KR20130105124A - Selenium-based differentiation method of human pluripotent stem cells into hematopoietic progenitor, vascular progenitor, endothelial and smooth muscle cells - Google Patents

Abstract

PURPOSE: A method for differentiating human pluripotent stem cells using a medium containing selenium is provided to selectively differentiate human pluripotent stem cells into mesenchymal stem cells and to differentiate the human pluripotent stem cells into hematopoietic progenitor cells, vascular progenitor cells, epithelial cells, and smooth muscle cells with high efficiency. CONSTITUTION: A method for differentiating human pluripotent stem cells into mesenchymal stem cells contains a medium containing selenium. The human pluripotent stem cells are differentiated into ectoderm, mesoderm, and endoderm. The human pluripotent stem cells are selected among human embryonic stem cells, human induced pluripotent stem cells, embryonic germ cells, and embryonic carcinoma cells. The mesenchymal stem cells are differentiated into one or more kinds of cells selected among hematopoietic progenitor cells, vascular progenitor cells, epithelial cells, and smooth muscle cells.

Description

Selenium-based differentiation method of human pluripotent stem cells into hematopoietic progenitor, vascular progenitor, endothelial and smooth muscle cells using SPE cells in blood cells, vascular progenitors, endothelial cells and smooth muscle cells

The present invention relates to a method for differentiating human pluripotent stem cells using selenium, and specifically, to a method for effectively differentiating human pluripotent stem cells into blood progenitor cells, vascular progenitor cells, vascular endothelial cells, and vascular smooth muscle cells.

Stem cells are cells that can be differentiated into various cells constituting biological tissues, which collectively refer to undifferentiated cells obtained from each tissue of embryo, fetus and adult. Stem cells are differentiated into specific cells by differentiation stimulation (environment), and unlike the cells in which cell division is stopped due to differentiation, proliferation is possible due to cell division (self-renewal). (Proliferation; expansion) is characterized by being able to differentiate into other cells by different environments or different differentiation stimulation, which is characterized by having plasticity (differentiation).

These stem cells can be classified in various ways. One of the most commonly used methods is according to an individual in which stem cells are separated. Embryonic stem cells (ES cells) isolated from embryos and adult stem cells isolated from adults Can be divided into Another common classification is according to the differentiation capacity of stem cells, which can be divided into pluripotency, multipotency and unipotency stem cells. Pluripotent stem cells are pluripotency cells that have the potential to differentiate into all cells. Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPSCs). ), Embryonic germ cells (EGC), embryonic carcinoma cells (ECC), and the like. Multipotent and / or unipotent stem cells may include, for example, adult stem cells.

Embryonic stem cells are formed from the inner cell mass of the blastocytes, which are the early stages of embryonic development, and have the potential to differentiate into all cells, which can differentiate into any tissue cell and also do not die. It can be cultured in undifferentiated state, and unlike adult stem cells, it is also possible to produce germ cells, so it can be inherited to the next generation (Thomson et al., Science, 282: 1145-1147, 1998; Reubinoff et al., Nat. Biotechnol., 18: 399-404, 2000). Human embryonic stem cells are prepared by separating and culturing only inner cell mass when forming human embryos. Currently, human embryonic stem cells made worldwide are obtained from frozen embryos remaining after infertility. Meanwhile, in addition to embryonic stem cells, iPSCs are included as a concept of pluripotent stem cells. Induced pluripotent stem cells (iPSCs) are cells which have redifferentiated differentiated adult cells in various ways and returned to embryonic stem cells, which are the early stages of differentiation. To date, dedifferentiated cells have been reported to exhibit almost the same characteristics as embryonic stem cells, pluripotent stem cells, in gene expression and differentiation capacity.

