KR20130022691A - A method for inducing vascular-lineage or myogenic-lineage differentiation of stem cells - Google Patents

Abstract

PURPOSE: A method for inducing differentiation of stem cells into a vascular system or a muscular system is provided to easily control a differentiation speed and a direction and to be used in a cell therapeutic adjuvant. CONSTITUTION: A composition for suppressing stem cell differentiation contains a blocker of a protein with an amino acid of sequence number 1 or an antibody or an aptamer which specifically binds to the protein. The stem cells are embryonic stem cells(ESCs) or induced pluripotent stem cells(iPSCs). A method for producing mesenchymal stem cells comprises: a step of forming an embryoid body; a step of culturing the embryoid body under a hypoxia condition; and a step of culturing the culture under a normoxia condition.

Description

A method for inducing vascular-lineage or myogenic-lineage differentiation of stem cells

The present invention relates to a method of maintaining the undifferentiated state of stem cells or inducing specific differentiation into vascular or muscular cells by using a novel target that controls the differentiation mechanism of stem cells.

Embryonic stem cells (ESCs) have self-renewal and pluripotency and can differentiate into various cell types (1). Various studies have been conducted to control ESCs to differentiate into specific types of cells, but there is little known mechanism for this. The concentration of oxygen, in particular hypoxia, is an important variable for the development of embryonic and stem cells.

Because of the limited diffusion of oxygen due to the increased size of the embryo and the formation of dense organs, oxygen concentration changes and hypoxia occur extensively during embryonic tissue development (2). Oxygen concentration changes during embryonic growth play an important role in the development of blood vessels in some tissues and organs (3-5). Thus, variations in oxygen tension appear to play an important role in embryonic development and vasculature formation.

Most stem cells belong to a complex microenvironment called niche (6). Contact of mesenchymal cells, extracellular matrix proteins, temperature and low oxygen concentrations can affect stem cell function and differentiation. In particular, the hypoxic microenvironment can be a key conducive factor between the stem cell phenotype of self-renewal ability and differentiation fate determination. The primary transcriptional regulator for hypoxic adaptation in mammals is HIF (hypoxia inducible factors) (2, 7). HIF-1α protein, a subunit of HIF1, is degraded as soon as it is produced at normal oxygen concentration (21% oxygen). In contrast, HIF-1b (ARNT) protein is stably expressed regardless of oxygen concentration. Under hypoxic conditions, HIF-1a protein is not degraded and forms a heterozygote with ARNT (HIF-1b), moves into the nucleus, and is located at a promoter site having a HRE (hypoxia-responsive element) sequence to which HIF1 can bind. In combination, transactivation of various genes involved in angiogenesis, vascular endothelial cell development and glycolysis (2, 7). The HIF1 system is important for the development of vascular structures, and therefore, mice without HIF-1α die and die normally due to vascular dysplasia and developmental arrest during embryonic development (8, 9).

The hypoxic microenvironment plays an important role in normal development, particularly in blood vessel formation, but it is still unclear and controversial how low oxygen concentrations affect the fate of ESCs. There are several reports that hypoxia inhibits differentiation, maintains the pluripotency of human embryonic stem cells (hESCs), and improves the survival rate of mouse embryonic stem cells (mESCs) (11). Hypoxic conditions inhibit osteogenic differentiation (12), and further, low oxygen pressure in the hypoxic region of the tumor triggers reverse differentiation and can be a factor in determining the phenotype of cancer stem cells (13). Cell culture in hypoxia promotes the production of pluripotent stem cells (iPSCs) in both mice and humans (14). On the other hand, it has been reported that low oxygen pressure facilitates differentiation. The hypoxic state appears to allow the cultured hESCs to differentiate into cardiomyocytes (15) and promote cartilage production of hESCs (16). Hypoxia promotes development into mesodermal tissue and production of hemato-endothelial progenitor cells (17) and affects the differentiation phenotype of the three germ layers of mESC (18).

When neural stem cells are cultured in a hypoxic state, production of dopaminergic neurons is increased (19). In the case of angiogenesis, low oxygen levels regulate the differentiation of human peripheral mononuclear cells, bone marrow CD133 ⁺ cells and mESCs (20-22), but their molecular mechanisms are not yet known.

The inventors investigated whether low oxygen concentration (1% O ₂ ) selectively induces mESCs to differentiate into the vascular system and attempted to elucidate their molecular mechanisms. Based on the fact that hypoxia plays an important role in proliferation, differentiation and maintenance of the vascular system in early embryonic development, we hypothesized that angiogenic differentiation is promoted when stem cells are exposed to hypoxia.

We spontaneously differentiated ESCs by forming an embryoid body (EB), which is similar to the process of embryonic gustrogenesis (1). For short-term cultures, the fate of stem cells was examined after exposure of EB to hypoxic (1% O ₂ ) (called “hypoxic priming”).

We have shown that priming of hypoxic EB increases differentiation into mesoderm-endodermal cells, and that these cells can differentiate into vascular cells more effectively than normal oxygen-EB. Particularly in the differentiation mechanism, the inventors have found for the first time that HIF-1 can act as an inhibitor of Oct-4 expression by binding to reverse HRE located in the Oct-4 promoter. In addition, hypoxic primed EB enhanced the development of the vascular system by increasing the expression of VEGF. Thus, the present inventors concluded that hypoxia promotes the differentiation of ESCs into the vascular system through reverse regulation of Oct4 and VEGF via HIF-1 and to induce the development of hypoxic priming of EB into specific tissues. It is suggested that it can be an effective means.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors made diligent research efforts to develop a method of inducing effective differentiation into specific cells or a method of continuously maintaining pluripotency by maintaining an undifferentiated state by revealing a differentiation regulating mechanism of pluripotent stem cells. As a result, it was found that reducing the activity or expression of HIF-1α inhibits the differentiation of stem cells, whereas increasing the activity or expression promotes differentiation into mesodermal cells, particularly vascular cells or muscle cells. Thus, the present invention has been completed.

It is therefore an object of the present invention to provide a composition for maintaining pluripotency of stem cells.

Another object of the present invention is to provide a composition for inducing differentiation of embryonic stem cells into mesodermal cells.

Still another object of the present invention is to provide a method for producing mesodermal cells.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a composition for inhibiting the differentiation of stem cells comprising a blocker of a protein consisting of the amino acid sequence of SEQ ID NO: 1 sequence or an antibody or aptamer specifically binding to the protein as an active ingredient. To provide.

The present inventors made diligent research efforts to develop a method of inducing effective differentiation into specific cells or a method of continuously maintaining pluripotency by maintaining an undifferentiated state by revealing a differentiation regulating mechanism of pluripotent stem cells. As a result, it was found that reducing HIF-1α activity or expression level inhibits the differentiation of stem cells, whereas increasing its activity or expression promotes differentiation into mesodermal cells, particularly vascular or muscular cells. .

As used herein, the term "protein" refers to a molecule formed by binding amino acid residues to each other by peptide bonds. The amino acid sequence of SEQ ID NO: 1 is an amino acid sequence of the HIF-1α protein.

According to the present invention, the inventors have found for the first time that HIF-1α can act as an inhibitor of expression of the Oct-4 gene, which determines the pluripotency of embryonic stem cells and regulates differentiation by binding to reverse HRE in the Oct-4 promoter. Found. Therefore, when the amount or activity of HIF-1α is decreased, pluripotency can be continuously maintained by maintaining stem cells in an undifferentiated state, thereby enabling efficient management of scarce therapeutic stem cell resources.

