation effects. Stem cells of the present invention are specifically derived from, for example, human, mouse, cow, horse, goat, sheep, dog, cat, rabbit or bird.
본 발명의 다른 구현예에 따르면， 본 발명의 조성물은 독시사이클린을 1 n -5000 yM 함량으로 포함한다. 본 명세서 상의 도 4 에는 MTT assay 결과가 표시되어 있고, 1 nM 의 독시사이클린의 첨가로도 배양 세포의 생존력이 향상되는 것을 관찰하였다. 또한, 도 32 에는 독시사이클린 무처리 대조군에 대한 상대 생존력을 나타내었으며, 도 29 의 According to another embodiment of the present invention, the composition of the present invention comprises doxycycline in the content of 1 n -5000 yM. The results of the MTT assay are shown in FIG. 4 of the present specification, and it was observed that the viability of the cultured cells was improved even by the addition of 1 nM of doxycycline. In addition, FIG. 32 shows the relative viability of the doxycycline untreated control group,
PI3 카이네이즈 활성도 측정을 나타내었는바， 이를 통해서도 상기 함량 범위의 독시사이클린을 처리시 배양 세포 생존력 증대 효과를 관찰할 수 있다. '더욱 구체적으로 본 발명의 조성물은 독시사이클린을 0.01 μΜ- 5000 μΜ 함량으로 포함하고, 더욱 더 구체적으로는 0.1 y M-5000 μΜ 함량으로 포함하며, 더욱 더 구체적으로는 1 μΜ-5000 μΜ 함량으로 포함하고， 더욱 더 구체적으로는 10 y M-5000 μΜ 함량으로 포함한다. 본 발명의 일 양태에 따르면， 본 발명은 다음 단계를 포함하는 줄기세포의 생존 및 증식을 증대시키는 줄기세포 배양 방법을 제공한다: The PI3 kinase activity measurement was shown, through which it is possible to observe the effect of increasing the culture cell viability upon treatment of the doxycycline in the above content range. More specifically, the composition of the present invention comprises doxycycline in an amount of 0.01 μΜ-5000 μΜ, even more specifically 0.1 y M-5000 μΜ, and even more specifically 1 μΜ-5000 μΜ. And even more specifically, 10 y M-5000 μΜ. According to one aspect of the present invention, the present invention provides a stem cell culture method for increasing the survival and proliferation of stem cells comprising the following steps:
(a) 줄기세포 배양용 배지를 준비하는 단계; (a) preparing a culture medium for stem cell culture;
(b) 상기 단계 (a)에서 준비된 배지에 독시사이클린을 처리하여 줄기세포를 배양하는 단계. (b) culturing stem cells by treating doxycycline in the medium prepared in step (a).
본 발명의 방법에 있어서, 사용 가능한 줄기세포 배양용 배지는 특별한 제한이 없고， 동물세포의 배양에 통상적으로 이용되는 어떠한 배지도 사용이 가능하다. 구체적으로 예를 들면， Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), a -MEM (Stanner, CP. et al. , Nat. New Biol. 230:52(1971))， Iscove's MEM (Iscove, N. et al . , J. Exp. Med. 147:923(1978))， 199 배지 (Morgan et al. , Proc. Soc. Exp. Bio. Med., 73:1(1950))， CMRL 1066, RPMI 1640 (Moore et al . , J. Amer. Med. Assoc. 199:519(1967)), F12 (Ham, Proc. Natl. Acad. Sci . USA 53:288(1965))， F10 (Ham, R.G. Exp. Cell Res. 29:515(1963)), DMEM (Dulbecco' s modification of Eagle's medium, Dulbecco, R. et al . , Virology 8:396(1959)), DMEM 과 F12 의 흔합물 (Barnes, D. et al . , Anal. Biochem. 102:255(1980))， Way-mouth's MB752/1
(Waymouth, C. J. Natl. Cancer Inst. 22:1003(1959)), McCoy's 5A (McCoy, T.A. , et al . , Proc. Soc. Exp. Biol. Med. 100： 115(1959)) 및 MCDB 시리즈 (Ham, R.G. et al., In Vitro 14:11(1978)) 등이 이용될 수 있다. 본 발명의 독시사이클린의 처리는 줄기 세포를 배양하는 동안 1 회 또는 2 회 이상 처리하는 가능하몌 바람직하게는 2 회 이상 처리할 수 있으며， 더 바람직하게는 일정 시간 간격으로 2 회 이상 처리할 수 있고， 구체적으로 예를 들면 24 시간 간격으로 2 회 이상 첨가할 수 있다. 독시사이클린을 최초 1 회 만 일반적으로 사용하는 인간배아줄기세포 배지, 구체적으로 예를 들어 시판되는 Stemcell Technology 사의 mTESR 배지, Millipore 사의 Pluristem 배지 둥에 섞어서 배양하더라도, 독시싸이클린을 첨가하지 않은 배지를 대조군으로 봤을 때， 대조군에 비하여 더욱 더 효율적으로 6 일간의 배지 교환없이 인간배아줄기세포의 배양이 가능하다 (참조: 도 19). 특히 독시싸이클린을 첨가하였을 때， 일주일간 배지 교환없이 사용가능한 제품으로 시판된 Millipore 사의 Pluristem 배지보다도 높은 생존율을 보인다. In the method of the present invention, the medium for stem cell culture that can be used is not particularly limited, and any medium conventionally used for culturing animal cells can be used. Specifically, for example, Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), a-MEM (Stanner, CP. Et al., Nat. New Biol. 230: 52 (1971) ), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc.Exp. Bio.Med., 73: 1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199: 519 (1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53: 288 (1965) ), F10 (Ham, RG Exp. Cell Res. 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)), DMEM and F12 Compound (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Waymouth, CJ Natl. Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, TA, et al., Proc. Soc.Exp. Biol. Med. 100: 115 (1959)) and the MCDB series (Ham , RG et al., In Vitro 14:11 (1978)) and the like. Treatment of the doxycycline of the present invention is possible to process once or twice or more during the culturing of stem cells, preferably two or more times, more preferably two or more times at regular time intervals, Specifically, for example, it may be added two or more times at intervals of 24 hours. Human embryonic stem cell medium which generally uses doxycycline only once, specifically, for example, cultured by mixing with commercially available Stemcell Technology's mTESR medium and Millipore's Pluristem medium, the medium without doxycycline was considered as a control. At this time, it is possible to culture human embryonic stem cells without changing medium for 6 days more efficiently than the control (see Fig. 19). In particular, when doxycycline is added, the survival rate is higher than that of Millipore's Pluristem medium, which is a product that can be used for one week without changing the medium.
본 발명의 일 구현예에 의하면， 본 발명의 단계 (b)는 Y-27632 를 독시사이클린과 함께 처리한다. 본 구현예의 Y— 27632 는 하기 화학식 2 에서 나타낸 화합물에 해당하며， ROCK (rh으 associated protein kinase) 억제제로서 구체적으로 pl60ROCK를 선택적으로 억제하는 특징이 있다. According to one embodiment of the invention, step (b) of the present invention treats Y-27632 with doxycycline. Y- 27632 of the present embodiment corresponds to the compound represented by the following Chemical Formula 2, and specifically has a characteristic of selectively inhibiting pl60ROCK as a ROCK (rh associated protein kinase) inhibitor.
상기 Y-27632 는 종래 줄기세포의 배양에 도움올주는 첨가제로서 알려져 있다. 그러나 Y-27632 를 사용하는 경우 배양 세포의 형태 (morphology)에 변형이 발생하는 문제가 있다 (참조: 도 16). 그러나 독시사이클린을 사용하는 경우에는 이러한 문제가 발생하지 않으며， 줄기세포의 생존이나 증식을 증대시키는 효능도 Y-27632 에 비하여 더욱
뛰어나다는 결과를 도출하였다 (참조: 도 13， 15 및 17) . 다만， Y-27632를 장기간 사용시 상술한 세포 형태의 변형 문제가 있음에도 불구하고 Y- 27632 를 독시사이클린과 병용 사용하는 경우， 줄기 세포의 배양에 가장 효과적임을 알 수 있다 (참조: 도 30) . Y-27632 is known as an additive to help the culture of conventional stem cells. However, when using Y-27632, there is a problem that deformation occurs in the morphology of cultured cells (see FIG. 16). However, the use of doxycycline does not cause this problem, and the effect of increasing the survival or proliferation of stem cells is more than that of Y-27632. Excellent results were obtained (see FIGS. 13, 15 and 17). However, even though Y-27632 has a problem with the above-described cell morphology when used for a long time, it can be seen that when Y-27632 is used in combination with doxycycline, it is most effective for culturing stem cells (see FIG. 30).
본 발명의 일 구체예에 의하면， 본 발명의 단계 (b)에서 Y-27632 는 매 계대 중 최초 1 회에만 처리한다. 구체적으로 예를 들면, 독시사이클린을 24 시간 간격으로 줄기세포 배양 배지에 처리하는 경우에 있어서, 독시사이클린의 최초 1 회 처리 시에만 Y-27632 를 함께 처리한다. 본 구체예에 의하면, Y-27632를 장기간 사용하는 경우 발생할 수 있는 세포 형태의 변형 문제의 발생 우려가 없으면서도， 독시사이클린 또는 Y— 27632 의 단독 사용시에 비하여 더욱 우수한 줄기 세포의 생존 및 증식 증대 효과를 얻올 수 있다 (참조: 도 30) . 본 발명의 다른 일 양태에 따르면, 본 발명은 줄기세포 배양올 위한 독시사이클린의 용도를 제공한다. According to one embodiment of the invention, in step (b) of the present invention Y-27632 is treated only the first time of each passage. Specifically, for example, when doxycycline is treated with stem cell culture medium at intervals of 24 hours, Y-27632 is treated together only during the first single treatment of doxycycline. According to this embodiment, there is no fear of occurrence of cell-type modification problems that may occur when Y-27632 is used for a long time, and even more excellent survival and proliferation effect of stem cells is compared with doxycycline or Y-27632 alone. Can be obtained (see Fig. 30). According to another aspect of the present invention, the present invention provides the use of doxycycline for stem cell culture.