As described above, human pluripotent stem cells have a pluripotency capable of differentiating into almost all somatic cells with self-renewal to constantly proliferate and maintain a constant number. The research is expected to bring radical advances in various academic fields such as embryology, regenerative medicine, and new drug development, and differentiates human pluripotent stem cells into desired cells, which can be used in various intractable patients such as diabetes, neurological diseases, and cardiovascular diseases. It is very useful in that it can be used as a therapeutic agent. In particular, an increasing number of people with ischemic cardiovascular disease and cell therapy using adult stem cells have been studied for patients who are inadequate to existing surgery or drug treatment, but have difficulty in securing a sufficient number of adult stem cells. Therefore, efforts are being actively made to differentiate human pluripotent stem cells, which have superior self-proliferation and differentiation ability to adult stem cells, into vascular endothelial cells and utilize them for treatment.

In order to use human pluripotent stem cells as a cardiovascular cell therapy, it is necessary to secure a sufficient number of differentiated cells necessary for transplantation.In order to do this, human pluripotent stem cells can be effectively induced into vascular progenitor cells or vascular endothelial cells and smooth muscle cells. Skill is essential.

In this regard, human embryonic stem cells can be differentiated into vascular endothelial cells using coculture with mouse-derived stromal cells, or using fetal bovine serum (FBS) or human recombinant growth factor / cytokines. It has been reported how. Among them, differentiation using co-culture with mouse bone marrow stromal matrix was performed by co-culture with human embryonic stem cells and 10% FBS culture using mouse bone marrow-derived stromal cells as a feeder layer. As a separate method, isolated KDR-positive cells can be further differentiated in culture containing 10% FBS and vascular endothelial growth factor A (VEGF) to obtain vascular endothelial cells (Pathway for differentiation of human embryonic stem cells to vascular cell components). and their potential for vascular regeneration.Sone M, Itoh H, Yamahara K, Yamashita JK, Yurugi-Kobayashi T, Nonoguchi A, Suzuki Y, Chao TH, Sawada N, Fukunaga Y, Miyashita K, Park K, Oyamada N, Sawada N , Taura D, Tamura N, Kondo Y, Nito S, Suemori H, Nakatsuji N, Nishikawa S, Nakao K. Arterioscler Thromb Vasc Biol. 2007 Oct; 27 (10): 2127-34).

In 2007, MIT's Robert Langer team reported how to induce differentiation of human embryonic stem cells into vascular cells using bovine serum. The method produces a three-dimensional embryonic body (EB) of human embryonic stem cells, incubates the embryonic body in a culture medium containing 20% FBS for 12 days, and isolates CD34 positive cells using a flow cytometer. In order to differentiate into vascular endothelial cells, CD31 positive vascular endothelial cells were obtained by incubating for another 10 to 15 days in EGM-2 (endothelial growth medium-2) to which 5% FBS was added (Vascular Progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle-like cells and form vascular networks in vivo.Ferreira LS, Gerecht S, Shieh HF, Watson N, Rupnick MA, Dallabrida SM, Vunjak-Novakovic G, Langer R. Circulation Research 2007 101: 286-94).

In addition, in 2007, Scadden's team reported a method of culturing human embryonic stem cells in a two-dimensional monolayer instead of three-dimensional embryoid bodies to differentiate into vascular endothelial cells. The method also induced differentiation of CD31 positive vascular endothelial cells by differentiating CD34 positive cells using 15% FBS and culturing for 7-10 days in EGM-2 added with 5% FBS (endothelial cells derived from human embryonic stem). cells form durable blood vessels in vivo.Wang ZZ, Au P, Chen T, Shao Y, Daheron LM, Bai H, Arzigian M, Fukumura D, Jain RK, Scadden DT.Nature Biotechnology 2007; 25: 317-8).

As described above, the conventional method of differentiating human embryonic stem cells into vascular cells uses co-culture with bovine serum or animal-derived cells, and thus in the clinical application of vascular endothelial cells differentiated from human embryonic stem cells. There are problems that can result in contamination by the source pathogen (eg, possible bacterial and viral infections) and thereby the patient's immune response.