As used herein, the term “stem cell” refers to an undifferentiated cell that is capable of differentiating into various cells constituting biological tissue, which can be reproduced without limitation to form specialized cells of tissues and organs. . Stem cells are developable pluripotent or pluripotent cells. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one derived (transit) cell, and then proliferate into mature, fully formed cells of the tissue.

According to a preferred embodiment of the present invention, the stem cells of the present invention are embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).

As used herein, the term “blocker” refers to any substance that reduces or eliminates the activity of the target protein HIF-1α, and includes both synthetic compounds and natural products. According to a preferred embodiment of the present invention, the blocker of the present invention is YC1 (5- [1- (phenylmethyl) -1H-indazol-3-yl] -2-furanmethanol) or digoxin.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to HIF-1α, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. According to the present invention, since HIF-1α specifically binds to the rHRE site on Oct-4 promoter and inhibits the expression of Oct-4 to promote differentiation of embryonic stem cells, the antibody specifically binding to HIF-1α is Differentiation of stem cells can be inhibited by interfering with the binding between HIF-1α and Oct-4 promoters.

Polyclonal antibodies can be produced by methods well known in the art for injecting HIF-1α antigen into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies are known in the art by the hybridoma method (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. The antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

As used herein, the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that exhibits a high affinity and specificity for binding to a specific target. For general information about aptamers, see Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med . 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA . 95 (24): 142727 (1998). For example, the process of screening for aptamers that bind to a particular molecule can include (i) DNA synthesis and in In vitro transcription methods (for RNA) are used to make nucleic acid libraries of various forms, and (ii) nucleic acids that are not bound by affinity chromatography or the like to select only nucleic acid constructs that can bind to target molecules. Selectively removing only the binding to the target molecule by washing the construct, and (iv) finally using the nucleic acid construct obtained by amplifying the nucleic acid after elution of the nucleic acid construct from the target molecule. Repeat 15 more times to find aptamers with excellent binding and specificity.

According to a preferred embodiment of the present invention, the antibody or aptamer of the present invention specifically binds to a site that binds to the Oct-4 promoter reverse hypoxia element (rHRE) of the protein consisting of the amino acid sequence of SEQ ID NO: 1.

According to a preferred embodiment of the invention, the protein consisting of the amino acid sequence of SEQ ID NO: 1 sequence of the present invention binds to the reverse hypoxia element (rHRE) of the Oct-4 promoter. Oct-4 is a marker for undifferentiated embryonic and embryonic stem cells, and is an important gene for determining pluripotency of embryonic stem cells and controlling differentiation. The inventors found that HIF-1α acts as an inhibitor of Oct-4 gene expression by binding to reverse HRE of the Oct-4 promoter, ultimately inhibiting stem cell differentiation.

According to a preferred embodiment of the present invention, the reverse hypoxia element (rHRE) of the present invention consists of the nucleotide sequence of SEQ ID NO: 3 sequence.

According to another aspect of the present invention, the present invention provides a differentiation of embryonic stem cells into mesodermal cells comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1 or a nucleotide encoding the amino acid sequence of SEQ ID NO: 1 as an active ingredient. It provides a composition for induction.

As used herein, the term “nucleotide” is a deoxyribonucleotide or ribonucleotide present in single- or double-stranded form and includes analogs of natural nucleotides unless otherwise specified (Scheit, Nucleotide). Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990). According to the present invention, HIF-1α, a protein consisting of the amino acid sequence of SEQ ID NO: 1, reduces the expression of Oct-4, increases the expression of mesodermal marker genes, and ultimately promotes specific differentiation into vascular cells. . Thus, a protein consisting of an amino acid sequence of SEQ ID NO: 1 or a nucleotide encoding an amino acid sequence of SEQ ID NO: 1 may be a protein or gene level regulator that induces specific differentiation into vascular cells. Induction of differentiation using a nucleotide encoding the amino acid sequence of SEQ ID NO: 1 may be carried out through various animal transformation methods known in the art. As used herein, the term “transformation” refers to the introduction of stem cells into which the nucleotides of the present invention are to be differentiated through appropriate gene carriers.

As used herein, the term "gene carrier" refers to any means of carrying a gene into a cell, and gene transfer has the same meaning as intracellular transduction of a gene. At the tissue level, the term gene delivery has the same meaning as spread of a gene. Thus, the gene delivery system of the present invention may be described as a gene penetration system and a gene diffusion system.

Gene carrier of the present invention is all used in conventional gene therapy, gene transfer, and the system can be applied, preferably a plasmid, adenovirus (Lockett LJ, et al., Clin. Cancer Res . 3: 2075-2080 (1997)), Adeno-associated viruses: AAV, Lashford LS., Et al., Gene Therapy Technologies , Applications and Regulations Ed. A. Meager, 1999), retroviral (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies , Applications and Regulations Ed. A. Meager, 1999), lentiviruses (Wang G. et al., J. Clin . Invest . 104 (11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc ... Natl Acad Sci USA 92 :. 1411-1415 (1995)), Bash Catania virus (Puhlmann M. et al, Human Gene Therapy 10: 649-657 (1999)), liposomes (Methods in Molecular Biology, Vol 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes.

The differentiation specificity obtained by the composition of the present invention enables efficient cell therapy by obtaining a high yield of cells suitable for the tissues in need of treatment, thereby lowering the possibility of teratoma formation and cancerization and minimizing wasted cellular resources.

According to a preferred embodiment of the invention, the mesoderm cells of the invention are vascular cells or muscle cells. More preferably, the vascular cells of the present invention are endothelial cells or smooth muscle cells. According to the present invention, the endothelial cell markers, PECAM and VE-cadherin, and SMA, which are markers of smooth muscle cells, were significantly increased by HIF-1α induced by hypoxia. Therefore, the composition of the present invention was confirmed to induce specific differentiation of embryonic stem cells into endothelial cells or smooth muscle cells. In addition, the present inventors found that efficient differentiation into muscle cells is induced by hypoxia-induced HIF-1α by using a medium for myocyte differentiation. 43) and immunofluorescent staining for N-cadherin. Therefore, the present invention can also be used for inducing differentiation into muscle cells by selecting the composition of the culture medium.

According to a preferred embodiment of the present invention, the nucleotide encoding the amino acid sequence of SEQ ID NO: 1 of the present invention is a nucleotide having the nucleotide sequence of SEQ ID NO: 2.

Since the protein or nucleotide used in the present invention is the same as described above, the description thereof is omitted to avoid excessive duplication.

According to another aspect of the present invention, the present invention provides a method for producing mesodermal cells, comprising the following steps:

(a) forming an embryonic body;

(b) culturing the embryoid body formed in step (a) in a hypoxia condition; And

(c) culturing the culture of step (b) in a normal oxygen condition.

As used herein, the term "embryoid body" refers to an aggregate of cells derived from embryonic stem cells. The method of forming the embryoid body may be carried out through various methods known in the art, including but not limited to, for example, a floating culture method and a hanging drop method. Preferably, the method for forming the embryoid body of step (a) of the present invention is carried out by a suspending method.

According to the present invention, the formed embryoid body is first incubated in a hypoxic state for a predetermined time (step (b)), and then incubated in a normal oxygen state (step (c)). We named this initially limited hypoxic culture “hypoxia-priming”. Compared with the conventional continuous hypoxic culture, the present invention performs only the hypoxic culture limited to the early stage of the culture, so that not only the survivability of stem cells is better, but also the long-term culture conditions (regulation of nitrogen and carbon dioxide, etc.) are unnecessary. Efficient obtaining of the differentiated cells needed is possible.

According to a preferred embodiment of the present invention, step (b) of the present invention is carried out by incubating for 24-72 hours in a hypoxic state, more preferably by incubating for 45-51 hours, most preferably 48 hours By incubating for a while.