본 발명은 상기 본 발명의 또 다른 일 양태인 독시사이클린을 유효성분으로 포함하는 줄기세포 배양용 조성물을 이용하는 것이고， 상기 본 발명의 또 다른 일 양태인 줄기세포의 생존 및 증식을 증대시키는 줄기세포 배양 방법에 따라 실시할 수 있는 것으로서， 중복되는 내용에 대해서는 본 명세서 기재의 과도한 복잡성을 피하기 위해 생략하도록 한다. 본 발명의 다른 일 양태에 따르면, 본 발명은 독시사이클린을 유효성분으로 포함하는 배지조성물의 줄기세포 배양을 위한 용도를 제공한다. The present invention is to use a stem cell culture composition comprising a doxycycline, which is another aspect of the present invention as an active ingredient, a stem cell culture method for increasing the survival and proliferation of stem cells is another aspect of the present invention As can be carried out according to, the duplicated content will be omitted in order to avoid excessive complexity of the description. According to another aspect of the present invention, the present invention provides a use for culturing stem cells of a medium composition comprising doxycycline as an active ingredient.
본 발명은 상기 본 발명의 또 다른 일 양태인 독시사이클린을 유효성분으로 포함하는 줄기세포 배양용 조성물을 이용하는 것이고， 상기 본 발명의 또 다른 일 양태인 줄기세포의 생존 및 증식올 증대시키는 줄기세포 배양 방법에 따라 실시할 수 있는 것으로서， 중복되는 내용에 대해서는 본 명세서 기재의 과도한 복잡성을 피하기 위해 생략하도록 한다. 【발명의 효과】 The present invention is to use a stem cell culture composition comprising a doxycycline as another active aspect of the present invention as an active ingredient, stem cell culture method for increasing the survival and proliferation of stem cells another aspect of the present invention As can be carried out according to, the duplicated content will be omitted in order to avoid excessive complexity of the description. 【Effects of the Invention】
본 발명의 특징 및 이점올 요약하면 다음과 같다:
(a) 본 발명은 줄기세포 배양용 조성물을 제공한다. In summary, features and advantages of the present invention are as follows: (a) The present invention provides a composition for stem cell culture.
(b) 본 발명은 줄기세포의 생존 및 증식을 증대시키는 줄기세포 배양 방법을 제공한다. (b) The present invention provides a stem cell culture method for increasing the survival and proliferation of stem cells.
(c) 본 발명은 독시사이클린 또는 독시사이클린을 유효성분으로 포함하는 배지조성물의 줄기세포 배양을 위한 용도를 제공한다. (c) The present invention provides a use for stem cell culture of a medium composition comprising doxycycline or doxycycline as an active ingredient.
(d) 본 발명의 줄기세포 배양용 조성물을 이용하는 경우， 다양한 줄기세포의 생존 (survival) 및 증식 (prol i ferat ion)을 모두 증대시킬 수 있다. (d) When using the stem cell culture composition of the present invention, it is possible to increase both the survival (survival) and proliferation (prol i ferat ion) of various stem cells.
(e) 본 발명의 줄기세포 배양용 조성물을 이용하는 경우, 특히 바이러스성 역분화 줄기세포로부터 유도된 다양한 줄기세포들의 장기간 계대배양에 따른 아팝토시스성 세포 사멸 및 조기 노화를 방지할 수 있다. (e) When using the composition for stem cell culture of the present invention, it is possible to prevent apoptotic cell death and premature aging due to long-term passage of various stem cells derived from viral dedifferentiated stem cells.
(f) 본 발명의 줄기세포 배양용 조성물을 이용하는 경우， 배지 교환 시기를 늘리는 효과가 있다. 특히 Millipore 사의 배지교환시기를 늘리는 시판배지인 Pluristem보다 더욱 높은 효율을 보이고， 일반적인 시판 배지에 첨가사용 시에 배지의 효율올 크게 증대하는 효과가 있다. (f) When the composition for stem cell culture of the present invention is used, there is an effect of increasing the medium exchange time. In particular, it shows higher efficiency than Pluristem, a commercial medium that increases the medium exchange time of Millipore, and when used in addition to a general commercial medium, the efficiency of the medium is greatly increased.
(g) 본 발명의 줄기세포 배양 방법을 이용하는 경우, 저렴한 비용으로 줄기세포를 안정적으로 배양할 수 있는 방법을 제공할 수 있다. (g) When using the stem cell culture method of the present invention, it is possible to provide a method for stably culturing stem cells at low cost.
(h) 본 발명의 줄기세포 배양 방법을 이용하는 경우， 다양한 즐기세포의 생존 및 증식올 획기적으로 증대시킬 수 있다. (h) When using the stem cell culture method of the present invention, it is possible to dramatically increase the survival and proliferation of various enjoy cells.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1 은 hNSC(h菌 an neural stem cell) 배양물에 있어서의 고농도의 인슐린에 의해 유발되는 아팝토시스성 세포사멸을 독시사이클린 첨가에 의해 억제할 수 있음을 나타낸다. FIG. 1 shows that apoptosis-induced apoptosis induced by high levels of insulin in hNSC culture can be inhibited by doxycycline addition.
도 2 는 hNSC human neural stem cell) 배양물에 있어서의 낮은 세포 농도에 의해 유발되는 아팝토시스성 세포사멸을 독시사이클린 첨가에 의해 억제할 수 있음을 나타낸다. 2 shows that apoptosis apoptosis caused by low cell concentration in hNSC human neural stem cell cultures can be inhibited by doxycycline addition.
도 3은 뉴론 생존상의 독시사이클린의 효과를 나타낸다. 3 shows the effect of doxycycline on neuronal survival.
도 4 및 도 5 는 독시사이클린， 이와 유사체 (미노사이클린， 테트라사이클린) 및 다른 항생제의 세포 ,생존 효과를 비교한 것을 나타낸다.
도 6， 7 은 독시사이클린 보충이 세포 계대배양 동안 바이러스성 hiPS—유래 NSCs 에 있어서의 이른 노화 및 아팝토시스성 세포 사멸을 예방하는 것을 나타낸다. 4 and 5 show a comparison of the cells, survival effects of doxycycline, analogs thereof (minocycline, tetracycline) and other antibiotics. 6 and 7 show that doxycycline supplementation prevents early aging and apoptotic cell death in viral hiPS-derived NSCs during cell passage.
도 7 은 독시사이클린 처리 (우측) 및 비처리 (좌측)의 레트로 -2-hiPS- NSC 배양물에 있어서， 계대수 4 의 마지막 날， 대표적인 β - 갈락토시다아제 (위， 세포 노화) 및 활성화 캐스페이즈 3(아래, 아팝토시스) -염색 세포를 나타낸다. FIG. 7 shows representative β-galactosidase (gastric, cell aging) and activation casing at the last day of passage 4 in retro-2-hiPS-NSC cultures treated with doxycycline (right) and untreated (left) Phase 3 (bottom apoptosis)-shows stained cells.
도 8 및 도 9는 ΑΡ+ 콜로니 형성 효율을 나타낸다. 8 and 9 show ΑΡ + colony formation efficiency.
도 10 은 독시사이클린 처리에 의해 수정된 항 -/프로-아팝토시스성 유전자에 대한 발현 어레이를 나타낸다. 10 shows expression arrays for anti- / pro-apoptotic genes modified by doxycycline treatment.
도 11 은 hESC 세포 확장에 대한 장기간 ( l ong-term) 배양에 있어서의 세포 성장 곡선을 나타낸다. FIG. 11 shows cell growth curves in long-term (l ong-term) culture for hESC cell expansion.
도 12 는 독시사이클린 -보층 배양물의 8 계대 배양 후 H9 세포꾀 수정되지 않은 핵형을 나타낸다. 12 shows the unmodified karyotype of H9 cells after 8 passages of doxycycline-supplemented cultures.
도 13 은 hESC 생존 및 유지에 있어서의 독시사이클린 및 Y-27632 의 효과 비교한 것으로서， 5일간의 세포 생장을 나타낸다. Figure 13 compares the effects of doxycycline and Y-27632 on hESC survival and maintenance, showing cell growth for 5 days.
도 14 는 hESC 생존 및 유지에 있어서의 독시사이클린 및 Y-27632 의 효과 비교한 것으로서, 형성된 콜로니의 수를 나타낸다. Figure 14 shows the number of colonies formed as a comparison of the effects of doxycycline and Y-27632 on hESC survival and maintenance.
도 15 는 hESC 생존 및 유지에 있어서의 독시사이클린 및 Y-27632 의 효과 비교한 것으로서, 형성된 콜로니의 크기를 나타낸다. Figure 15 shows the size of colonies formed as a comparison of the effects of doxycycline and Y-27632 on hESC survival and maintenance.
도 16 은 무처리， Y-27632-처리， 및 독시사이클린-처리한 배양물로부터의 대표적인 콜로니의 위상차 이미지를 나타낸다. FIG. 16 shows phase contrast images of representative colonies from untreated, Y-27632-treated, and doxycycline-treated cultures.
도 17은 AP+ 미분화된 콜로니의 %비율을 나타낸다. 17 shows the% ratio of AP + micronized colonies.