In order to overcome the above problems, researches on new differentiation methods that do not include serum and animal sources have been actively conducted in recent years. Specifically, human recombinant growth factor / cytokines required for differentiation into vascular cells, in particular human recombinant vascular endothelial growth factor A, human recombinant bone morphogenic protein-4, human Using serum-free and xeno-free media containing recombinant recombinant fibroblast growth factor (bFGF), or human recombinant Activin A We tried to secure the safety of clinical application.

However, in order to use vascular cells differentiated from human pluripotent stem cells as a cardiovascular cell therapy, it is most important to improve the differentiation methods developed to date to increase the differentiation efficiency and secure sufficient number of differentiated cells for transplantation. .

In order to solve the problems of the prior art, the present inventors conducted various studies to develop a technique for improving the efficiency of vascular endothelial cell differentiation method that does not use serum and animal-derived materials or animal-derived cells, and as a result selenium Using human recombinant growth factor / cytokine medium comprising the human pluripotent stem cells were found to be able to differentiate into high efficiency blood progenitors, vascular progenitors, vascular endothelial and vascular smooth muscle cells and came to complete the present invention.

Accordingly, the present invention selectively differentiates human pluripotent stem cells into mesoderm using selenium-added human recombinant growth factor / cytokine differentiation medium to increase the differentiation efficiency into blood and vascular progenitor cells or vascular endothelial and smooth muscle cells. It is an object to provide a method.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a method for differentiating human pluripotent stem cells into mesodermal stem cells using a medium containing selenium (selenium). Human pluripotent stem cells used in the present invention include human embryonic stem cells, human induced pluripotent stem cells, embryonic germ cells, and embryonic tumor cells.

In an embodiment of the present invention, the mesoderm stem cells may be differentiated into blood progenitor cells, vascular progenitor cells, vascular endothelial cells, and vascular smooth muscle cells.

In another embodiment of the present invention, the medium may further contain human recombinant growth factor or cytokine. The human recombinant growth factor or cytokine used in the present invention is not limited, but may include human recombinant BMP-4, human recombinant bFGF, and / or human recombinant VEGF.

When using the differentiation medium containing selenium according to the method of the present invention, there is an effect that can selectively differentiate human pluripotent stem cells into mesodermal stem cells, ultimately blood precursor cells, vascular precursor cells, vascular endothelial cells with high efficiency Cells, vascular smooth muscle cells and the like. Such excellent efficient differentiation induction technology of the present invention is essential for the practical application of human pluripotent stem cells as a cardiovascular disease cell therapy, the present invention is expected to contribute greatly to the practical use of cardiovascular cell therapy. In addition, selenium promotes the differentiation of hematopoietic progenitor cells, CD34-positive cells, into the treatment of malignant diseases such as immunodeficiency diseases, aplastic anemia, hemoglobin disease, myeloid leukemia, and malignant lymphoma that can be treated by hematopoietic stem cell transplantation. It is expected to contribute greatly.