As used herein, the term "hypoxic state" refers to a condition in which the oxygen concentration in the atmosphere in the culture environment is lowered overall. Preferably, the oxygen concentration in the atmosphere is 0-5%, more preferably 0-3%, and most preferably 1%. Hypoxic conditions can be established by a variety of methods known in the art, it is possible to use a variety of commercially available low oxygen microbial culture apparatus. According to the present invention, when the embryonic stem cells are cultured in a hypoxic state, the expression of SMA and Desmin, which are mesodermal gene markers, is increased, so that specific differentiation into mesodermal cells, more specifically vascular endothelial cells or smooth muscle cells It was confirmed that possible.

Throughout this specification, unless otherwise indicated, “%” used to indicate the concentration of a particular substance is solid / solid (weight / weight)%, solid / liquid (weight / volume)%, liquid / Liquid is (volume / volume)% and gas / gas is (volume / volume)%.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for maintaining pluripotency of stem cells, a composition for inducing differentiation of embryonic stem cells into mesodermal cells, and a method for producing mesodermal cells.

(b) The present invention can be usefully used for efficient cell therapy aids by easily controlling the differentiation rate and differentiation direction of pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells.

1 is a diagram showing that naturally occurring hypoxic sites occur in EB (embryoli) during spontaneous differentiation. After producing EB (embryoform) by conventional method, ESCs gather in round water droplets (500 cells per 20 μl) to form a cell population (ploid), and then after 3 days, EB is hypoxic or Cultured in normal oxygen conditions (Fig. 1a). The hypoxic site was detected using an anti-pimonidazole adduct antibody, a hypoxic marker (FIG. 1B). Pimonidazole hydrochloride was added to the culture 1 hour before EB was collected. EB was collected on day 5 (a, b) and day 10 (c) . Positive immune activity was detected by red fluorescence (b, c) . Negative controls were treated with mouse isotype IgG instead of hypoxic markers and no negative staining was found (d). Magnification = x100. Scale bar = 100 μΜ.
Figure 2 shows the differentiation into mesoderm-endodermal tissue and further into the vascular system due to hypoxic priming of EB. EB was produced by incubating for 3 days in the normal oxygen state, and then incubated for a predetermined period in the normal oxygen or hypoxic state, respectively, and the EB was collected to separate total RNA (FIG. 2A). The EB produced by the suspension method was exposed to hypoxia for 2 days. Expression of pluripotency (Oct4, Nanog), ectoderm (Ncam, Nestin), mesoderm (SMA, Desmin), endoderm (Troma-1) and hypoxic reactive gene (VEGF) were examined by real-time PCR (n = 4). , * p <0.05) (FIG. 2B). Hypoxic reactive genes (VEGF) and vascular marker genes (VEGFR2, PECAM, SMA, VE-cadherin) increased in 1% oxygen environment and Oct4 significantly decreased compared to normal oxygen state (FIG. 2C). The graph shows the rate of change relative to ESC (n = 4, * p <0.05).
Figure 3 shows that HIF-1α inhibits Oct4 transcription by binding to the rHRE of the Oct4 promoter in a hypoxic state. 3A is a plot (left) and a quantitative graph (right, n = 4, * p <0.05) showing Western blotting results of HIF-1α and Oct4 in a hypoxic state. 3B is a diagram showing the upstream sequence of Oct4 promoter (GenBank Accession Number S58422) (Verena Nordhoff et al. Mammalian Genome 12: 309-317 (2001). Nucleotides were numbered starting from the translation start point and four potential rHREs (5'-TGCA (C / G) -3 ') are indicated by squares. E14 cells were screened with 1 μg pGL3-Oct4-rHRE (FIG. 3C), pGL3-EPO-HRE (FIG. 3D), 0.2 μg pCMV-β-gal, 0.5 μg pEGFP-HIF-1α and 0.5 μg pEGFP-HIF-1b. Co-transformation for hours followed by incubation for 16 hours under 21% and 1% 0 ₂ (n = 4). HIF-1 decreased Oct4 promoter activity by binding to Oct4-rHRE (FIG. 3E). ChIP analysis shows that HIF-1α binds to the Oct4 promoter site. E14- or C57-stem cells exposed to hypoxia for 16 hours were immunoprecipitated with an antibody against HIF-1α. Precipitated DNA was analyzed by PCR using primers specific for rHRE (rHRE1-rHRE4) on Oct4 promoter (n = 3, respectively).
Figure 4 shows that the vascular differentiation potential of hypoxic primed EB increased. The EB-generated EBs were incubated in normal or hypoxic states for 2 days, respectively, and attached to 0.3% gelatin coated 24-well plates (2-3 EB per well) in DMEM medium containing 10% FBS, the next day. Cells were transferred to vascular differentiation medium and further cultured until day 14 (FIG. 4A). The morphology of the differentiated EBs after 1 and 7 days of reattachment revealed that the cells spread around the reattached cell population (left and center figure), and hypoxic primed EB (HyEB) was normal oxygen EB (NorEB). It is wider than that (FIG. 4B), which means that the differentiation is more accelerated. 4B is a quantitative graph showing the distance from the center of the colony on day 7 of reattachment. One EB was measured at three different points and a total of 10 EBs were measured. Magnification = x40, scale bar = 300 μm (FIGS. 4C and 4D). Figure 4c is the result of immunofluorescence staining for PECAM (red) 7 days after reattachment, Figure 4d is the result of immunofluorescence staining for PECAM (red) / a-SMA (green) on day 14. PECAM and a-SMA were very strong in hypoxic primed EB, which means that hypoxic primed EB differentiates into the vascular system more efficiently. Confocal microscopy showed no fluorescence signal in the isotype IgG control and the negative control. Magnification = x400, Scale bar = 30 mm.
Figure 5 shows the results mean that HIF-1-mediated and YC1 illustration shows the impact on the expression of VEGF and vascular markers play an important role in vascular differentiation of the HIF-1 bits. EB was treated with designated concentrations of YC1 under hypoxic or normal oxygen and Western blotting for HIF-1α (top 5A). MRNA expression of VEGF, a HIF-1 reactive gene, was dose dependent in cells treated with YC1. There was a decrease in phosphorus and expression of vascular genes (PECAM, VEGFR2, VE-cadherin) was also dose-dependently reduced for YC1. HIF-1α levels were indicated by standardization on α-tubulin in quantitative graphs of five independent experiments (* p <0.05) (bottom of FIG. 5A). Expression levels of hypoxic reactive gene (VEGF) and vascular marker genes (VEGFR2, PECAM, VE-cadherin) were analyzed by real-time PCR (n = 4, * p <0.05) (FIG. 5B).
Figure 6 shows the effect of neutralizing anti-mouse VEGF antibody on endothelial differentiation potential in hypoxic primed EB. The expression of endothelial markers PECAM or VE-cadherin in hypoxic primed EB was significantly increased compared to Nor-EB. In contrast, it can be seen that the number of endothelial cells decreases when the neutralizing antibody against VEGF is added. The EB formed by the topical method was incubated in normal or hypoxic state for 2 days, respectively, and attached to 0.3% gelatin coated 24-well plates (2-3 EB per well) in DMEM medium containing 10% FBS, the next day. Transfer to vascular differentiation EGM medium and incubated for 3 more days (FIG. 6A). Neutralizing antibody (50 mg / ml) or control isotype IgG antibody against mouse VEGF was administered daily into the medium. Figure 6b-c shows the results of FACS analysis on PECAM, VE-cadherin (endothelial cells) and a-SMA (smooth muscle cells) C57 cells 3 days after EB reattachment. Nor, normal oxygen-EBs; Hy, hypoxic primed EBs. nVEGF, enhanced anti-mVEGF antibody; nIgG, isotype IgG antibody. Quantitative graphs of FACS analysis of E14 cells (FIG. 6B) and C57 cells (FIG. 6C) are shown (n = 4, respectively).
FIG. 7 shows that hypoxic priming of EB in in vivo ischemic tissues increases engraftment rate and differentiation into endothelial cells in ischemic tissues. BALB / c-nude mice underwent lower limb ischemia surgery and intramuscular injection of NorEB or HyEB, followed by differentiating EBs labeled with DiI (red) (FIG. 7A). Figure 7b is a picture showing the mouse after three days of surgery. The ischemic lower limb (arrow) was relatively good in the HyEB injection group, but loss of the lower limb was observed in the NorEB-injecting group (arrowhead). 7C is a quantitative graph of leg length after ischemia surgery (n = 5 per group). Figure 7d is a picture showing the results of observation in a confocal microscope for PECAM (green) in the longitudinal longitudinal incision of the adductor 7 days after transplantation. Capillary endothelial cells stained with PECAM were located around skeletal muscle cells (bright phase) and localized with DiI immunofluorescence (red), indicating that differentiated vascular cells are well integrated with existing tissues. There was no fluorescence signal in the IgG control group. Magnification = x400, scale bar = 20 um. At least three different cross sections were analyzed from each individual (n = 5). Figure 7e is a graph showing the number of DiI ⁺ / PECAM ⁺ cells injected into the ischemic lower extremity muscle. The number of cells at 7 different microscopic observation sites was measured (n = 5, * p <0.05, respectively). Figure 7f is a figure suggesting a mechanism for promoting blood vessel differentiation by hypoxic primed EB.
8 is a diagram showing the results of quantification of vascular differentiation of E14 and C57 cells by FACS analysis. It can be seen that hypoxic-primed EB accelerates endothelial differentiation without the addition of exogenous VEGF.
FIG. 9 shows the results of tracing differentiated cells from DiI-labeled EB after implanting mEB into BALB / c-nude mouse lower limb ischemia model, removing femoral artery, and injecting NorEB or HyEB into the adductor. Capillaries stained with VE-cadherin (white) and another endothelial marker BS1-lectin (green) also appear as DiI-immunofluorescence (red), whereby the implanted EBs perform angiogenic differentiation in vivo . And hypoxic primed EB differentiates into blood vessels more efficiently than enteric oxygen EB.
10 is a diagram showing the results of examining the effect of digoxin on the expression of HIF-1-mediated VEGF and myogenic-differentiation markers. Digoxin, a HIF-1 blocker, was treated in EB at normal or hypoxic conditions at the indicated concentrations. Expression of hypoxic reactive genes (VEGF) and myogenic differentiation genes (GATA4, Nkx2.5) were examined by real-time PCR. In hypoxic state, the expression of myogenic differentiation gene was increased, and digoxin treatment decreased the expression of myogenic differentiation gene.
FIG. 11 shows an increase in myocyte differentiation potential in hypoxic primed EB. FIG. EB cultured in hypoxic or normal oxygen for 2 days was transferred to myocyte differentiation medium one day later, and after 4 days of reattachment, 5ng TGF-b and 5ng BMP2 were added to the culture medium, and then cultured up to 14 days. Subsequently, immunofluorescence staining was performed on connectin-43 and N-cadherin, which are marker genes of myoblasts. Rat cardiomyocyte line H9C2 cells were used as a positive control. Nuclei were stained with SytoxBlue (blue). Representative confocal micrographs were disclosed and no fluorescence signal was detected in isotype IgG control and negative control. Nor, normal oxygen-EB; Hy, hypoxic primed-EB, scale bar = 50 μm. Magnification = x400.
12 is a diagram showing the results of observing the number of beating foci (beating foci) and the number of cells while culturing under the conditions of myocyte culture medium after attaching the hypoxic primed EB to the cell culture plate. EB can differentiate into cardiac cells under myocyte culture medium, and the pulsating cell population appears. This beating foci appears faster in hypoxic primed EB, and the number is also remarkable.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Stem Cell Culture and In vitro Differentiation