도 18 은 독시사이클린으로 처리한 H9 세포 내에서의 인간 줄기 세포 전사 인자를 코딩하는 유전자의 발현 수준을 무처리 배양에서의 발현 수준과 비교한 것을 나타낸다. Figure 18 shows the expression level of genes encoding human stem cell transcription factors in H9 cells treated with doxycycline compared to the expression level in untreated culture.
도 19 는 인간 배아 줄기세포 배양시 독시사이클린의 처리만으로 배지 교환 시기를 늘리는 효과가 있음을 나타낸다. 19 shows the effect of increasing the medium exchange time only by treatment with doxycycline in culturing human embryonic stem cells.
도 20 내지 22 은 세포 해리-유발의 엑토미오신 과활성화가 Y- 27632 에 의해 방지되었으나， 독시사이클린 처리에 의해서는 방지되지 않는 것을 나타낸다.
도 23 은 독시사이클린에 의해 활성화된 세포 내부 신호의 추정을 나타낸다. 20-22 show that ectomycin overactivation of cell dissociation-induced was prevented by Y-27632, but not by doxycycline treatment. Figure 23 shows the estimation of the intracellular signal activated by doxycycline.
도 24 는 독시사이클린 처리 0 분， 10 분 및 30 분 후의 상기 신호 활성화를 웨스턴블랏 분석으로 더 측정한 결과를 나타낸다. 24 shows the results of further measurement of the signal activation after 0, 10 and 30 minutes of doxycycline treatment by Western blot analysis.
도 25 내지 27 은 AP+ 콜로니의 수 (도 25)， 크기 (도 26) , 및 퍼센트 비율 (도 27)에 대한 독시사이클린 -유발 효과가 PI3K-Akt 신호에 특이적인 차단제에 의해 차단되었음을 나타낸다. 25-27 show that the doxycycline-induced effect on the number (FIG. 25), size (FIG. 26), and percentage ratio (FIG. 27) of AP + colonies was blocked by blockers specific for PI3K-Akt signal.
도 28은 일반적인 방사능 사진 기반 PI3K 활성 분석을 나타낸다. 도 29 는 PI3K 활성에 대한 독시사이클린의 효과를 PI3 kinase act ivi ty/ inhi bi ter assay ki tR를 이용하여 축정한것을 나타낸다. 28 shows a general radiographic based PI3K activity assay. 29 shows that the effect of doxycycline on PI3K activity chukjeong using the PI3 kinase act ivi ty / inhi bi ter assay ki t R.
도 30은 독시사이클린과 함께 매 계대의 첫날에 Y-27632를 처리하여 준 경우에 줄기세포의 생존 및 증식이 증대되는 결과를 나타낸다. Figure 30 shows the result of increased survival and proliferation of stem cells when Y-27632 was treated on the first day of each passage with doxycycline.
도 31 은 독시싸이클린을 첨가하여 20 계대까지 배양하여도 기본적인 인간배아줄기세포의 성질이 변하지 않음을 나타낸다. Figure 31 shows that even when cultured up to 20 passages with doxycycline does not change the properties of the basic human embryonic stem cells.
도 32 는 독시사이클린 무처리 대조군에 대한 독시사이클린 처리 실시예에 있어서의 배양 줄기세포의 상대 생존력을 나타낸다. Fig. 32 shows the relative viability of cultured stem cells in the doxycycline treated example to the doxycycline untreated control.
【발명을 실시하기 위한 구체적인 내용】 [Specific contents to carry out invention]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. 실시예 Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . Example
실험 재료 및 방법 Experimental Materials and Methods
세포 배양 Cell culture
HYE-08-02 하의 한양대학교 (서울， 대한민국)의 IRB( inst i tut ional review board)에 의해 승인된 hESC 리서치 가이드라인을 기초로 hESCs 및
hiPSCs 를 배양하였다. 본 실험에서 사용된 hESCs 및 hiPSCs 는 하기 표 1 및 표 2에 나타내었다. HESCs and hESCs based on hESC research guidelines approved by the Inst i tut ional review board (IRB) of Hanyang University (Seoul, Korea) under HYE-08-02. hiPSCs were cultured. HESCs and hiPSCs used in this experiment are shown in Tables 1 and 2 below.
【표 1】 Table 1
【표 2] [Table 2]
인간배아줄기세포 및 역분화 줄기세포 (표 1 및 표 2 참조)에 동일한 효과를 보이나， 구체적으로 H9(WA-09) 인간 배아줄기세포를 계대배양하면서 하나의 그룹에는 독시사이클린을 처리하지 않았고 (대조구)， 또 다른 하나의 그룹에는 매일 독시사이클린을 첨가하여 배양하였다. 계대 배양은 콜라지네이즈를 이용하여 세포를 떼어내고， 중력에 의해 배아줄기세포를 가라앉히는 방법올 사용하여 1:5 로 분할 계대배양하였다. Accutase™ 나 트립신을 이용하여 떼어내어도 독시싸이클린의 세포 생존의 효과를 볼 수 있었다. 매 계대 배양시 동일한 효과가 관찰되었고 8 계대까지 관찰하였으며, 계대 배양 후 6 일째에 그 효과를 비교 관찰하였다. 향후 20 계대까지의 실험에서도 세포 생존의 지속적인 효과를 볼 수 있었고, 세포의 특성이 변하지 않았다.
독시사이클린 사용으로 배지 교환없이 배양 효과 분석 It has the same effect on human embryonic stem cells and dedifferentiated stem cells (see Tables 1 and 2), but in particular when passaged H9 (WA-09) human embryonic stem cells, one group was not treated with doxycycline (control) Another group was incubated with doxycycline daily. Subculture was subcultured by 1: 5 using the method of detaching cells using collagenase and using the method of sinking embryonic stem cells by gravity. When removed using Accutase ™ or trypsin, the effect of doxycycline cell survival was observed. The same effect was observed at each passage and up to 8 passages were compared and observed at day 6 after passage. In future experiments up to 20 passages, there was a sustained effect of cell survival, and the characteristics of the cells did not change. Analysis of culture effect without changing medium by using doxycycline
구체적으로 H9(WA-09) 인간 배아줄기세포를 배양하면서 하나의 그룹에는 독시사이클린을 처리하지 않았고 (대조구)， 또 다른 하나의 그룹에는 독시사이클린을 첨가하여 배지 교환 없이 6 일간 배양하였다. 배양은 콜라지네이즈를 이용하여 세포를 떼어내고， 중력에 의해 배아줄기세포를 가라앉히는 방법올 사용하여 1:5 로 분할 배양시 배지를 첨가하여 교환 없이 6 일간 배양하였다. Accutase™ 나 트립신을 이용하여 떼어내어도 독시싸이클린의 세포 생존의 효과를 볼 수 있었다. 피더층없이 , TESR 배지 (STEMCELL technology)를 이용하여， 혹은 Pluristem 배지 (MiUipore)를 이용하여 Matrigel™ 상에서 배양하거나 마우스 피더층에 보통의 배아줄기세포 배지를 사용하여 배양하여 세포 생존 효과를 비교하였다. 면역 염색법 Specifically, while culturing H9 (WA-09) human embryonic stem cells, one group was not treated with doxycycline (control), and another group was incubated for 6 days without medium exchange by adding doxycycline. Cultivation was carried out for 6 days without the exchange by adding a medium in 1: 5 divided culture using a method of detaching cells by using collagenase, and by dividing the embryonic stem cells by gravity. When removed using Accutase ™ or trypsin, the effect of doxycycline cell survival was observed. Cell viability was compared by culturing on Matrigel ™ using TESR medium (STEMCELL technology) or on Pluristem medium (MiUipore) or using normal embryonic stem cell medium on mouse feeder layer. Immunostaining
배양된 세포를 PBS 내의 4% 파라포름알데히드로 고정하였고， 표 3 에 기재된 1 차 항체와 함께 4°C에서 하투 동안 배양하였다. 시각화를 위해 적절한 형광—표지된 2 차 항체 (Jackson Immunoresearch Laboratories)를 이용하였다. 염색된 샘플을 DAPI 마운트 용액 (Vector Laboratories)을 이용한 VECTASHIELD 내에 마운트 하였다. Cultured cells were fixed in 4% paraformaldehyde in PBS and incubated at 4 ° C. with the primary antibodies described in Table 3. Appropriate fluorescently-labeled secondary antibodies (Jackson Immunoresearch Laboratories) were used for visualization. Stained samples were mounted in VECTASHIELD using DAPI mount solution (Vector Laboratories).