1 is a graph illustrating the gene expression patterns of cells in the selenium-treated group (white) and the treated group (black: selenium 20 ng / ml, gray: selenium 50 ng / ml) during differentiation of human embryonic stem cells. As a result, real-time RT-PCR of the gene expression of cells before and after differentiation (H9) and 8 and 15 days after differentiation was shown in real time (A: undifferentiated human embryonic stem cell marker gene, B: mesoderm specific marker gene). , C: endoderm specific marker gene, D: ectoderm specific marker gene).
Figure 2 shows the results of immunofluorescence staining of the cell differentiation in the selenium-treated group and the treated group (50 ng / ml) during the differentiation of human embryonic stem cells, 9 and 15 days after differentiation, respectively. Cell differentiation into mesoderm, endoderm, and ectoderm are shown (CD34: mesoderm marker, AFP (a-fetoprotein): endoderm marker, b III-tubulin: ectoderm marker).
Figure 3 shows the gene expression patterns of cells in the selenium-free group (white) and the treated group (black: selenium 20 ng / ml, gray: selenium 50 ng / ml) during the differentiation of human embryonic stem cells As a result, gene expression of cells 8 and 15 days after differentiation (H9) and after differentiation was confirmed by real-time RT-PCR. EC (endothelial cell) and SMC (smooth muscle cell) were used as positive controls for the expression of vascular endothelial cells and vascular smooth muscle cell specific genes respectively (A: vascular endothelial cell specific marker gene, B: vascular smooth muscle cell specific marker gene) ).
Figure 4 is an immunofluorescence staining showing the differentiation of vascular endothelial cells and vascular smooth muscle cells in the group treated with selenium (50 ng / ml) during the differentiation process of human embryonic stem cells (vascular endothelial cell specific markers : CD31, VE-cad, vascular smooth muscle cell specific marker: SMA).
5 is a flow cytometry analysis of the differentiation of human embryonic stem cells into vascular precursors and vascular endothelial cells in the selenium-treated group and the treated group (20 or 50 ng / ml) during the differentiation of human embryonic stem cells. One result is shown. Cells recovered after 15 days of differentiation from human embryonic stem cells were reacted with CD31 (vascular endothelial cell specific marker) and CD34 (vascular progenitor cell specific marker) antibodies for flow cytometry.

As used herein, the term 'stem cells' refers to master cells that can be regenerated 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 cells, or one daughter cell and one derived ('transit') cell, and then proliferate into mature, fully formed cells of the tissue. As used herein, the term 'pluripotent stem cell' refers to a stem having pluripotency capable of differentiating into three germ layers constituting a living body, that is, endoderm, mesoderm, and ectoderm. It refers to cells, and generally corresponds to embryonic stem cells, induced pluripotent stem cells, embryonic germ cells, embryonic tumor cells.

As used herein, the term 'embryonic stem cell' is a cell cultured by separating and cultured from an inner cell mass (inner cell mass) of the blastocyst, which is an early development after fertilization. Refer. As used herein, the term 'adult cell' refers to a cell derived from an adult that is born and alive as opposed to an embryonic cell. As used herein, the term 'induced pluripotent stem cell (iPSC)' is a cell induced by artificially dedifferentiating (reprogramming) the already differentiated adult cells to the pluripotency (pluripotency) Have The term 'embryonic germ cell' used in the present invention refers to a cell derived from primordial germ cells occurring in the gonadal ridge region of the fetus and has characteristics similar to embryonic stem cells but embryonic stem. It has weaker differentiation and division ability than cells. The term 'embryonic tumor cell' used in the present invention is an undifferentiated stem cell established from teratocarcinomas formed by the tumorization of primordial germ cells, and the main material for multipotent research before embryonic stem cells are established. Was used. Embryonic tumor cells, although having problems such as chromosomal abnormalities, have pluripotency.

As used herein, the term "differentiation" refers to a phenomenon in which structures or functions are specialized to each other during cell division and proliferation. It means to change. In general, a relatively simple system is separated into two or more qualitatively different systems. For example, there may be qualitative or quantitative differences between parts of a biological system that were initially homogeneous, such as head or trunk distinction between eggs that were initially homogeneous in origin, or distinctions of cells such as muscle cells or neurons The state in which there is a difference or as a result is divided into qualitative or inferior parts or partial systems is called differentiation.

As used herein, the term 'cell therapeutic agent' is a cell and tissue prepared by separating, culturing and special manipulation from a human being and used as a medicine used for the purpose of treatment, diagnosis and prevention, and to restore the function of the cell or tissue. Or a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise modifying a cell's biological properties in vitro. Cell therapy drugs are classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  One. selenium  Differentiation of Human Pluripotent Stem Cells Using Containing Medium

<Step I>

Human embryonic stem cells (H9, WiCell, USA) in undifferentiated state were removed using a cell detachment solution (ReproCell, Japan) according to a known method (Takahashi K et al. Cell (2007) 131 , 861-872). Formed.