Undifferentiated E14 mouse embryonic stem cells (mESC) and C57BL / 6-background mESCs (C57-mESCs, ATCC Accession No. SCRC-1002) were added to 10% FBS (Hyclone), 1% penicillin / streptomycin (GIBCO), 0.1 mM β DMEM (Dulbecco's modified Eagle's medium, GIBCO, Grand Island, supplemented with mercaptoethanol (Sigma), 1% non-essential amino acid (GIBCO), 2 mM L-glutamine and 1000 U / ml leukemia inhibitory factor, Millipore) Cultured in mitomycin C (Sigma-Aldrich) -treated mouse fibroblast (MEF) trophoblasts (NY, USA). To induce differentiation, EBs were formed via a hanging drop method in the absence of LIF and feeder cells. In summary, one drop containing 500 cells per 20 μl was applied to the cover of a 100 mm culture dish (SPL) using surface tension. Round water droplets collected ESCs and induced cell aggregation (embryoderm, embryoid body). After 3 days, EBs were cultured in normal or hypoxic state for spontaneous differentiation. To measure vascular differentiation in vitro , EBs collected in hypoxic or normal oxygen incubators were plated and cultured in 0.3% gelatin coated tissue culture 24-well plates in DMEM / 10% FBS. After one day, cells were transferred to EGM2 medium (Lonza) supplemented with 5% FBS and incubated for 3, 7 and 14 days for vascular differentiation.

Hypoxic states and hypoxic markers

For hypoxic composition, cells were cultured in a low oxygen chamber (Forma Scientific) maintaining low oxygen pressure (balanced with 1% O ₂ and 5% CO ₂ , N ₂ ). The Forma Hypoxia Chamber (anaerobic system) provides tighter control of hypoxia through a closed hypoxic workspace. Mouse monoclonal antibodies against the hypoxic markers pimonidazole hydrochloride and pimonidazole adducts were purchased from Hypoxyprobe ™, 0 Inc. Pimonidazole hydrochloride was added one hour before EB collection and EB fixed with 4% PFA for immunofluorescence staining.

Real time PCR

Total RNA was prepared using the QIAshredder and RNeasy mini kit (Qiagen Inc.) according to the manufacturer's instructions. 1 μg of RNA was converted to cDNA via PrimeScript ™ 1st strand cDNA Synthesis Kit (Takara). PCR was performed using SYBR Green PCR Master Mix (Roche) through specific primers (Supplementary Table 1). Real time samples were reacted with the ABI PRISM-7500 Sequence Detection System (Applied Biosystems). GAPDH was reacted together as a control and used for standardization.

Western blotting

Normal or hypoxic EBs were collected and lysed in Lysis buffer containing protease inhibitors (Roche). Total protein (10-30 μg) was immunoblotted with primary antibodies against HIF-1α (31) and Oct-4 (SantaCruz Biotechnology). HIF-1α inhibitor YC1 ( Mol cancer Ther 7 (12): 2008) was purchased from Sigma. α-tubulin (Calbiochem) was used as an internal control. Band intensities were quantified using TINA 2.0 (RayTest) and normalized to the intensity of α-tubulin.

Luciferase Analysis

A partial sequence of the genome DNA containing the promoter portion of Oct-4 having four sites estimated to be rHRE was obtained by genome PCR and cloned into an enhancer-free luciferase pGL3 promoter vector (Promega). E14 cells were plated at a density of 2 × 10 ⁵ cells / well and transformed with a combination of various effector plasmids. After transformation, luciferase analysis was performed using Luciferase Assay System kit (Promega) and Luminometer (Turner Design). Relative luciferase activity was normalized to light unit / β-galactosidase activity.