【표 3] [Table 3]
세포 주기 및 사멸 분석 Cell cycle and killing assay
세포 생존능력은 종래 알려진 ΜΊΤ 분석 (28)에 의해 결정하였다. 아팝토시스성 (apopt otic) 세포 사멸은， 아팝토시스성 세포-특이적 표지자인 절단된 캐스페이즈 -3(cleaved Caspase-3) 및 아넥신 (Annexin) V/PI 에 대하여 양성인 세포의 현미경 상에서의 직접 계수 (counting)에 의한 백분율에 의해 추정하였다. 아넥신 V/PI-염색된 세포를 Cell viability was determined by the known ΜΊΤ assay (28). Apoptotic cell death was observed under the microscope of cells positive for cleaved Caspase-3 and Annexin V / PI, which are apoptotic cell-specific markers. Estimated by percentage by direct counting. Annexin V / PI-stained Cells
FACS(Canto™I I ) (Chemomet ec ) 및 Nuc 1 eoCount er ® (NC-3000™) (Chemomet ec )를 이용하여 더 계수하였다ᅳ 각각의 세포 주기 단계 상에 축적된 세포 분포를 결정하기 위하여 FACS 분석을 적용하였다. 노화 - 갈락토시다아제 염색 킷 (Senescence β-Galactosidase Staining Kit(Cell Signaling)를 이용하여 제조자의 지침에 따라 세포 노화를 결정하였다. 알칼리 포스파타아제 활성 분석 FACS (Canto ™ II) (Chemomet ec) and Nuc 1 eoCounter ® (NC-3000 ™) (Chemomet ec) were further counted for FACS analysis to determine the cell distribution accumulated on each cell cycle step. Was applied. Aging-Galactosidase staining kit (Senescence β-Galactosidase Staining Kit (Cell Signaling) was used to determine cell aging according to the manufacturer's instructions Alkaline phosphatase activity assay
알칼리 포스파타아제 기질 킷 1(백터)을 제조자의 지침에 따라 집락 내의 알칼리 포스파타아제 활성을 분석하기위해 사용하였다. 전분화능의 측정 Alkali phosphatase substrate kit 1 (vector) was used to analyze alkaline phosphatase activity in colonies according to the manufacturer's instructions. Measurement of starch capacity
2 주간 피더 -프리 (feeder-free) 배양물로부터 dFGF 를 제거하는것에 의해 3 개의 생식 계통으로의 hESCs 의 In vitro분화를 유도하였고, 그 후 세포를 내배엽 (Foxa2, α-태아 단백질 (Feroprotein) , 중배엽 (smooth muscle
actin(SAM)) 및 , 외배엽 (Tujl, GFAP)에 대해 특이적인 마커를 위해 면역 염색하였다. 6 주령 NOD/SCID 마우스의 생식선 하에 1X106 H9 hESCs (독시사이클린의 존재하에 8 계대수로 56 일간 배양)를 주사하는 것에 의해, In vivo 기형종 (teratoma) 형성을 유도하였다. 8 주 후， 얻어진 종양을 제거하고, 파라핀에 침지시켰다. 구획은 해마특실린 및 에오신 (H&E)을 이용하여 염색하였다. 염색체분석 (Karyotying) In vitro differentiation of hESCs into three germ lines was induced by removing dFGF from feeder-free cultures for 2 weeks, after which the cells were excreted with endoderm (Foxa2, α-Feroprotein, Mesoderm muscle actin (SAM)) and, immunostained for markers specific for ectoderm (Tujl, GFAP). In vivo teratoma formation was induced by injection of 1 × 10 6 H9 hESCs (cultured for 8 days in 8 passages in the presence of doxycycline) under the gonad of 6-week-old NOD / SCID mice. After 8 weeks, the obtained tumor was removed and immersed in paraffin. Compartments were stained using hippocampus and eosin (H & E). Chromosomal Analysis
대한민국 서울의 네오딘 의학 연구소 (NEODIN medical institute)에서의 표준 G 밴딩 (standard G banding)에 의해 , 독시사이클린 (1 yg/niL)의 존재하에서 보존된 hESCs(H9) 상에서 염색체분석이 수행되었다. 정량적 PCR 분석 Chromosomal analysis was performed on conserved hESCs (H9) in the presence of doxycycline (1 yg / niL) by standard G banding at the NEODIN medical institute, Seoul, Korea. Quantitative PCR Analysis
공지된 방법을 통해 (29) 총 RNA 준비 및 cDNA 합성을 수행하였다. (29) Total RNA preparation and cDNA synthesis were performed via known methods.
RT2 Profiler PCR ArrayR(Qiagen)를 사용하는 세포 아팝토시스—관련 단백질 (cat. 330231 PAHS-012ZA) 및 인간 줄기 세포 전사인자 (cat. 330231 PAHS-501ZA)에 대하여 고-처리율 유전자 발현 프로파일링을 수행하였다. 일반적인 리얼 -타임 PCR 분석을 이용하여 결과를 더 확인하였다. 세포 내의 신호 활성화의 측정 High-throughput gene expression profiling for cellular apoptosis—associated protein (cat. 330231 PAHS-012ZA) and human stem cell transcription factor (cat. 330231 PAHS-501ZA) using RT 2 Profiler PCR Array R (Qiagen) Was performed. The results were further confirmed using normal real-time PCR analysis. Measurement of Signal Activation in Cells
인간 포스포-키나아제 분석 (Proteome Profiler™ 분석) (R&D systems) 및 CBA 복합화 비드 -기반 면역 분석 (560005， 560010, 560012, 560013, 560015, 560059, 560065, 560093, 560150, 560792, BD)을 이용하여 제조자의 지침에 따라， 독시사이클린의 처리에 의해 활성화된 세포 내의 신호를 검출하였다. 상기 검출에 의해 얻은 결과를 확인하거나, 엑틴미오신 과활성화와 관련된 Eᅳ캐드헤린 및 pMLC와 같은 단백질의 수준을 확인하기 위해， 공지된 방법을 통해 (30) 웨스턴 블랏 분석을 수행하였다. Using human phospho-kinase assay (Proteome Profiler ™ assay) (R & D systems) and CBA complexed bead-based immunoassay (560005, 560010, 560012, 560013, 560015, 560059, 560065, 560093, 560150, 560792, BD) According to the manufacturer's instructions, signals in activated cells were detected by treatment with doxycycline. (30) Western blot analysis was performed by known methods to confirm the results obtained by the detection or to confirm the levels of proteins such as Eukcadherin and pMLC associated with actinomycin overactivation.
PI3 키나아제 활성 분석
공지된 방법에 의해 (31) 일반적인 방사선사진 -기반 PI3K 활성 분석을 수행하였다. 요컨대， 독시사이클린의 존재 또는 부재 하에 배양된 H9 hESCs 로부터의 총 세포 추출물로부터， 항 -PI3K 항체 (antiP85)를 이용하여 PI3K 를 면역 침강시켰다. 효소 반응은 [γ-32Ρ]ΑΤΡ 및 L-al7 포스파티딜이노시를 (기질)을 침전물에 첨가하는 것에 의해 개시하였고， 반응에 의해 방사능표지된 물질은 방사능 사진을 이용하여 검출하였다. PI3K 활성에 대한 독시사이클린의 효과는， 세포 추출물의 부재에서 재조합 클래스 I PI3K 효소 단백질 및 기질 (PIP2)이 결합되는 상업적으로 이용가능한 키트 (PI3 kinase activity/inhibitor assay ki tR(Mi U ipore))를 이용하여 더 확인하였다. PI3K 활성자 /억제자 활성은 약간 변형된 제조자의 지침에 따라 측정하였다. 독시사이클린과 Y-27632 병용 사용에 따른 효과 분석 PI3 Kinase Activity Assay (31) General radiographic-based PI3K activity assays were performed by known methods. In short, PI3K was immunoprecipitated with anti-PI3K antibody (anti P 85) from total cell extracts from H9 hESCs cultured with or without doxycycline. The enzymatic reaction was initiated by adding [γ-32Ρ] ΑΤΡ and L-al7 phosphatidylinosi (substrate) to the precipitate, and the radiolabeled material by the reaction was detected using radiographs. The effect of doxycycline on PI3K activity was determined by using a commercially available kit (PI3 kinase activity / inhibitor assay ki t R (Mi U ipore)) to which recombinant Class I PI3K enzyme protein and substrate (PIP2) are bound in the absence of cell extracts. It was further confirmed using. PI3K activator / inhibitor activity was measured according to the slightly modified manufacturer's instructions. Effect analysis of combination with doxycycline and Y-27632
인간배아줄기세포 및 역분화 줄기세포 (표 1 및 표 2 참조)에 동일한 효과를 보이나 구체적으로 H9(WA-09) 인간 배아줄기세포를 계대배양하면서 하나의 그룹에는 독시사이클린과 Y27632 를 처리하지 않았고 (대조구), 다른 하나의 그룹에는 독시사이클린 (1 yg/ml)만을 매일 처리하였으며， 또 다른 하나의 그룹에는 Υ-27632(10 μΜ)를 매일 처리하였고， 또 다른 하나의 그룹에는 매 계대 첫날에 독시사이클린과 Υ-27632를 동시에 첨가하고, 다음 날부터는 독시사이클린 만을 첨가하여 배양하였다. 통계 분석 It has the same effect on human embryonic stem cells and dedifferentiated stem cells (see Tables 1 and 2), but specifically, passages of H9 (WA-09) human embryonic stem cells were not treated with doxycycline and Y27632 in one group ( Control), the other group received only doxycycline (1 yg / ml) daily, the other group received Υ-27632 (10 μΜ) daily, and the other group received doxycycline on the first day of each passage. Υ-27632 was added at the same time, and from the next day, only doxycycline was added and incubated. Statistical analysis
두 그룹의 통계적인 비교는 스튜던트 양측 (2-tailed) t 검정 또는 양측 짝지은 (2-tailed paired) t 검정에 의해 수행하였다. 둘 이상의 그룹을 비교하기 위해， 셰페 (Scheffe) 또는 투키 사후 분석 (Tukey post -hoc analysis)(SPSS Inc)을 이용한 1원 분산분석 (one-way AN0VA)을 적용하였다. 실험 결과 Statistical comparisons of the two groups were performed by Student's two-tailed t test or two-tailed paired t test. To compare two or more groups, one-way AN0VA using Scheffe or Tukey post-hoc analysis (SPSS Inc) was applied. Experiment result
인간 NSCs(hNSCs)가 hESC/iPSCs 의 in vitro분화에 의해 유래되었다. hESC/iPSC-유래의 NSCs(hES/iPS-NSCs)는 대표적인 NSC-특이적 표지자 (13)인 네스틴 (nestin)을 균질하게 발현하였다. 상기 네스틴 +hNSCs 는 bFGF(basic
f ibrobl ast growth factor )에 의해 확장될 수 있고， bFGF의 제거 및 결합성 신경영양인자의 존재하에서 뉴론 세포로 분화된다 ( 13 , 14) . 도 1 내지 도 7 Human NSCs (hNSCs) were derived by in vitro differentiation of hESCs / iPSCs. hESC / iPSC-derived NSCs (hES / iPS-NSCs) homogeneously expressed nestin, a representative NSC-specific marker (13). The nestin + hNSCs are bFGF (basic f ibrobl ast growth factor) and differentiate into neuronal cells in the absence of bFGF and in the presence of binding neurotrophic factors (13, 14). 1 to 7
도 1 내지 도 7 은 hNSCs 의 배양물 및 hESC/ iPSCs 로부터 유도된 뉴론 상의 독시사이클린 보층의 효과를 나타낸다. 독시사이클린은 hNSC 배양물에 있어서의 고농도의 인슐린 (참조: 도 1) 또는 낮은 세포 농도 (참조: 도 2)에 의해 유발되는 아팝토시스성 세포 사멸올 방지한다. (a) 1.4 x i05/cm2 또는 (b) 3.4 x i04/cm2로 도말된 H9 hESC-유래의 NSCs 를 독시사이클린 보층 ( : g/mL)과 함께 또는 보충 없이 배양하였다. 생존가능한 세포 계수 및 활성화된 (절단된) 캐스페이즈 3 에 대해 양성인 세포에 의해 아팝토시스성 세포 사멸을 측정하였다. 높은 인슐린 농도를 갖는 배양물에서의, 대표적인 위상차 및 절단된 캐스페이즈 3 염색된 이미지는 왼쪽 패널에 나타내었다. 스케일바는 50 μ ηι 를 나타낸다. 1-7 show the effect of doxycycline complement on neurons derived from culture of hNSCs and hESC / iPSCs. Doxycycline prevents apoptosis cell death caused by high levels of insulin (see FIG. 1) or low cell concentrations (see FIG. 2) in hNSC cultures. H9 hESC-derived NSCs plated with either (a) 1.4 × IO 5 / cm 2 or (b) 3.4 × IO 4 / cm 2 were incubated with or without supplementation of doxycycline complement (: g / mL). Apoptotic cell death was measured by viable cell counts and cells positive for activated (cut) caspase 3. In cultures with high insulin concentrations, representative phase differences and truncated Casphase 3 stained images are shown in the left panel. Scale bars represent 50 μηη.
