MARKER FOR DETECTING PROLIFERATION OF STEM CELL AND HIGH-EFFICIENCY PROLIFERATION METHOD OF STEM CELL USING SAME

Abstract

The present application relates to a marker gene for detecting the proliferative ability of stem cells and uses thereof, and provides a marker detection composition for detecting the proliferative ability of stem cells, a kit for detecting the proliferative ability of stem cells, and a composition for improving the proliferative ability of stem cells, etc. According to the composition or method according to an aspect, stem cells having a high proliferative ability may be easily selected, and the proliferative ability of stern cells may be significantly improved.

Claims

1. A marker detection composition for detecting the proliferative ability of stem cells, comprising a formulation for measuring the expression level of a developmental arteries and neural crest epidermal growth factor-like (DANCE) gene.

2. The marker detection composition of claim 1, wherein the expression level is a level of a DANCE protein or an mRNA encoding the protein.

3. The marker detection composition of claim 1, wherein the formulation comprises a substance that specifically binds to a DANCE protein or an mRNA encoding the protein.

4. The marker detection composition of claim 3, wherein the substance is a primer, a probe, a nucleotide, an antibody or an antigen-binding fragment thereof, a ligand, a receptor, an agonist or an antagonist, or a combination thereof, each of which specifically binds to a DANCE protein or an mRNA encoding the protein.

5. The marker detection composition of claim 1, wherein the stem cells are embryonic stem cells or adult stem cells.

6. The marker detection composition of claim 5, wherein the adult stem cells are mesenchymal stem cells derived from a tissue selected from umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

7. A kit for detecting the proliferative ability of stem cells, the kit comprising the marker detection composition of any one of claims 1 to 6.

8. A method of providing information to predict the proliferative ability of stem cells, comprising: contacting stern cells isolated from an individual with a developmental arteries and neural crest epidermal growth factor-like (DANCE) protein or a substance that specifically binds to mRNA encoding the protein, to form a complex thereof; measuring a level of the complex to measure an expression level of the DANCE gene; and comparing the measured expression level with that of the DANCE gene of a control group.

9. The method of claim 8, further comprising determining that the stem cells have high proliferative ability when the expression level of the DANCE gene measured from the stern cells is higher than that of the control group.

10. The method of claim 8, wherein the measuring of the expression level is performed by RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, nucleic acid microarray including DNA, Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, ouchterlony immune diffusion, rocket immunity electrophoresis, tissue immunostaining, Immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, magnetic bead-antibody immunoprecipitation, protein chip, or a combination thereof.

11. A composition for improving the proliferative ability of stem cells, the composition comprising Developmental arteries and neural crest epidermal growth factor-like (DANCE) protein or a gene encoding the protein

12. The composition of claim 11, wherein the composition is provided in the form of a medium composition containing a DANCE protein.

13. The composition of claim 11, wherein the stem cells are embryonic stem cells or adult stem cells.

14. The composition of claim 13, wherein the adult stern cells are mesenchymal stem cells derived from a tissue selected from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

15. A method of producing stem cells having a high proliferative ability, the method comprising culturing stem cells in a medium containing the composition of claim 11.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0053] FIG. 1 shows results of observation of the effect of DANCE expression on the adhesion ability of stern cells, as observed by a phase contrast microscope.

[0054] FIGS. 2A and 2B show results of confirming the effect of DANCE expression on the proliferative ability of stem cells, specifically FIG. 2A shows the result of the CCK-8 assay and FIG. 2B shows the result of quantifying and evaluating ATP levels in mesenchymal stem cells.

[0055] FIGS. 3A and 3B show results of confirming the effect of DANCE expression on apoptosis of stem cells, as confirmed by FITC Annexin V/7-AAD kit, specifically FIG. 3A shows a result of flow cytometry, and FIG. 3B shows a result of quantifying and comparing the result obtained by flow cytometry.

[0056] FIGS. 4A and 46 show results of comparison of the expression levels of DANCE of a total of four stern cell groups, specifically FIG. 4A shows a result of confirming the expression level of DANCE by Western blot, and FIG. 4B shows a result of quantifying and comparing the result obtained by Western blot.

[0057] FIGS. 5A and 5B show comparison results of the proliferative ability of a total of four stem cell groups, specifically FIG. 5A shows a result of comparing the number of mesenchymal stem cells when harvested after seeding the same amount of cells, and FIG. 5B shows a result of comparing the doubling time of mesenchymal stem cells based on the result shown in FIG. 5A.

[0058] FIG. 6 shows a result of observation of the treatment effect on improvement of the adhesion ability of stern cells before or during culture of DANCE, as observed by a phase contrast microscope.

[0059] FIGS. 7A and 7B show results of confirmation of the effect of treatment on improvement of the proliferative ability of stem cells before or during culture of DANCE, specifically FIG. 7A shows a result of comparing the number of mesenchymal stem cells, and FIG. 7B shows a result of comparing the doubling times of mesenchymal stem cells.