Chromatin immunoprecipitation (ChIP) analysis

ChIP (Chromatin immunoprecipitation) was performed using the ChIP Assay Kit (Upstate) according to the manufacturer's instructions. Anti-HIF-1α antibody (31) was used for immunoprecipitation against DNA fragments. PCR primers were designed to amplify Oct-4 promoters (-1303 ~ -286) containing four rHRE estimation sites.

rHRE-1 (-1293 to -1288): forward, 5'-GTTCAGAGCATGGTGTAGGAGCA-3 ';

Reverse, 5’- GACACTAAGGAGACGGGATTAGG-3 ’

rHRE-2 (-1176 to -1172): forward, 5'-CAGAAAACCACTCTAGGGAAGTT-3 ';

Reverse, 5’- TCTCCACCTCTCCTCAAAGCAG-3 ’

rHRE-3 (-622 to -618): forward, 5'- CCTTAACTGTGAGGGGATGG-3 ';

Reverse, 5’- CCAGAGTTTAGAGGCTCTACACC-3 ’

rHRE-4 (-290 to -286): forward, 5'-CTCCAGAGGATGGCTGAGTGGGC-3 ';

Reverse, 5′-TCTGGACAGGACAACCCTTAGGACG-3 ′.

FACS ( Fluorescence - activated cell sorter ) analysis

Vascular differentiation of E14 and C57 cells was quantified via FACS analysis. Differentiated cells were eliminated with 0.05% trypsin-EDTA. Single cells (at least 1 × 10 ⁴ ) were placed in FACS tubes (BD Falcon) and fixed with 1% PFA on ice for 20 minutes. In order to inhibit the VEGF effect, neutralizing antibodies against mouse VEGF were used. For FACS analysis, cells were treated with anti-PECAM (SantaCruz biotechnology), anti-VE-cadherin (Santa Cruz Biotechnology) or anti-SMA (Abcam) antibody and anti-mouse Alexa 660 or anti-rabbit Alexa 555 secondary antibody ( Incubated with Molecular Probes). Flow cytometry was performed using BD CantoII.

Immunofluorescence  dyeing

In order to analyze the tissue of the lower limb ischemic mouse, the endogenous muscle at 7 days was extracted, washed with PBS and cooled with liquid nitrogen. 10 mm thick histological sections were prepared from rapidly cooled tissue samples, fixed with 4% PFA, blocked with 1% BSA and incubated with anti-PECAM (BD Pharmingen) or anti-VE-cadherin antibody (SantaCruz). It was then incubated with Alexa 488 or Alexa 647 secondary antibody (Molecular Probes). FITC-labeled BS-1-lectin (Sigma) staining was performed for capillary detection. EB or differentiated cells on coverslips were fixed with 4% PFA and blocked with blocking buffer (0.5% goth serum, 0.1% Triton-x100 / 1% BSA-PBS) followed by anti-pimonidazole adduct (Hypoxyprobe ™, Inc), anti-PECAM (BD Pharmingen) or anti-SMA antibody (Abcam) followed by Alexa 488 or Alexa 555 secondary antibody. Nuclei were stained with SytoxBlue (Molecular probe) and mounted using fluorescent mounting medium (DAKO). Fluorescence images were obtained using a fluorescence microscope (Olympus IX71, Japan) and a confocal microscope (Carl Zeiss LSM710).

mouse Ischemia  Model

All animal experiments were conducted under the approval of the Animal Experiment Ethics Committee of Seoul National University Hospital. Male BALB / c-nude mice (8 weeks old) were anesthetized and one femoral artery was ligated and removed. Since the low-oxygen or oxygen-EB of the normal (the total number of cells ^{5 x 10 4/100 ul PBS} ) were injected muscles. To track the differentiating cells, E14 EB was labeled with 2 μg / ml DiI (Molecular Probes Inc.) for 30 minutes before forming EB via suspension method. To determine the differentiation of EBs into endothelial cells, the endogenous muscle was extracted 7 days after transplantation and fixed with 4% PFA for immunofluorescence staining.

Muscle cells  Measurement of differentiation

Were grown in tissue culture coated 24-well plates - for in vitro measuring the differentiation of muscle cells at 2 days hypoxic or gelatin with a normal to the plated EB culture in an oxygen incubator DMEM / 10% FBS. One day later, cells were cultured for myocyte differentiation (IMDM (Iscove's Modified Dulbecco's Medium, Gibco), 15% calf fetal serum (Lonza), 0.2 mmol / L L-glutamine (Gibco), 0.1 mmol / L β-mer). Captoethanol (Sigma), 0.1 mmol / L non-essential amino acid (Gibco)]. After 4 days of reattachment, 5ng TGF-b and 5ng BMP2 were added to the culture medium and incubated for up to 14 days. Thereafter, immunofluorescence staining was performed on connexin-43 or N-cadherin, a marker gene of myoblasts.

Statistical analysis

The experimental results are expressed as mean ± standard deviation. Differences between the groups were compared by unpaired t-test or analysis of variance (ANOVA), followed by post-test using Bonferroni's test. If the p value was less than 0.05, it was considered to have statistical significance. All statistical analyzes were performed using SPSS 17.0 (SPSS Inc., Chicago, US).

Experiment result

Hypoxia  Status is mESC -origin EB To accelerate differentiation

We investigated the effects of hypoxia on the culture and transcription of mESC-derived cell aggregates called EBs. EB formation is commonly used to induce spontaneous differentiation of embryonic stem cells due to their similarity to the embryonic gastulation process (1). Embryonic stem cells can be differentiated into various types of cells by forming EBs. Can be. We first examined whether hypoxic sites appeared in EB during spontaneous differentiation in normal oxygen (21% O ₂ ) conditions (FIGS. 1A and 1B). EB was formed by incubation for 3 days through the hanging drop method, and hypoxic sites were measured using pimonidazole hydrochloride. The hypoxic marker pimonidazole hydrochloride is converted into an intermediate activated by a hypoxic-activated nitro reductase, which forms a covalent bond with the cellular components of the hypoxic site (23).

After 3 days of EB formation, EB (full EB incubation period, 5 days) incubated in hypoxic condition (1% O ₂ ) for ₂ days showed strong staining results for the pimonidazole hydrochloride adduct (FIG. 1B). EBs cultured for 5 days in normal oxygen (21% O ₂ ) were not stained. Interestingly, even in EB cultured under normal oxygen conditions, positive immune responses to pimonidazole hydrochloride adducts were weakly detected after 7 days and markedly detected on day 10 (FIG. 1B), so that the hypoxic environment was reduced to EB spheres (FIG. 1B). It supported the hypothesis of the present inventors that they can appear naturally in the embryonic body).

In order to investigate whether EB culture in low oxygen environment affects differentiation into specific tissues, we exposed EB to 1% oxygen environment for 3 days (total EB culture period, 6 days) Collected at defined time points were monitored for expression of genes that are markers of pluripotency and differentiation (FIG. 2A). On day 2 of hypoxic exposure, the expression of VEGF, which is one of the major subfactors whose transcription is primarily regulated in response to hypoxia, was examined (24) (FIG. 2B). VEGF mRNA was significantly increased compared to the normal oxygen control group, indicating that EB is sensitive to hypoxic stimulation. Interestingly, expression of Oct4 and Nanog genes, markers of undifferentiated state maintenance, was significantly reduced in hypoxic EB. Differentiation marker genes were then monitored for each germ layer: ectoderm (Ncam, Nestin), mesoderm (SMA, Desmin) and endoderm (Troma-1). The expression levels of Ncam and Nestin were very low and did not change due to hypoxic exposure. SMA, Desmin, and Troma-1 showed large changes, and the expression levels were 7.8, 2.6, and 2.4 times higher than those of normal oxygen EB (FIG. 2B). These results indicate that hypoxia promotes differentiation and in particular leads to mesoderm-endodermal cells expressing markers including SMA, Desmin and Troma-1.