도 3 은 뉴론 생존상의 독시사이클린의 효과를 나타낸다. 뉴론의 하위유형은 9 일간 H9 hESC-NSCs 의 분화에 의하여 유도되었다. 뉴론 배양물은 5 일간 독시사이클린의 보층에 의해 또는 보충 없이 유지하였다. 배양 마지막 날， 도파민 활성 (TH) , 세로톤 활성 ( serotoni n) 및 GABA 활성 (GABA) 뉴론을 구별하기 위하여 면역 염색을 수행하였다. 스케일바는 50 μ ηι를 나타낸다. 3 shows the effect of doxycycline on neuronal survival. Subtypes of neurons were induced by differentiation of H9 hESC-NSCs for 9 days. Neuronal cultures were maintained for 5 days with or without supplementation of doxycycline. On the last day of culture, immunostaining was performed to distinguish dopamine activity (TH), serotoni n and GABA activity (GABA) neurons. Scale bar represents 50 μηι.
도 4 및 도 5 는 독시사이클린， 이와 유사체 (미노사이클린， 테트라사이클린) 및 다른 항생제의 세포 생존 효과를 비교한 것을 나타낸다. Η9 hESCs 유래 hNSCs 의 분화 동안， 정해진 농도의 항생제를 배양배지에 보층하였다. 세포 생존력은 ΜΠ 분석에 의해 분화 5 일째날 측정하였다. 각각의 항생제-처리된 배양물로부터의 가장 높은 Μπ 값 (세포 생존능)은 도 4 and 5 show a comparison of the cell survival effects of doxycycline, its analogs (minocycline, tetracycline) and other antibiotics. During differentiation of hNSCs derived from ƒ9 hESCs, antibiotics of defined concentrations were complemented in the culture medium. Cell viability was measured on day 5 of differentiation by Μπ assay. The highest Μπ value (cell viability) from each antibiotic-treated culture is shown in FIG.
5 에서 비교하였다. 무처리 대조구 (점선)와 ρ<0.01 에서 매우 달랐다. 미노사이클린， 테트라사이클린과 같은 유사체에 비하여 독시사이클린의 세포 생존 증진 효과가 더욱 우수하였으며， 다른 항생제의 경우에는 이러한
효과를 발휘하지 못하였다. 이는 독시사이클린-매개의 세포 생존 증대 효과가 그것의 항생제 효과와 연관되어 있지는 않은 것을 보여준다. Compared at 5. It was very different in untreated control (dotted line) and ρ <0.01. Doxycycline was more effective in promoting cell survival than analogs such as minocycline and tetracycline. For other antibiotics, It did not work. This shows that the doxycycline-mediated cell survival enhancing effect is not associated with its antibiotic effect.
도 6 , 7 은 독시사이클린 보층이 세포 계대배양 동안 바이러스성 hiPS-유래 NSCs 에 있어서의 이른 노화 및 아팝토시스성 세포 사멸올 예방하는 것을 나타낸다. 레트로바이러스성 (Retro-2 , 가운데 도)， 렌티바이러스성 hiPSC 주 ( l ineKLent i— 1， 우측 도) 및 H9 hESCs (대조구， 좌측 도)로부터 유래된 NSCs 는 독시사이클린 보층의 존재 또는 부존재 하에서 매 7 일마다 계대배양 하는 것에 의해 확장되었다. 도 6 에 나타낸 세포 생장 곡선은 매 계대의 마지막 날에 총 생존 가능한 세포의 수를 계수하는 것에 의해 표시하였다. p<0.01 에서의 독시사이클린-보층된 배양물에 있어서， 생존 가능한 세포 수의 대폭적인 증가를 보였다. 6, 7 show that the doxycycline complement prevents early aging and apoptotic cell death in viral hiPS-derived NSCs during cell passage. NSCs derived from retroviral (Retro-2, middle degree), lentiviral hiPSC strain (l ineKLent i— 1, right side) and H9 hESCs (control, left side) were found every 7 hours in the presence or absence of doxycycline strata. Expanded by subculture every day. The cell growth curves shown in FIG. 6 are indicated by counting the total number of viable cells at the end of each passage. In doxycycline-layered cultures at p <0.01, there was a significant increase in viable cell numbers.
도 7 은 독시사이클린 처리 (우측) 및 비처리 (좌측)의 레트로 -2-hiPS- NSC 배양물에 있어서， 계대수 4 의 마지막 날, 대표적인 β - 갈락토시다아제 (위, 세포 노화) 및 활성화 캐스페이즈 3(아래, 아팝토시스) -염색 세포를 나타낸다. 스케일바는 50 μ ηι를 나타낸다. FIG. 7 shows representative β-galactosidase (gastric, cell aging) and activation casing at the last day of passage 4 in retro-2-hiPS-NSC cultures treated with doxycycline (right) and untreated (left) Phase 3 (bottom apoptosis)-shows stained cells. Scale bar represents 50 μηι.
바이러스 유래 iPSCs 의 NSC 로의 분화는 7 계대 이상의 생존이 어려운 제약이 있었으나, 독시사이클린의 처리로 이 문제를 해결하였다. 도 8 내지 도 18 Differentiation of virus-derived iPSCs into NSCs was difficult to survive for 7 passages or more, but treatment with doxycycline solved this problem. 8 to 18
독시사이클린 보층은, 배양에 있어서 세포 생존 및 미분화 hESC/iPSCs의 유지를 강화하였다. Doxycycline complement layer enhanced cell survival and maintenance of undifferentiated hESC / iPSCs in culture.
도 8 및 도 9 는 AP+ 콜로니 형성 효율을 나타낸다. 해리된 hESC/ iPSCs 는 4X 104 세포 /6 cm 접시로 도말하고, 독시사이클린 ( 1 y g/ml )의 존재 또는 부존재 하에서, mTESR 배지를 이용한 피더 -프리 매트리겔에서 배양하였다. 5 일 후 배양물을 염색하고， 알칼리 포스파테이즈 (alkal ine phosphatase , AP)， 미분화 hESC/ iPSC 표지자， 및 AP+ 콜로니를 계수하였다. ρθ .01 , n=5-8 , 스튜던트 양측 t 검정을 이용하였다. 도 9 는 대표적인 H9 hESCs 로부터의 AP+ 콜로니이다, 스케일바는 100 μ ιτι이다. 8 and 9 show AP + colony formation efficiency. Dissociated hESC / iPSCs were plated in 4 × 10 4 cells / 6 cm dishes and cultured in feeder-free Matrigel with mTESR medium in the presence or absence of doxycycline (1 yg / ml). After 5 days the cultures were stained and counted alkaline phosphatase (AP), undifferentiated hESC / iPSC markers, and AP + colonies. ρθ .01, n = 5-8, Student's two-sided t-test was used. 9 is AP + colony from representative H9 hESCs, scale bar is 100 μιτι.