The embryoid body obtained from the above was human recombinant BMP-4 (10 ng / ml, Prospec), human recombinant bFGF (5 ng / ml, Prospec), human recombinant VEGF (10 ng / ml, R & D), sodium selenite (20-50). ng / ml, Sigma), and DMEM / F12 (Gibco, composition: http://www.atcc.org/Portals/1/Pdf/30-) supplemented with 10% serum replacement (Knockout ™ Serum Replacement xenofree, Gibco) 2006.pdf) was suspended in the low oxygen partial pressure (3% oxygen concentration) conditions for 8 days. In the control group, sodium selenite was differentiated under the same conditions without addition to the medium.

<Step II>

The embryoid body differentiated in step I was transferred to a culture plate using a pipette, attached to the dish bottom, and then human recombinant BMP-4 (20 ng / ml, Prospec) and human recombinant bFGF (5 ng / ml , Prospec), endothelial cell Basal Medium-2 (Lonza) supplemented with human recombinant VEGF (50 ng / ml, R & D), sodium selenite (20-50 ng / ml, Sigma) Ie, 20% oxygen concentration) for an additional 7 days. In the control group, sodium selenite was differentiated under the same conditions without addition to the medium.

Example  2. selenium  Confirmation of mesoderm-related gene expression increase by treatment

Cells were harvested after 8 and 15 days of differentiation from Example 1, and the differentiation patterns into ectoderm, endoderm, and mesoderm were examined by real-time RT-PCR.

Specifically, RNA of the cell was extracted using Trizol (Invitrogen), and expression of genes was observed using Real-time PCR. To this end, 1 μg of RNA was first synthesized into cDNA using a Superscript first-strand synthesis system (Invitrogen), and the amplified gene expression was amplified using the primers and real time PCR SYBR-Green PCR master mix (Applied Biosystems) of each gene. Measured by Step One PlusTM Real-time PCR system (Applied Biosystems), the results are shown in FIG.

At this time, the primers used are shown in Table 1 below.

Cover factor order SEQ ID NO: Oct4 forward 5'-gacagggggaggggaggagctagg-3 ' One reverse 5'-cttccctccaaccagttgccccaaac-3 ' 2 Nanog forward 5'-tttggaagctgctggggaag-3 3 reverse 5'-gatgggaggaggggagagga-3 ' 4 Brachyury forward 5'-acccagttcatagcggtgac-3 ' 5 reverse 5'-ccattgggagtacccaggtt-3 ' 6 Mesp1 forward 5'-ctcgtctcgtccccagactc-3 ' 7 reverse 5'-gcagtttctcccgctcactg-3 ' 8 CD34 forward 5'-tggaccgcgctttgct-3 ' 9 reverse 5'-ccctgggtaggtaactctggg-3 ' 10 Sox17 forward 5'-cgctttcatggtgtgggctaaggacg-3 ' 11 reverse 5'-tagttggggtggtcctgcatgtgctg-3 ' 12 Gata6 forward 5'-taattccattcccatgactc-3 ' 13 reverse 5'-cctatgtagagcccatcttg-3 ' 14 Pax6 forward 5'-tgtccaacggatgtgtgagt-3 ' 15 reverse 5'-tttcccaagcaagagtggac-3 ' 16 Nestin forward 5'-cctgtcagaagaatttgagg-3 ' 17 reverse 5'-actttcttcctcatctgcaa-3 ' 18 CD31 forward 5'-aggtgttggtggaaggagtg-3 ' 19 reverse 5'-cgtgtagttgccactgtgct-3 ' 20 VEcadherin forward 5'-cggtcaaactgcccatactt-3 ' 21 reverse 5'-cagcccaaagtgtgtgagaa-3 ' 22 SMA forward 5'-agaacatggcatcatcacca-3 ' 23 reverse 5'-tacatggctgggacattgaa-3 ' 24 Myocardin forward 5'-ctgctgtaaagtccaaatcc-3 ' 25 reverse 5'-taggtagctgaatcggtgtt-3 ' 26 GAPDH forward 5'-aagggtcatcatctctgccc-3 ' 27 reverse 5'-gtgatggcatggactgtggt-3 ' 28