BEST MODE

[0060] Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

EXAMPLE 1

Changes in Adhesion and Proliferation Abilities of Stem Cells according to Expression of DANCE

[0061] In this example, it was attempted to confirm the effect of DANCE expression on the adhesion and proliferation abilities of stem cells. Specifically, the mesenchymal stem cells were treated with siRNA targeting DANCE (hereinafter referred to as siDANCE) to suppress the DANCE expression in mesenchymal stem cells, and then changes in the adhesion and proliferation abilities of the mesenchymal stem cells were observed. In addition, when the siDANCE-treated mesenchymal stem cells were again treated by adding DANCE 10 ng/ml to the culture medium (siDANCE+DANCE), changes in the adhesion and proliferation abilities of the mesenchymal stern cells were observed. Specifically, the adhesion ability of mesenchymal stem cells was evaluated using images taken with a phase contrast microscope, and the proliferative ability of mesenchymal stem cells was evaluated by a cell counting kit-8 (CCK-8) and measurement of intracellular ATP levels. Here, the DANCE protein, and the like, were obtained from bio-techne (USA) (Catalog Number: 9006-FB) and used. Meanwhile, in this example, a group (Serum(−)) in which mesenchymal stern cells were cultured in a serum-free medium and a group (siCTRL) treated with scrambled siRNA were set as a control group and a comparison group, respectively.

[0062] FIG. 1 shows an observation result of the effect of DANCE expression on the adhesion ability of stem cells, as observed by a phase contrast microscope, and FIGS. 2A and 2B show confirmation results of the effect of DANCE expression on the proliferative ability of stem cells. As a result, the adhesion ability of mesenchymal stem cells was decreased by inhibition of the expression of the DANCE gene, and the mesenchymal stern cells with decreased adhesion ability were restored to a similar level as before by treatment with DANCE. In addition, the proliferative ability of mesenchymal stern cells was also lowered by inhibition of the expression of the DANCE gene, similarly to the above, and this change was recovered by the treatment with DANCE.

[0063] Meanwhile, FIGS. 3A and 3B show confirmation results of the effect of DANCE expression on apoptosis of stem cells, as confirmed by using an FITC Annexin V/7-AAD kit. As a result, the relationship between the apoptosis of mesenchymal stem cells and the expression of the DANCE gene could not be confirmed. These experimental results indicate that the changes in the adhesion and proliferation abilities of the mesenchymal stem cells identified in this example are not the results caused by apoptosis but the results caused by changes in the intrinsic characteristics of cells.

EXAMPLE 2

Change in Doubling Time of Stem Cells According to Expression of DANCE

[0064] In this example, it was attempted to confirm the effect of the DANCE expression on the doubling time of stem cells. Specifically, the obtained mesenchymal stem cells were randomly classified into four mesenchymal stem cell groups (MSC A, MSC B, MSC C, and MSC D), and then the expression levels of DANCE in the respective classified mesenchymal stem cell groups were measured by Western blot. In addition, it was also attempted to verify again the relationship between the DANCE expression and the proliferative ability of the mesenchymal stern cells by calculating the number of cells harvested after seeding the same amount of cells in the mesenchymal stem cell group, and the doubling time.

[0065] FIGS. 4A and 4B show comparison results of the expression levels of DANCE of a total of four stem cell groups, in which DANCE expression levels were observed to be high in the order of MSC D, MSC A, MSC B, and MSC C. In addition, FIGS. 5A and 5B show comparison results of the proliferative ability of a total of four stem cell groups. As a result, the number of mesenchymal stem cells showed a similar tendency to the expression level of DANCE, and the doubling time of the mesenchymal stern cells showed a tendency opposite to the expression level of DANCE. These experimental results indicate that the proliferative ability of mesenchymal stem cells is closely related to the DANCE expression.

EXAMPLE 3

Improvement of Adhesion or Proliferative Ability of Stem Cells According to Treatment with DANCE

[0066] In this example, it was attempted to confirm the effect of DANCE treatment on improvement of the adhesion or proliferative ability of stem cells. Specifically, with respect to mesenchymal stem cells with low proliferative ability, etc., changes in their adhesion or proliferative ability were confirmed when 10 ng/ml of DANCE protein was added to the culture medium and treated 1 hour before seeding (Pretreat), or when DANCE proteins were added to on the culture medium during the cultivation process (Treat). The adhesion or proliferative ability of mesenchymal stem cells was evaluated in the same manner as in Example 1 or Example 2, respectively. Meanwhile, in this example, the DANCE protein untreated group (CTRL) was set as a control.

[0067] FIG. 6 shows an observation result of the effect of treatment on improvement of the adhesion ability of stem cells before or during culture of DANCE, as observed by a phase contrast microscope, and FIGS. 7A and 7B show confirmation results of the effect of treatment on improvement of the proliferative ability of stem cells before or during culture of DANCE. As a result, the adhesion ability of mesenchymal stem cells was significantly improved in all groups treated with the DANCE protein. In addition, the proliferative ability of mesenchymal stem cells was significantly improved by the pretreatment of the DANCE protein or treatment in the culture process, specifically, the doubling time in P2 was significantly shortened from 87 hours of the prior art to 49 hours and the doubling time in P3 from 84 hours of the prior art to 50 hours. These experimental results indicate that treatment with the DANCE protein before or during the cultivation of mesenchymal stem cells can improve the adhesion or proliferative ability of mesenchymal stem cells.

[0068] The above-described present invention has been provided for illustrative purposes only, and a person skilled in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative and non-limiting in all respects.