도 10 은 독시사이클린 처리에 의해 수정된 항 -/프로-아팝토시스성 유전자에 대한 발현 어레이를 나타낸다. Η9 hESCs를 독시사이클린의 존재
또는 부존재하에서 배양하였고， RT2 PCR Prof i l er Arra 를 이용하여 항- /프로-아팝토시스성 유전자의 발현 수준을 추정하였다. 좌측에 나타낸 것은 독시사이클린-처리된 배양물에 있어서의 증가된 항-아팝토시스성 (빨간 점들) 및 감소된 프로-아팝토시스성 (파란 점들) 전사의 경향을 보여주는 ' vol cano plot '이다. 수직의 분흥색 선은 유전자 발현 역치 (threshold)에서의 1.5 배 변화를 나타낸다. p<0.05(수평의 파란선)에서 독시사이클린 처리에 '의해 상당히 증가- 또는 감소-조절된 유전자는 각각의 막대 그래프로 나타내었다. 10 shows expression arrays for anti- / pro-apoptotic genes modified by doxycycline treatment. Η9 hESCs Presence of Doxycycline Or incubated in the absence, the expression level of the anti- / pro-apoptotic gene was estimated using RT 2 PCR Prof Arra. Shown on the left is the 'vol cano plot' showing the trend of increased anti-apoptotic (red dots) and reduced pro-apoptotic (blue dots) transcription in doxycycline-treated cultures. Vertical redundancy lines represent 1.5-fold change in gene expression threshold. p <0.05 (in a horizontal blue line) to doxycycline in the treatment, a significant increase in by-or reduced-regulated genes is shown in each histogram.
도 11 은 hESC 세포 확장에 대한 장기간 ( long-term) 배양에 있어서의 세포 성장 곡선을 나타낸다. 독시사이클린 ( 1 y g/ml )이 포함된 또는 포함되지 않은 bFGF-보층된 KSR 배지 내의 MEF 피더 상에서， 56 일 동안 8 계대수로， H9 hESCs 를 배양하였다. 곡선은 매 계대의 마지막 날에 총 생존 가능한 세포 수를 계수하는 것에 의해 나타내었다. p<0.001 에서의 동일한 세포 계대에 있어서의， 각각의 무처리 대조구와 상당히 달랐다ᅳ 독시사이클린의 처리에 의하여 세포 수의 뚜렸한 증가를 확인하였다. 11 shows cell growth curves in long-term culture for hESC cell expansion. H9 hESCs were incubated in 8 passages for 56 days on MEF feeder in bFGF-supplemented KSR medium with or without doxycycline (1 y g / ml). The curve is shown by counting the total viable cell number on the last day of each passage. Significantly different cell numbers were observed with treatment of doxycycline in the same cell passage at p <0.001.
도 12 는 독시사이클린 -보층 배양물의 8 계대 배양 후 H9 세포의 수정되지 않은 핵형을 나타낸다. 20 개의 무작위-선정한 핵 내에서 염색체 분석을 수행하였다. 독시사이클린에 의하여 염색체의 손상이나 변화가 유발되지 않았다. 12 shows unmodified karyotypes of H9 cells after 8 passages of doxycycline-supplemented cultures. Chromosomal analysis was performed in 20 randomly-selected nuclei. Doxycycline did not cause chromosomal damage or alteration.
도 13 내지 18 은 hESC 생존 및 유지에 있어서의 독시사이클린 및 Y- Figures 13-18 show doxycycline and Y- in hESC survival and maintenance
27632 의 효과 비교한 것을 나타낸다. 해리된 H9 세포를 낮은 세포 농도 (500 세포 /웰， 96ᅳ웰 플레이트)에서 도말하였고， 메트리겔 -mTeSR 에서, 독시사이클린 ( 1 u g/ml ) 또는 Y-27632 10 u M)를 사용하거나 사용함이 없이 5 일 동안 배양하였다. 5 일간의 세포 생장은 도 13 에 나타내었다. 독시사이클린을 처리하는 경우， 무처리의 대조구는 물론이고， Y-27632 에 비하여도 확연한 세포 수의 증가를 확인할 수 있었다. 형성된 콜로니의 수 (참조: 도 14) 및 크기 (참조: 도 15)를 비교하였다. 클론의 수는 세포 생존과 관계되어 있으며， 콜론의 크기는 세포의 증식과 관계되어 있다. 독시사이클린을 처리하는 경우 무처리 대조군에 비하여 세포의 생존 및 증식이 상당히 증대됨을 확인하였고， Y-27632 에 비하여 세포의 증식 증대 효과가 월등함을 확인하였다. *p<0.001 대조구와 비교， #p<0.05 Y-27632
처리군과 비교, 투키 포스트 -호크 분석을 이용한 1 원 분산분석 (One-way ANOVA) ( n=5 독립 연구를 수행하였다 (도 13 내지 도 15) . The effect of 27632 is compared. Dissociated H9 cells were plated at low cell concentrations (500 cells / well, 96 well plates) and in metrigel-mTeSR, with or without doxycycline (1 ug / ml) or Y-27632 10 uM). Incubated for days. Cell growth for 5 days is shown in FIG. 13. In the case of treatment with doxycycline, a clear increase in the number of cells was observed as well as in the untreated control as compared to Y-27632. The number of colonies formed (see FIG. 14) and the size (see FIG. 15) were compared. The number of clones is related to cell survival and the size of the colon is related to cell proliferation. When treated with doxycycline it was confirmed that the survival and proliferation of the cells significantly increased compared to the untreated control, it was confirmed that the effect of increasing the proliferation of the cells compared to the Y-27632. * p <0.001 compared with control, #p <0.05 Y-27632 One-way ANOVA ( n = 5 independent studies using Tukey Post-hawk analysis, compared to treatment groups) was performed (FIGS. 13-15).
도 16 은 무처리, Y-27632-처리, 및 독시사이클린-처리한 배양물로부터의 대표적인 콜로니의 위상차 이미지를 나타낸다. 지정된 세포의 고성능 이미지이며， 스케일바는 50 μ ηι 를 나타낸다. Υ-27632 를 처리하는 경우 세포의 형태 (morphology)에 변화가 발생함을 관찰하였고， 이는 세포에 좋지 않은 영향을 미침을 의미한다. 그러나 독시사이클린을 처리하는 경우에 이러한 형태의 변화가 관찰되지 않았으며， 이는 독시사이클린의 처리가 Y-27632 처리로 인한 문제를 해결할 수 있음을 의미한다. 16 shows phase contrast images of representative colonies from untreated, Y-27632-treated, and doxycycline-treated cultures. High performance image of the designated cells, scale bar represents 50 μηι. When 을 -27632 was treated, the morphology of the cells was observed to change, which means that the cells had an adverse effect. However, this type of change was not observed when treating doxycycline, which means that the treatment of doxycycline may solve the problems caused by Y-27632 treatment.
도 17 은 AP+ 미분화된 콜로니의 %비율을 나타낸다. *p<0.01 대조구와 비교， #p<0.05 Y-27632 처리군과 비교, 각각의 그룹에서 n=9 , 분산분석 (ANOVA)올 수행하였다. 독시사이클린을 처리하는 경우 미분화된 클론의 %비율이 무처리 대조구 및 Y-27632 를 처리하는 경우에 비하여 높게 측정되었다. 17 shows the% ratio of AP + micronized colonies. * p <0.01 compared with control, #p <0.05 compared with Y-27632 treated group, n = 9 in each group, analysis of variance (ANOVA) was performed. The percentage of undifferentiated clones treated with doxycycline was higher than that of untreated control and treated with Y-27632.
도 18 은 독시사이클린으로 처리한 H9 세포 내에서의 인간 줄기 세포 전사 인자를 코딩하는 유전자의 발현 수준을 무처리 배양에서의 발현 수준과 비교한 것을 나타낸다. 산포도에서, 분흥색 직선은 유전자 발현 역치 (threshold)에서의 2 배 변화를 나타낸다. 전분화능 (빨간 점 )과 연관된 유전자의 발현을 선정하고, 대조구와 상대되는 독시사이클린 처리 배양물 내의 배율 변화 ( fold change)는 오른쪽 그래프에 나타내었다. Figure 18 shows the expression level of genes encoding human stem cell transcription factors in H9 cells treated with doxycycline compared to the expression level in untreated culture. In the scatter plot, the dichroic straight line represents a 2-fold change in the gene expression threshold. Expression of genes associated with pluripotency (red dot) was selected and the fold change in doxycycline treated culture relative to the control is shown in the graph to the right.
