COMPOSITION FOR ENHANCING REPROGRAMMING EFFICIENCY FROM SOMATIC CELL TO INDUCED PLURIPOTENT STEM CELL, COMPRISING MTOR ACTIVATOR, AND METHOD FOR ENHANCING REPROGRAMMING EFFICIENCY BY USING SAME

Abstract

The present invention relates to a composition for enhancing reprogramming efficiency from somatic cells to induced pluripotent stem cells, comprising an mTOR activator, and a method for enhancing reprogramming efficiency by using same. In the method, reprogramming factors including OCT4, SOX2, c-Myc, and KLF4 are transduced into somatic cells, followed by treatment with an mTOR activator, thereby remarkably increasing reprogramming efficiency into induced pluripotent stem cells. Therefore, the composition and method can be used for effectively inducing the reprograming of somatic cells into induced pluripotent stem cells.

Claims

1. A composition for enhancing reprogramming efficiency from somatic cells to induced pluripotent stem cells (iPSCs), the composition comprising an mTOR activator.

2. The composition of claim 1, wherein the mTOR activator is 4,6-di-4-morpholinyl-N-(4-nitrophenyl)-1,3,5-triazin-2-amine or a derivative thereof.

3. The composition of claim 1, further comprising a nucleic acid sequence encoding for at least one protein selected from the group consisting of OCT4, SOX2, c-Myc, and KLF4.

4. The composition of claim 1, wherein the somatic cells are at least one selected from the group consisting of human umbilical vein endothelial cells (HUVEC), human dermal fibroblasts (HDF), and human placenta derived cells (HPC).

5. The composition of claim 1, further comprising a CXCR2 activator.

6. The composition of claim 1, further comprising a placenta-derived cell conditioned medium (PCCM).

7. A method for enhancing reprogramming efficiency from somatic cells to induced pluripotent stem cells, the method comprising: a somatic transformation step of transducing a nucleic acid sequence encoding for at least one protein consisting of OCT4, SOX2, c-Myc, and KLF4 into somatic cells; and an incubating step of incubating the transformed somatic cells with an mTOR activator.

8. The method of claim 7, wherein the mTOR activator is 4,6-di-4-morpholinyl-N-(4-nitrophenyl)-1,3,5-triazin-2-amine or a derivative thereof.

9. The method of claim 7, wherein the somatic cells are at least one selected from the group consisting of human umbilical vein endothelial cells (HUVEC), human dermal fibroblasts (HDF), and human placenta derived cells (H PC).

10. The method of claim 7, wherein the incubating step is carried out in presence of a CXCR2 activator.

11. The method of claim 7, wherein the incubating step is carried out in a placenta-derived cell conditioned medium (PCCM).

12. The method of claim 7, further a cell isolation step of isolating induced pluripotent stem cells from a colony formed in the incubating step.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1a is a scheme illustrating a procedure of transducing reprogramming factors into somatic cells and incubating the somatic cells with 4,6-di-4-morpholinyl-N-(4-nitrophenyl)-1,3,5-triazin-2-amine.

[0038] FIG. 1b shows western blot analysis results illustrating protein expression levels in primary umbilical vein endothelial cells (HUVEC), primary human dermal fibroblasts (HDF), and human placenta derived cells (HPC) according to the presence or absence of MHY1485.

[0039] FIG. 1c shows immunofluorescence assay images of HUVEC, HDF, and HPC cells demonstrating that treatment with MHY1485 inhibits autophagy.

[0040] FIG. 2a shows images comparing differences in reprogramming efficiency of HUVEC cells between treatment with and without MHY1485 and between placenta derived cell conditioned medium (PCCM) and growth medium.

[0041] FIG. 2b shows images comparing differences in reprogramming efficiency of HDF cells between treatment with and without MHY1485 and between placenta-derived cell conditioned medium (PCCM) and growth medium.

[0042] FIG. 2c shows images comparing differences in reprogramming efficiency of HPC cells between treatment with and without MHY1485 and between placenta-derived cell conditioned medium (PCCM) and growth medium.