As a result, as shown in FIG. 1, the expression of Oct4 and Nanog genes, which are markers of undifferentiated stem cells, was significantly reduced during differentiation compared to undifferentiated human embryonic stem cells (H9), whereas ectoderm (Pax6, Nestin), Mesoderm (Brachyury, Mesp1, CD34), endoderm (Sox17, Gata6) It can be seen that the expression of various genes are increased. In particular, it can be seen that the expression of mesoderm marker genes is significantly increased when using a medium containing selenium during differentiation compared to ectoderm and endoderm.

Example  3. selenium  Selective Differentiation of Human Pluripotent Stem Cells into Mesodermal Progenitor Cells by Treatment

To verify that Selenium selectively increases mesodermal differentiation of human pluripotent stem cells, immunofluorescence staining using CD34, alpha-fetoprotein (AFP), or bIII-tubulin antibody was performed on 9 and 15 days of differentiation.

Specifically, the differentiated cells obtained from Example 1 were fixed in 4% paraformaldehyde for 10 minutes and reacted for 10 minutes at 0.5% Triton X-100. Block in phosphate buffered saline (PBS) containing 10% goat serum for 30 minutes and block CD31, VE-cadherin, SMA, CD34, AFP, or bIII-tubulin primary The antibody was reacted at 4 ° C. for 24 hours. After the reaction, the resultant was washed with PBS, and reacted with fluorescent labeled secondary antibody at room temperature for about 2 hours, followed by washing with PBS. Finally, the nuclei were stained with 4,6-diamino-2-phenylindole (DAPI) and observed using a Nikon ECLIPSE Ti-U inverted microscope (Nikon Instruments Inc.).

As a result, as shown in Figure 2, the number of CD34 (blood and vascular precursor cell-specific marker) positive cells was significantly increased by selenium treatment, while selenium treatment was positive for AFP (endodermal cell marker) or It can be seen that the number of bIII-tubulin (ectoderm cell marker) positive cells does not increase.

The results indicate that the selective differentiation of human pluripotent stem cells into mesodermal cells containing CD34 positive cells is promoted by selenium.

Example  4. selenium  Endothelial and Endothelial of Human Pluripotent Stem Cells by Treatment Vascular Smoothing Confirmation of gene expression promotion to myocytes

Cells after 8 and 15 days of differentiation obtained from Example 1 were collected and examined for differentiation into vascular endothelial cells and vascular smooth muscle cells through real-time RT-PCR. RT-PCR method was performed in the same manner as in Example 2.

As a result, as shown in FIG. 3, the expression of PECAM (CD31) and VE-cadherin genes, which are vascular endothelial cell-specific markers, as well as specific markers of vascular smooth muscle cells during differentiation compared to undifferentiated human embryonic stem cells (H9) It can be seen that the expression of SMA and Myocardin genes are also increased.

In particular, in the case of using a medium containing selenium during differentiation, the expression of PECAM, VE-cadherin, SMA, and Myocardin genes was significantly increased after 15 days of differentiation compared with the control group not treated with selenium.

Example  5. selenium  Endothelial and Endothelial of Human Pluripotent Stem Cells by Treatment Vascular Smoothing Confirmation of selective differentiation into myocytes

To verify that Selenium selectively increases the differentiation of human pluripotent stem cells into vascular endothelial and vascular smooth muscle cells, immunofluorescence staining with CD31, VE-cadherin, or SMA antibodies was performed on cells 9 and 15 of differentiation. And the result is shown in FIG.