hESC 와 iPSCs 의 배양시 독시사이클린의 처리만으로 줄기세포의 생존율을 높일 수 있음을 확인하였다. 특히， 종래의 배아줄기세포 생존 향상에 우수한 효과가 알려져 있고 상용화된 Y-27632 와 비교하는 경우에도 월등한 효과를 확인할 수 있었다. 도 19 When culturing hESCs and iPSCs, it was confirmed that treatment of doxycycline could increase the survival rate of stem cells. In particular, excellent effects are known to improve the survival of conventional embryonic stem cells, and even when compared to the commercialized Y-27632 was able to confirm the superior effect. Figure 19
인간 배아 줄기세포는 매일 싸이토카인의 첨가와 배지 교환을 해 줘야하는 불편으로 많은 연구자들이 배양이 어려움을 겪고 있으나, 단순히 독시사이클린의 처리만으로 배지 교환 시기를 늘리는 효과를 확인하였다 (참조: 도 19) . 이는 배지를 교환하기 어려운 실험 조건, 예를
들면 배지교체 없이 지속적인 세포 성장 관측이 필요한 실험에 있어서 매우 유용하며， 배지를 교체해야하는 실험자의 수고를 덜 수 있다. 현재 배지교환 없이 배아줄기세포를 7 일간 생존시킬수 있다고 알려진 Mi l l i pore 사의 P lur i st em 배지에 독시싸이클린을 첨가하여도 월등한 배양향상 효과를 볼 수 있으며， 다른 배지에 독시싸이클린을 첨가하는 것만으로도 Plur i stem 배지에 비해 더욱 효율적인 배아줄기세포 배양이 가능하다. 도 20 내지 도 29 Human embryonic stem cells are difficult to cultivate due to the inconvenience of daily cytokine addition and medium exchange, but many researchers have confirmed the effect of increasing the time of medium exchange simply by treatment with doxycycline (see FIG. 19). This is an experimental condition that is difficult to change the medium, For example, it is very useful for experiments that require continuous cell growth observation without changing the medium, and saves the labor of the experimenter who needs to change the medium. The addition of doxycycline to the P lur i st em medium of Mi lli pore, which is known to be able to survive embryonic stem cells for 7 days without changing the medium, can provide a superior culture improvement effect, and only by adding doxycycline to another medium. In addition, it is possible to culture embryonic stem cells more efficiently than Plur i stem medium. 20 to 29
독시사이클린은 PI3K-Akt 신호 활성화를 통해 미분화 hESCs 의 세포 생존과 유지를 가능하게 한다. 도 20 내지 22 은 세포 해리-유발의 엑토미오신 과활성화가 Y- 27632 에 의해 방지되었으나, 독시사이클린 처리에 의해서는 방지되지 않는 것올 보여준다. H9 hESCs 를 독시사이클린 또는 Y-27632 와 함께 또는 상기 화합물 없이 (대조군)， 세포 해리 전 1 시간 동안 배양하였다. 세포는 세포 해리 0 분, 10 분 및 30 분 후 회수하였고， 세포—해리-유발의 아팝토시스와 연관된 단백질을 검출하기 위하여 웨스턴블랏을 실시하였다 (참조: 도 20) . 도 21 및 22 은 Y-27632 또는 독시사이클린 처리된 배양물 내에서의 대표적인 pMLC-면역염색 이미지 및 해리된 hESCs 의 위상차 이미지를 나타낸다. Y-27632 또는 독시사이클린으로 전처리된 세포를 해리시켰고， 직접 mTESR 를 갖는 메트리겔 상에 도말하였으며, 상기 화학물질의 계속적인 존재하에 배양하였다. 상기 이미지는 도말 (해리) 후 지정된 시간에 수집하였다. 스케일바는 10 u m 이다. 도 20 에서 볼 수 있듯이， Υ-27632 를 전처리하는 경우에는 Ε-캐드헤린은 감소하지 않고 pMLC 는 감소하는 것을 관찰할 수 있으나, 대조적으로 독시사이클린을 전처리하는 경우에는 무처리의 대조구와 마찬가지로 E-캐드헤린은 감소하고 pMLC 는 증가하는 것올 확인할 수 있다. 이는 독시사이클린이 Y-27632 와 같은 ROCK 억제제가 아님을 보여준다. Doxycycline enables cell survival and maintenance of undifferentiated hESCs through PI3K-Akt signal activation. 20-22 show that ectomycin overactivation of cell dissociation-induced was prevented by Y-27632 but not by doxycycline treatment. H9 hESCs were incubated for 1 hour prior to cell dissociation with or without doxycycline or Y-27632 (control). Cells were harvested after 0, 10 and 30 minutes of cell dissociation and Western blot was performed to detect proteins associated with cell-dissociation-induced apoptosis (see FIG. 20). 21 and 22 show representative pMLC-immunostained images and phase contrast images of dissociated hESCs in Y-27632 or doxycycline treated cultures. Cells pretreated with Y-27632 or doxycycline were dissociated and plated directly on metrigel with mTESR and incubated in the continued presence of the chemical. The images were collected at designated times after smearing (dissociation). The scale bar is 10 u m. As can be seen in Figure 20, when Υ-27632 pretreatment, Ε-cadherin does not decrease and pMLC decreases, in contrast, when doxycycline pretreatment, as in the untreated control E-cad Herrin decreases and pMLC increases. This shows that doxycycline is not a ROCK inhibitor like Y-27632.
도 23 은 독시사이클린에 의해 활성화된 세포 내부 신호의 추정을 나타낸다. H9 hESCs 는 획득 전에 독시사이클린 ( 1 w g/ml )과 함께 또는
독시사이클린 없이 (대조군) 30 분간 배양하였고， 46 인산화된 세포내 단백질을 검출하기 위해 고안된 인간 포스포키나아제 블랏 어레이 R 를 이용하여 면역블랏 분석하는데 이용하였다. 상기 어레이 분석은 3 회 수행되었다. 무처리 대조구 및 독시사이클린 처리 세포의 블랏의 대표 쌍을 나타내었다. 블랏에 있어서， 독시사이클린-처리군 중 대조구와 비교하여 그 강도가 1.5 배 이상인 점 들은 점선 원으로 표시하였다. 참조 점은 밑줄로 표시하였다. 오른쪽의 그래프는 세 개의 블랏으로부터 표시된 점의 강도의 수치적으로 분석된 값을 나타낸다. ρθ.001** 및 P<0.05* 에서의 짝 비교 t 검정 (pai red t-test ) 결과가 대조구와 상당히 달랐다. 독시사이클린 처리 0분, 10분 및 30분 후의 상기 신호 활성화를 웨스턴블랏 분석으로 더 측정하였다 (참조: 도 24) . 도 20 에서도 독시사이클린 처리시 pAKT 신호의 증가를 관찰할 수 있었듯이ᅳ 도 23 에 있어서 독시사이클린-매개의 생존 및 미분화 hESCs 의 유지에 대하여 어떠한 세포 내부 신호 단백질이 영향을 미치는가 결정하기 위하여 46 개 세포 내부 신호 단백질의 활성을 인간 포스포-키나아제 분석 (Proteome Prof i l erTM , R&D Systems)를 이용하여 스크린하였다 (참조: 도 23) . 그 결과 독시사이클린 처리 배양물에 있어서 pAKT 의 두드러진 활성화를 관찰할 수 있었다. PI3K-AKt 세포 내부 신호가 hESC 다능성 및 자기ᅳ재생의 유지에 대한 가장 중요한 신호 경로로서 밝혀졌다. Figure 23 shows the estimation of the intracellular signal activated by doxycycline. H9 hESCs may be combined with doxycycline (1 wg / ml) prior to acquisition or The cells were incubated for 30 min without doxycycline (control) and used for immunoblot analysis using human phosphokinase blot array R designed to detect 46 phosphorylated intracellular proteins. The array analysis was performed three times. Representative pairs of blots of untreated control and doxycycline treated cells are shown. In the blot, the dots in the doxycycline-treated group whose intensity is 1.5 times or more compared to the control were indicated by dotted circles. Reference points are underlined. The graph on the right shows the numerically analyzed values of the intensity of the points shown from the three blots. The results of the pai red t-test at ρθ.001 ** and P <0.05 * were significantly different from the control. The signal activation after 0, 10 and 30 minutes of doxycycline treatment was further measured by Western blot analysis (see Figure 24). As shown in FIG. 20, an increase in pAKT signal during doxycycline treatment was observed in FIG. 23. In order to determine which intracellular signal proteins influence on doxycycline-mediated survival and maintenance of undifferentiated hESCs in FIG. The activity of the protein was screened using human phospho-kinase assay (Proteome Prof. ™, R & D Systems) (see FIG. 23). As a result, significant activation of pAKT was observed in doxycycline treated cultures. PI3K-AKt intracellular signals have been found to be the most important signal pathway for the maintenance of hESC pluripotency and self-renewal.
본 발명자들은 hESC 생존 (참조: 도 25) , 자기-재생 (참조: 도 26)， 및 미분화 상태의 유지 (참조: 도 27)에 대한 독시사이클린의 효과가 PI3K 억제제인 LY29007 및 워트만닌 OVortmannin)에 의해 감소 또는 제거됨을 확인하였다. 도 25 내지 27 은 AP+ 콜로니의 수 (도 25)， 크기 (도 26)， 및 퍼센트 비율 (도 27)에 대한 독시사이클린 -유발 효과가 PI3K-Akt 신호에 특이적인 차단제에 의해 차단되었음올 나타낸다. PI3K-Akt 차단제인 LY29002(20 μ Μ) 또는 워트만닌 (wortmanninX lOO μ Μ) 또는 Raf-Erk 차단제인 PD98059( 10 μ Μ)를 포함하거나 또는 포함하지 않는 독시사이클린 ( 1 g/ml ) 또는 비히클 (DMS0)의 존재 하에서 5 일 동안 해리된 H9 세포를 배양하는 것에 의해 콜로니 형성 분석을 수행하였다. n=3 독립 실험이며， 투키
포스트 -호크 분석을 이용한 1 원 분산분석 (one-way AN0VA)에 있어서 , *p<0.001 에서 비히클로 처리한 대조구 배양물 및 #p<0.005 에서 독시사이클린 단독으로 처리한 배양물과 상당히 달랐다. We found that the effect of doxycycline on hESC survival (see Figure 25), self-renewal (see Figure 26), and maintenance of undifferentiated status (see Figure 27) on PI3K inhibitors LY29007 and Wortmannin OVortmannin) Confirmed to be reduced or eliminated. 25-27 show that doxycycline-induced effects on the number (FIG. 25), size (FIG. 26), and percentage ratio (FIG. 27) of AP + colonies were blocked by blockers specific for PI3K-Akt signal. Doxycycline (1 g / ml) or vehicle (with or without PI3K-Akt blocker LY29002 (20 μΜ) or wortmannin (wortmanninX LOO μΜ) or Raf-Erk blocker PD98059 (10 μΜ) Colony formation assays were performed by culturing dissociated H9 cells for 5 days in the presence of DMS0). n = 3 independent experiments, Tucky In one-way AN0VA using post-hawk analysis, it was significantly different from control cultures treated with vehicle at * p <0.001 and cultures treated with doxycycline alone at #p <0.005.