We investigated whether apoptosis occurs in EB exposed to hypoxia by measuring the number of cells that caused apoptosis using Annexin V-PE. It was confirmed by Annexin V immunofluorescence staining and FACS analysis that measurable apoptosis did not occur when EB was exposed to hypoxia for 3 days. As shown in FIG. 2B, since the hypoxic exposure during the first few days of differentiation resulted in increased differentiation to mesoderm-endoderm, the inventors are interested in vascular differentiation (endothelial and smooth muscle cells) in the mesoderm system, resulting in vascular differentiation. Expression of marker genes was examined (FIG. 2C). VEGF, a hypoxic response gene, was overexpressed compared to normal oxygen postnatal at each defined time point (FIG. 2C). Oct4 mRNA was significantly decreased during the differentiation period, and the expression level in the hypoxic state was more significantly decreased than the normal oxygen. The expression levels of vascular genes (VEGFR2, PECAM, SMA, VE-cadherin mRNA) were similarly increased in the differentiation stage of normal oxygen EB, and higher in hypoxic EB than in normal oxygen EB (FIG. 2C). These results show that stem cell differentiation into the vasculature increased due to exposure of EB to hypoxia (ie, "hypoxic priming of EB").

HIF -1α is Oct4  Promoter rHREs By binding to Oct4  Reduces Warrior

One of the new findings of the inventors is that EB differentiation is promoted during the hypoxic culture process. In order to analyze the mechanism of EB differentiation by hypoxia, we investigated the direct effect of hypoxia on Oct4 expression. Oct4 is known to be an essential regulator of maintaining stem cell self-renewal capacity and pluripotency (25). Levels of Oct4 mRNA transcripts (FIG. 2) and their protein levels decreased dramatically in hypoxic states (FIG. 3A). In contrast, the HIF-1α protein increased significantly in the hypoxic state (FIG. 3A), supporting the possibility that HIF-1α would be involved in the inhibition of transcription of Oct4.

We then investigated the mechanism by which HIF-1α regulates Oct4 transcription. HIF-1 is a major transcription factor that activates the transcription of several genes in response to hypoxia and ischemia. These genes include VEGF and erythropoietin (EPO), and HIF-1 activates transcription by binding to conservative hypoxia-responsive elements (HREs) in the promoters of these genes (7, 26-27).

Interestingly, mouse Oct4 promoter 30 contains four sequences that are likely to act as rHRE (FIG. 3B). The conservative HRE sequence to which HIF-1 binds is 5'-ACGT (G / C) -3 '(26, 27) and thus the corresponding rHRE is 5'-TGCA (C / G) -3' (Fig. 3b).

We performed a promoter luciferase assay to investigate the effect of HIF-1α on the transcriptional activity of Oct4 promoter including four rHREs (Oct4-rHREs) (FIGS. 3C and 3D). Hypoxia significantly reduced reporter gene activity in E14 cells transduced with Oct4-rHRE. Co-transformation with HIF-1α / 1b vector also supported the fact that both hypoxic and normal oxygen states reduced luciferase activity (FIG. 3C) and that HIF-1α was involved in inhibition of Oct4 promoter activity by binding to rHRE. . Since EPO transcription was known to be increased by HIF-1 (24), the same experiment was performed with an EPO reporter vector containing HREs (EPO-HREs). EPO-HRE reporter gene activity was significantly increased in cells co-transformed with HIF-1α / 1b (FIG. 3D). Thus, HIF-1 appears to enhance EPO promoter activity by binding to EPO-HRE, while reducing Oct4 promoter activity by binding to Oct4-rHRE (FIG. 3E). Through these results, the inventors performed a ChIP analysis to investigate the binding of HIF-1α to four sites estimated as rHRE of Oct4 promoter (FIG. 3F). We used specific primers to detect each potential rHREs (rHRE1-rHRE4) (FIG. 3B). In E14 and C57 stem cells, three of the four potential rHREs acted as binding sites for HIF-1α but no binding of HIF-1α to the second rHRE site (rHRE2) was observed (FIG. 3G). As a result, it was again confirmed that Oct4 transcription was inhibited through direct binding between the Oct4 promoter and HIF-1α under hypoxia.

EB of Hypoxia Priming is  Efficiently differentiate into the vascular system.

Because hypoxic priming of EB increases differentiation and expression of vascular marker genes, we measured the differentiation potential of hypoxic primed EB into endothelial and smooth muscle cells. EBs were incubated for 2 days in normal or hypoxic state respectively and attached to 0.3% gelatin-coated 24-well plates. The next day, the culture of cells was replaced with a culture for vascular differentiation and cultured for 14 days (FIG. 4A). After attaching to the gelatin-coated plate, the EB unfolded for 24 hours after plating. The cells spread around the reattached EB showed the appearance of differentiated cells. In particular, hypoxic primed EB (HyEB) is more prevalent, meaning that differentiation is accelerated (FIG. 4B). We measured HyEB's vascular (endothelial and smooth muscle cell) differentiation potential through FACS analysis on PECAM, VE-cadherin and SMA 3 days after reattachment. In HyEB, cells marked with PECAM or VE-cadherin, which are vascular endothelial cell markers, increased significantly compared to normal oxygen-EB.

The ratio of SMA, a smooth muscle marker, did not show similar or significant differences in both normal and hypoxic groups. We confirmed the differentiation potential into the vascular system through staining with anti-PECAM and anti-SMA antibodies (FIGS. 4C and 4D). After 3 days of resorption, PECAM was stained very weakly in HyEB, while it was not detected in normal oxygen EB (NorEB). Over time, PECAM staining (red) became very strong in HyEB after 7 days of reattachment and showed a stable junction structure (FIG. 4C). Interestingly, PECAM ⁺ staining appeared to vary by location. At the center of the colony, PECAM ⁺ cells were clustered and showed a honeycomb with a small nucleus (FIG. 4C; HyEB). The nuclei of PECAM ⁺ cells in the swollen periphery of the colonies were larger than the nuclei of cells in the center of the colony, and PECAM ⁺ cells formed HyEBs by forming tubular structures (HyEB-a) and tubular branching (HyEB-b). Showed efficient differentiation into endothelial cells. In contrast, little PECAM ⁺ cells were detected at the colony center of NorEB and stained very weakly at the colony outgrowth. However, this is a stain in the form of dots and not a conjugated form (FIG. 4C NorEB), which means that they are not fully differentiated endothelial cells. SMA and VE-cadherin immunofluorescence were not detected after 7 days of reattachment in both NorEB and HyEB, and after 14 days of reattachment, strong PECAM ⁺ staining appeared in the cluster center of HyEB (FIG. 4D). PECAM staining around the colony was weak compared to the center. SMA ⁺ cells were detected high around the cluster periphery of HyEB (FIG. 4D). VE-cadherin was also high around the colony of HyEB on day 14. PECAM ⁺ -cells, which have the characteristics of vascular primitive cells, are located in the colony of EB, and they are found to move radially around the colony as they differentiate into vascular cells (SMA ⁺ , VE-cadherin ⁺ ). From these data, we deduced that hypoxic primed EB differentiated into the vasculature more efficiently.