[0043] FIG. 3 is a graph comparing differences in reprogramming efficiency of HUVEC, HDF, and HPC cells between treatment with and without MHY1485 and between placenta-derived cell conditioned medium (PCCM) and growth medium.

[0044] FIG. 4 is a plot comparing differences in reprogramming efficiency of HUVEC, HDF, and HPC cells between treatment with MHY1485 and employment of PCCM.

DETAILED DESCRIPTION

[0045] A better understanding of the present disclosure may be obtained through the following examples, which are set forth to illustrate, but are not to be construed to limit the present disclosure.

[0046] Throughout the description, the term “%” used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid.

EXAMPLE 1

Assay for Ability of MHY1485 to Increase Reprogramming Efficiency

[0047] In order to examine whether 4,6-di-4-morpholinyl-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485, CAS Number 326914-06-1), which is an mTOR (mammalian target of rapamycin) activator, has an influence on reprogramming efficiency, reprogramming factors (OCT4, SOX2, c-Myc, KLF4; OSKM) were transduced into primary umbilical vein endothelial cells (HUVEC, ATCC #PCS-100-010), primary human dermal fibroblasts (HDF, ATCC # PCS-201-012), and human placenta derived cells (HPC) via the Sendai virus (SeV) system as shown in FIG. 1a.

[0048] Twenty-four hours after transduction, the cells were cultured for one week in a placenta-derived cell conditioned medium (PCCM) or growth medium (GM). MHY1485 was added at a dose of 2 μg/mL once per 24 hours for three weeks. After one week of the culturing in a placenta-derived cell conditioned medium (PCCM) or growth medium (GM), the cells were induced to undergo reprogramming for two weeks in Matrigel-coated culture dishes containing mTeSR medium together with MHY1485 under a general pluripotent stem cell culturing condition.

[0049] Western blotting identified the expression of CXCR2, mTOR, and cMYC. Autophagic flux activity was identified by staining with Tra-60 specific for stem cell markers.

[0050] As can be seen in FIG. 1b, treatment with MHY1485 increased the expression of CXCR2, mTOR, and cMYC in somatic cells, but decreased the expression of LC3a/b. An increased expression level of p62 indicated inactivation of autophagic flux.

[0051] In an immunofluorescence assay, treatment with MHY1485 decreased the expression of LC3a/b in somatic cells, thus demonstrating the inhibition of autophagy, with the mTOR inhibitor rapamycin serving as a control, as shown in FIG. 1 c.

EXAMPLE 2

Relationship of MHY1485 and CXCR2 Stimuli in Effect of Enhancing Reprogramming Efficiency

[0052] As shown in FIGS. 2a to 2c, reprogramming efficiency was increased in all the cells treated with MHY1485, compared to untreated cells. In addition, higher reprogramming efficiency was observed in PCCM than GM.

[0053] Therefore, treatment with MHY1485 was found to inactivate autophagy, leading to an increase in reprogramming efficiency.

[0054] As is understood from FIG. 3 and Table 1, significantly higher reprogramming efficiency was detected from MHY1485-treated groups than untreated groups.

TABLE-US-00001 TABLE 1 Reprogramming efficiency (%) GM GM + hPCCM − hPCCM + MHY1485 MHY1485 MHY1485 MHY1485 HPC 0.08 0.35 0.30 0.92 HDF 0.01 0.04 0.02 0.07 HUVEC 0.05 0.13 0.10 0.39

[0055] HUVEC, HDF, and HPC cells were induced to undergo reprogramming by treatment with MHY1485 or in PCCM in three dishes for each cell line. Comparison of a total of 9 dishes therebetween exhibited a significantly high reprogramming efficiency in the MHY1485-treated groups, as shown in FIG. 4 and Table 2.

TABLE-US-00002 TABLE 2 GM + MHY1485 hPCCM − MHY1485 Reprogramming 0.17 0.14 efficiency (%)

[0056] Therefore, the data obtained above indicate that treatment with MHY1485 brings about a significantly higher effect of increasing reprogramming efficiency, compared to the employment of PCCM medium.