도 28 및 29 는 독시사이클린에 의해 강화된 PI3K 효소 활성을 나타낸다. 도 28 은 일반적인 방사능 사진 기반 PI3K 활성 분석을 나타낸다. 분석을 위한 양성 대조구로서， PI3K 활성은， 알려진 PI3K 억제제 (액콜 ) (31) (왼쪽 도)를 포함하는 또는 포함하지 않는 대조세포주 (인간 유방 암 세포 MDA-MB-231)에서 동시에 결정하였다. 도 29 는 PI3K 활성에 대한 독시사이클린의 효과를 PI3 kinase act ivi ty/inhibi ter assay ki tR를 이용하여 측정한것을 나타낸다. 분석은 또한 다른 항생제 또는 알려진 PI3k 억제제 (대조구)의 존재하에서 수행되었다: 독시사이클린 (Dox) ; 테트라사이클린 (Tet ) ; 미노사이클린 (Mino) ; 카나마이신 (Kan) ; Chlorampheni col (Chi or )；28 and 29 show PI3K enzyme activity enhanced by doxycycline. 28 shows a general radiographic based PI3K activity assay. As a positive control for analysis, PI3K activity was simultaneously determined in control cell lines (human breast cancer cell MDA-MB-231) with or without a known PI3K inhibitor (Accol) (31) (left diagram). 29 shows that the effect of doxycycline on PI3K activity as measured with a PI3 kinase act ivi ty / inhibi ter assay ki t R. The assay was also performed in the presence of other antibiotics or known PI3k inhibitors (control): doxycycline (Dox); Tetracycline (Tet); Minocycline (Mino); Kanamycin (Kan); Chlorampheni col (Chi or);
Blastcidine(Bl as) . 그래프는 억제제 /활성제 없는 활성을 제외한 후의 계산된 PI3K 효소 활성을 나타낸다. 독시사이클린 복용량 -의존 PI3K 활성 곡선도 표시하였다. Blastcidine (Bl as). The graph shows the calculated PI3K enzyme activity after excluding the activity without inhibitor / activator. Doxycycline dose-dependent PI3K activity curves are also indicated.
독시사이클린 처리에 의해 직접적으로 포스포 -MT 가 증가되는 PI3- 키나아제를 활성화 시킴으로서 인간 줄기세포 생존력 증대 등의 주역할을 야기하게 됨을 확인하였다. 도 30 It was confirmed that activating PI3-kinase, which directly increases phospho-MT by doxycycline treatment, causes a major role such as increasing human stem cell viability. Figure 30
독시사이클린과 함께 매 계대의 첫날에 Y-27632 를 처리하여 준 경우에 줄기세포의 생존 및 증식이 증대됨을 관찰할 수 있었다 (참조: 도 When Y-27632 was treated on the first day of each passage with doxycycline, it was observed that the survival and proliferation of stem cells were increased.
30) . ' 도 31 , 30). "Fig. 31,
독시사이클린과 함께 20 계대 배양시에도 인간배아줄기세포의 마 알칼라인포스포테이즈 (AP) , TRA 1-60， 0ct4 가 발현됨을 관찰할 있었다 (참조: 도 31) .
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바， 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서， 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. It was also observed that Maal alkaline phosphate (AP), TRA 1-60, and 0ct4 were expressed in human embryonic stem cells even when cultured with doxycycline (see FIG. 31). Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the invention will be defined by the appended claims and equivalents thereof.
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Claims
【청구항 1】 【Claim 1】
독시사이클린을 유효성분으로 포함하는 줄기세포 배양용 조성물. A composition for stem cell culture containing doxycycline as an active ingredient.
【청구항 2】 【Claim 2】
제 1 항에 있어서， 상기 줄기세포 배양용 조성물은 줄기세포의 생존 (survival) 및 증식 (proli ferat ion)을 증대시키는 것을 특징으로 하는 조성물. The composition according to claim 1, wherein the composition for culturing stem cells increases survival and proliferation of stem cells.
【청구항 3】 【Claim 3】
제 1 항에 있어서， 상기 줄기세포는 전능성 줄기세포 (totipotent stem cell), 다능성 줄기세포 (pluripotent stem cell), 중복성 The method of claim 1, wherein the stem cells are totipotent stem cells, pluripotent stem cells, or redundant stem cells.
줄기세포 (Multipotent stem cell) 또는 단분화성 줄기세포 (Unipotent stem cell)인 것을 특징으로 하는 조성물. A composition characterized in that it is a stem cell (multipotent stem cell) or unipotent stem cell (Unipotent stem cell).
【청구항 4] [Claim 4]
제 3 항에 있어서, 상기 전능성 줄기세포는 배아줄기세포 (embryonic stem cell), 배아생식세포 (embryonic germ eel Is) 또는 The method of claim 3, wherein the totipotent stem cells are embryonic stem cells, embryonic germ cells, or
배아종양세포 (embryonic carcinoma eel Is)인 것을 특징으로 하는 조성물. A composition characterized in that it is embryonic tumor cells (embryonic carcinoma eel Is).
【청구항 5】 【Claim 5】
제 4 항에 있어서， 상기 전능성 줄기세포는 배아줄기세포인 것을 특징으로 하는 조성물. The composition according to claim 4, wherein the totipotent stem cells are embryonic stem cells.
【청구항 6】 【Claim 6】
제 3 항에 있어서， 상기 다능성 줄기세포는 역분화 다능성 The method of claim 3, wherein the pluripotent stem cells are dedifferentiated and pluripotent.
줄기세포 (induced Pluripotent stem cell)인 것을 특징으로 하는 조성물. A composition characterized in that it is a stem cell (induced pluripotent stem cell).
【청구항 7】
제 3 항에 있어서， 상기 중복성 줄기세포는 신경 줄기세포 (neural stem cell), 조혈 줄기세포 (hematopoietic stem cell) 또는 간엽 줄기세포 (mesenchymal stem cell)인 것을 특징으로 하는 조성물. 【Claim 7】 The composition according to claim 3, wherein the redundant stem cells are neural stem cells, hematopoietic stem cells, or mesenchymal stem cells.
【청구항 8】 【Claim 8】
제 7 항에 있어서, 상기 중복성 줄기세포는 신경 줄기세포인 것을 특징으로 하는 조성물. The composition according to claim 7, wherein the overlapping stem cells are neural stem cells.
【청구항 9] [Claim 9]
제 8 항에 있어서, 상기 신경 줄기세포는 바이러스성 역분화 줄기세포 유래의 신경 줄기세포인 것을 특징으로 하는 조성물. The composition according to claim 8, wherein the neural stem cells are neural stem cells derived from viral pluripotent stem cells.
【청구항 10】 【Claim 10】
제 1 항에 있어서, 상기 독시사이클린은 1 nM -5000 μΜ 함량으로 포함되는 것올 특징으로 하는 조성물. The composition according to claim 1, wherein the doxycycline is contained in an amount of 1 nM -5000 μΜ.
【청구항 11】 【Claim 11】
다음 단계를 포함하는 줄기세포의 생존 및 증식을 증대시키는 즐기세포 배양 방법: A cell culture method to increase survival and proliferation of stem cells comprising the following steps:
(a) 줄기세포 배양용 배지를 준비하는 단계; (a) preparing a medium for stem cell culture;
(b) 상기 단계 )에서 준비된 배지에 독시사이클린을 처리하여 줄기세포를 배양하는 단계. (b) Culturing stem cells by treating the medium prepared in step) with doxycycline.
【청구항 12] [Claim 12]
제 11 항에 있어서， 상기 단계 (b)는 Y-27632를 독시사이클린과 함께 처리하는 것을 특징으로 하는 방법. The method of claim 11, wherein step (b) involves treating Y-27632 with doxycycline.
【청구항 13】 【Claim 13】
제 12 항에 있어서, 상기 Y-27632는 매 계대 중 최초 1회에만 처리하는 것을 특징으로 하는 방법 .
The method of claim 12, wherein Y-27632 is treated only at the first time of each passage.
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| US20050176140A1 (en) * | 2004-02-10 | 2005-08-11 | Benedict Daniel J. | Method and apparatus for cell culture using a two liquid phase bioreactor |
| EP2103685A1 (en) * | 2008-03-20 | 2009-09-23 | Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Vectors and methods for generating vector-free induced pluripotent stem (iPS) cells using site-specific recombination |
| US20100062534A1 (en) * | 2008-09-09 | 2010-03-11 | The General Hospital Corporation | Inducible lentiviral vectors for reprogramming somatic cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20050176140A1 (en) * | 2004-02-10 | 2005-08-11 | Benedict Daniel J. | Method and apparatus for cell culture using a two liquid phase bioreactor |
| EP2103685A1 (en) * | 2008-03-20 | 2009-09-23 | Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Vectors and methods for generating vector-free induced pluripotent stem (iPS) cells using site-specific recombination |
| US20100062534A1 (en) * | 2008-09-09 | 2010-03-11 | The General Hospital Corporation | Inducible lentiviral vectors for reprogramming somatic cells |
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| Title |
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| MALIK, YS. ET AL.: "Doxycycline can stimulate cytoprotection in neural stem cells with oxygen-glucose deprivation-reoxygenation injury: A potential approach to enhance effectiveness of cell transplantation therapy", BIOCHEMACAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 432, 2013, pages 355 - 358, XP028991000 * |
| WATANABE, K. ET AL.: "A ROCK inhibitor permits survival of dissociated human embryonic stem cells", NATURE BIOTECHNOLOGY, vol. 25, 6 June 2007 (2007-06-06), pages 681 - 686, XP002458303 * |
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