HIF -1 parameters VEGF The Hypoxia Primed EB Important for vascular system differentiation

Since hypoxic priming of EB increases vascular differentiation, it was further investigated whether HIF-1α is involved in gene expression of vascular markers (FIG. 5). We treated EB with HIF-1 blocker YC1 under hypoxic conditions (31). Protein levels of HIF-1α were increased in EBs cultured in hypoxia without YC1 treatment, but dose-dependent decrease was observed in EBs treated with YC1 (FIG. 5A). MRNA expression of VEGF, a HIF-1 reactive gene, showed a dose dependent decrease in cells treated with YC1 (FIG. 5B). Interestingly, expression of vascular genes (PECAM, VEGFR2, VE-cadherin) was also dose dependently reduced for YC1 (FIG. 5B). This observation suggests that an important role in vascular differentiation of the HIF-1 bits.

In particular, because VEGF is a well known endothelial growth and differentiation factor (32), we focused on the role of VEGF in endothelial cell differentiation in both E14 and C57 stem cells (Figure 6). To induce differentiation from stem cells to endothelial cells, methods of adding exogenous VEGF are often used (33-34). We found that low oxygen-primed EB accelerated endothelial differentiation without the addition of exogenous VEGF (FIGS. 6 and 8), and VEGF increased significantly in hypoxic primed EB (FIGS. 2 and 5). In order to suppress the VEGF effect on hypoxic primed EB, neutralizing antibodies against mouse VEGF were added to the cell culture (FIGS. 6B-6D). The expression of endothelial markers PECAM or VE-cadherin in hypoxic primed EB was significantly increased compared to Nor-EB. In contrast, the addition of neutralizing antibodies to VEGF reduced the number of endothelial cells (Hy + nVEGF) (FIGS. 6B-6D), resulting in hypoxic conditions leading to the production of VEGF from EB, which in turn resulted in Form activates endothelial differentiation.

EB of Hypoxia Priming is In Inbo  Increases differentiation into endothelial cells

Next, we implanted mEB into BALB / c-nude mouse lower limb ischemia model to investigate whether hypoxic primed EBs perform vascular differentiation in ischemia damaged tissues (FIG. 7). After removing the femoral artery of BALB / c-nude mice, NorEB or HyEB was injected into the adductor muscle. Cells differentiated from DiI-labeled EB showed red DiI-fluorescence for easy tracking and quantification.

The NorEB-injected group showed progressive lower limb injury, while the ischemic lower extremity of the HyEB-injected group remained relatively good (FIGS. 7B and 7C). Interestingly, in histological analysis, the inventors observed that the engraftment rate of HyEB injected into the lower extremities of mice was higher than NorEB, and many capillary (PECAM ⁺ / DiI ⁺ ) structures derived from HyEB were observed. As shown in FIG. 7D, capillaries (PECAM ⁺ ) stained with PECAM are located between skeletal myocytes (bright image). In particular, in the ischemic lower limb tissue of the HyEB-injected group, capillaries (PECAM ⁺ / DiI ⁺ ) differentiated from injected embryos It has also been observed among skeletal muscle cells, indicating that it is well fused with existing tissues. The number was much higher than the NorEB-group (FIG. 7E). Furthermore, we found clumps formed in the lower extremities of the NorEB-injected group and metastasized in liver tissue, but no clumps in the HyEB-injected group. This suggests that the hypoxic-primed EB has a more advanced stage than the normal oxygen EB. Capillaries stained with VE-cadherin (white) and another endothelial marker, BS1-lectin (green), also appear as DiI-immunofluorescence (red) (FIG. 9), so that the implanted EB is a blood vessel in vivo . Formation differentiation is performed, showing that hypoxic primed EBs differentiate into blood vessels more efficiently than enteric oxygen EBs.

EB of Hypoxia Priming is Myocyte  Increases eruption

EB was hypoxic primed, transferred to muscle differentiation medium, and immunofluorescence staining was performed on Connexin-43 (Cx43) and N-cadherin (N-cad) 14 days after reattachment. Cx43 and N-cad were very strong in hypoxic primed EB (FIG. 11). In addition, after attaching the hypoxic primed EB to the cell culture plate, the number of beating foci (beating foci) and the number of cells was observed while culturing in the myocyte culture medium conditions. EB can differentiate into cardiac cells under myocyte culture medium, and the pulsating cell population appears, and this beating foci appears faster in hypoxic primed EB, and the number was also significantly higher (FIG. 12). ). Therefore, it was confirmed that hypoxic-priming of EB also induces efficient differentiation into muscle cells.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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<110> SNU R & DB Foundation <120> A Method for Inducing Vascular-lineage or Myogenic-lineage          Differentiation of Stem Cells <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 826 <212> PRT <213> Homo sapiens <400> 1 Met Glu Gly Ala Gly Gly Ala Asn Asp Lys Lys Lys Ile Ser Ser Glu   1 5 10 15 Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Lys              20 25 30 Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro Leu Pro His          35 40 45 Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met Arg Leu Thr Ile      50 55 60 Ser Tyr Leu Arg Val Lys Leu Leu Asp Ala Gly Asp Leu Asp Ile  65 70 75 80 Glu Asp Asp Met Lys Ala Gln Met Asn Cys Phe Tyr Leu Lys Ala Leu                  85 90 95 Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp Met Ile Tyr Ile             100 105 110 Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr         115 120 125 Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Met     130 135 140 Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys Lys Gly Lys Glu 145 150 155 160 Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys Cys Thr Leu Thr                 165 170 175 Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr Trp Lys Val Leu             180 185 190 His Cys Thr Gly His Ile His Val Tyr Asp Thr Asn Ser Asn Gln Pro         195 200 205 Gln Cys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu Val Leu Ile Cys     210 215 220 Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro Leu Asp Ser Lys 225 230 235 240 Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys Asp                 245 250 255 Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu Leu Gly             260 265 270 Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr         275 280 285 Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr Thr Gly Gln     290 295 300 Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp Val Glu Thr Gln 305 310 315 320 Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro Gln Cys Ile Val                 325 330 335 Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His Asp Leu Ile Phe             340 345 350 Ser Leu Gln Gln Thr Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp         355 360 365 Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp Thr Ser     370 375 380 Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu Thr Leu Leu 385 390 395 400 Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser Asn                 405 410 415 Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu Glu Val Pro Leu Tyr Asn             420 425 430 Asp Val Met Leu Pro Ser Pro Asn Glu Lys Leu Gln Asn Ile Asn Leu         435 440 445 Ala Met Ser Pro Leu Pro Thr Ala Glu Thr Pro Lys Pro Leu Arg Ser     450 455 460 Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Pro 465 470 475 480 Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp                 485 490 495 Gln Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser G             500 505 510 Pro Asn Ser Ser Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val         515 520 525 Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp Thr     530 535 540 Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu 545 550 555 560 Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ser                 565 570 575 Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser             580 585 590 Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Thr Gln Ile Gln         595 600 605 Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp Glu Leu     610 615 620 Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys Ile Leu Ile Ala 625 630 635 640 Ser Pro Ser Pro Thr His Ile His Lys Glu Thr Thr Ser Ala Thr Ser                 645 650 655 Ser Pro Tyr Arg Asp Thr Gln Ser Arg Thr Ala Ser Pro Asn Arg Ala             660 665 670 Gly Lys Gly Val Ile Glu Gln Thr Glu Lys Ser His Pro Arg Ser Pro         675 680 685 Asn Val Leu Ser Val Ala Leu Ser Gln Arg Thr Thr Val Pro Glu Glu     690 695 700 Glu Leu Asn Pro Lys Ile Leu Ala Leu Gln Asn Ala Gln Arg Lys Arg 705 710 715 720 Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Val Gly Ile Gly Thr                 725 730 735 Leu Leu Gln Gln Pro Asp Asp His Ala Ala Thr Thr Ser Leu Ser Trp             740 745 750 Lys Arg Val Lys Gly Cys Lys Ser Ser Glu Gln Asn Gly Met Glu Gln         755 760 765 Lys Thr Ile Ile Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly     770 775 780 Gln Ser Met Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys 785 790 795 800 Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu                 805 810 815 Glu Leu Leu Arg Ala Leu Asp Gln Val Asn             820 825 <210> 2 <211> 2481 <212> DNA <213> Homo sapiens <400> 2 atggagggcg ccggcggcgc gaacgacaag aaaaagataa gttctgaacg tcgaaaagaa 60 aagtctcgag atgcagccag atctcggcga agtaaagaat ctgaagtttt ttatgagctt 120 gctcatcagt tgccacttcc acataatgtg agttcgcatc ttgataaggc ctctgtgatg 180 aggcttacca tcagctattt gcgtgtgagg aaacttctgg atgctggtga tttggatatt 240 gaagatgaca tgaaagcaca gatgaattgc ttttatttga aagccttgga tggttttgtt 300 atggttctca cagatgatgg tgacatgatt tacatttctg ataatgtgaa caaatacatg 360 ggattaactc agtttgaact aactggacac agtgtgtttg attttactca tccatgtgac 420 catgaggaaa tgagagaaat gcttacacac agaaatggcc ttgtgaaaaa gggtaaagaa 480 caaaacacac agcgaagctt ttttctcaga atgaagtgta ccctaactag ccgaggaaga 540 actatgaaca taaagtctgc aacatggaag gtattgcact gcacaggcca cattcacgta 600 tatgatacca acagtaacca acctcagtgt gggtataaga aaccacctat gacctgcttg 660 gtgctgattt gtgaacccat tcctcaccca tcaaatattg aaattccttt agatagcaag 720 actttcctca gtcgacacag cctggatatg aaattttctt attgtgatga aagaattacc 780 gaattgatgg gatatgagcc agaagaactt ttaggccgct caatttatga atattatcat 840 gctttggact ctgatcatct gaccaaaact catcatgata tgtttactaa aggacaagtc 900 accacaggac agtacaggat gcttgccaaa agaggtggat atgtctgggt tgaaactcaa 960 gcaactgtca tatataacac caagaattct caaccacagt gcattgtatg tgtgaattac 1020 gttgtgagtg gtattattca gcacgacttg attttctccc ttcaacaaac agaatgtgtc 1080 cttaaaccgg ttgaatcttc agatatgaaa atgactcagc tattcaccaa agttgaatca 1140 gaagatacaa gtagcctctt tgacaaactt aagaaggaac ctgatgcttt aactttgctg 1200 gccccagccg ctggagacac aatcatatct ttagattttg gcagcaacga cacagaaact 1260 gatgaccagc aacttgagga agtaccatta tataatgatg taatgctccc ctcacccaac 1320 gaaaaattac agaatataaa tttggcaatg tctccattac ccaccgctga aacgccaaag 1380 ccacttcgaa gtagtgctga ccctgcactc aatcaagaag ttgcattaaa attagaacca 1440 aatccagagt cactggaact ttcttttacc atgccccaga ttcaggatca gacacctagt 1500 ccttccgatg gaagcactag acaaagttca cctgagccta atagtcccag tgaatattgt 1560 ttttatgtgg atagtgatat ggtcaatgaa ttcaagttgg aattggtaga aaaacttttt 1620 gctgaagaca cagaagcaaa gaacccattt tctactcagg acacagattt agacttggag 1680 atgttagctc cctatatccc aatggatgat gacttccagt tacgttcctt cgatcagttg 1740 tcaccattag aaagcagttc cgcaagccct gaaagcgcaa gtcctcaaag cacagttaca 1800 gtattccagc agactcaaat acaagaacct actgctaatg ccaccactac cactgccacc 1860 actgatgaat taaaaacagt gacaaaagac cgtatggaag acattaaaat attgattgca 1920 tctccatctc ctacccacat acataaagaa actactagtg ccacatcatc accatataga 1980 gatactcaaa gtcggacagc ctcaccaaac agagcaggaa aaggagtcat agaacagaca 2040 gaaaaatctc atccaagaag ccctaacgtg ttatctgtcg ctttgagtca aagaactaca 2100 gttcctgagg aagaactaaa tccaaagata ctagctttgc agaatgctca gagaaagcga 2160 aaaatggaac atgatggttc actttttcaa gcagtaggaa ttggaacatt attacagcag 2220 ccagacgatc atgcagctac tacatcactt tcttggaaac gtgtaaaagg atgcaaatct 2280 agtgaacaga atggaatgga gcaaaagaca attattttaa taccctctga tttagcatgt 2340 agactgctgg ggcaatcaat ggatgaaagt ggattaccac agctgaccag ttatgattgt 2400 gaagttaatg ctcctataca aggcagcaga aacctactgc agggtgaaga attactcaga 2460 gctttggatc aagttaactg a 2481 <210> 3 <211> 5 <212> DNA <213> Homo sapiens <400> 3 tgcag 5

Claims (16)

Composition for inhibiting the differentiation of stem cells comprising a blocker of a protein consisting of the amino acid sequence of SEQ ID NO: 1 sequence or an antibody or aptamer specifically binding to the protein as an active ingredient.
The composition of claim 1, wherein the antibody or aptamer specifically binds to a site that binds to the Oct-4 promoter reverse hypoxia element (rHRE) of the protein consisting of the amino acid sequence of SEQ ID NO: 1.
According to claim 1, wherein the protein consisting of the amino acid sequence of SEQ ID NO: 1 sequence is a composition, characterized in that binding to the reverse hypoxia element (rHRE) of the Oct-4 promoter.
The composition of claim 2 or 3, wherein the reverse hypoxia element (rHRE) comprises a nucleotide sequence of SEQ ID NO: 3.
The composition of claim 1, wherein the stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).
A composition for inducing differentiation of embryonic stem cells into mesodermal cells comprising a protein consisting of an amino acid sequence of SEQ ID NO: 1 or a nucleotide encoding a amino acid sequence of SEQ ID NO: 1 as an active ingredient.
The composition of claim 6, wherein the mesoderm cells are vascular cells or muscle cells.
8. The composition of claim 7, wherein said vascular cells are endothelial cells or smooth muscle cells.
The composition of claim 6, wherein the nucleotide encoding the amino acid sequence of SEQ ID NO: 1 is a nucleotide having a nucleotide sequence of SEQ ID NO: 2.
The composition of claim 6, wherein the protein consisting of the amino acid sequence of the first sequence of SEQ ID NO: 1 binds to the reverse hypoxia element (rHRE) of the Oct-4 promoter.
Method for producing mesodermal cells comprising the following steps:
(a) forming an embryonic body;
(b) culturing the embryoid body formed in step (a) in a hypoxia condition; And
(c) culturing the culture of step (b) in a normal oxygen condition.
12. The method of claim 11, wherein the hypoxic state has an oxygen concentration of 0-5%.
12. The method of claim 11, wherein step (b) is incubated for 24-72 hours in a hypoxic state.
The method of claim 13, wherein step (b) is incubated for 45-51 hours in a hypoxic state.
12. The method of claim 11, wherein said mesodermal cells are vascular cells or muscular cells.
The method of claim 15, wherein the vascular cells are endothelial cells or smooth muscle cells.

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