As a result, the number of CD31 and VE-cadherin (vascular endothelial specific marker) positive cells was significantly increased by selenium (50 ng / ml) treatment, and SMA (vascular smooth muscle cell specific marker) by selenium (50 ng / ml) treatment. ) The number of positive cells also increased significantly.

From the above results, it can be seen that selenium promotes the selective differentiation of human pluripotent stem cells into vascular endothelial and vascular smooth muscle cells.

In addition, in order to quantitatively confirm the immunofluorescence staining results, cells were harvested from human embryonic stem cells at day 15 of differentiation and subjected to flow cytometry (FACS, Aria flow cytometry, BD Bioscience). 5 is shown.

Specifically, the cells were separated by treatment with Accutase (Innovative cell technologies, Inc.) for 15 minutes, and then reacted with an antibody (anti-human CD31, CD34 antibody) having a complex of fluorescent materials (anti-human CD31, CD34 antibody) at 4 ° C. for 1 hour, followed by PBS (phosphate buffered). saline).

As a result, as shown in Figure 5, the number of CD34 positive cells, blood and vascular progenitor cell-specific markers among the cells on the 15th day of differentiation, 3.6% of the total is increased by about 5% by selenium treatment. In addition, it can be seen that the number of CD31 positive cells, vascular endothelial cell specific markers, increased to 19-20% by selenium treatment.

The results indicate that selenium selectively increases the differentiation of human pluripotent stem cells into blood progenitors, vascular progenitors, vascular endothelial and vascular smooth muscle cells.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Selenium-based differentiation method of human pluripotent stem          cells into hematopoietic progenitor, vascular progenitor,          endothelial and smooth muscle cells <130> PB12-10439 <160> 28 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oct4 forward primer <400> 1 gacaggggga ggggaggagc tagg 24 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Oct4 reverse primer <400> 2 cttccctcca accagttgcc ccaaac 26 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog forward primer <400> 3 tttggaagct gctggggaag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog reverse primer <400> 4 gatgggagga ggggagagga 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Brachyury forward primer <400> 5 acccagttca tagcggtgac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Brachyury reverse primer <400> 6 ccattgggag tacccaggtt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mesp1 forward primer <400> 7 ctcgtctcgt ccccagactc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mesp1 reverse primer <400> 8 gcagtttctc ccgctcactg 20 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> CD34 forward primer <400> 9 tggaccgcgc tttgct 16 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD34 reverse primer <400> 10 ccctgggtag gtaactctgg g 21 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Sox17 forward primer <400> 11 cgctttcatg gtgtgggcta aggacg 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Sox17 reverse primer <400> 12 tagttggggt ggtcctgcat gtgctg 26 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gata6 forward primer <400> 13 taattccatt cccatgactc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gata6 reverse primer <400> 14 cctatgtaga gcccatcttg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pax6 forward primer <400> 15 tgtccaacgg atgtgtgagt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pax6 reverse primer <400> 16 tttcccaagc aagagtggac 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nestin forward primer <400> 17 cctgtcagaa gaatttgagg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nestin reverse primer <400> 18 actttcttcc tcatctgcaa 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD31 forward primer <400> 19 aggtgttggt ggaaggagtg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD31 reverse primer <400> 20 cgtgtagttg ccactgtgct 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> 223 VEcadherin forward primer <400> 21 cggtcaaact gcccatactt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEcadherin reverse primer <400> 22 cagcccaaag tgtgtgagaa 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMA forward primer <400> 23 agaacatggc atcatcacca 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMA reverse primer <400> 24 tacatggctg ggacattgaa 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myocardin forward primer <400> 25 ctgctgtaaa gtccaaatcc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myocardin reverse primer <400> 26 taggtagctg aatcggtgtt 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 27 aagggtcatc atctctgccc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 28 gtgatggcat ggactgtggt 20

Claims (6)

Method of differentiating human pluripotent stem cells into mesodermal stem cells using a medium containing selenium. The method of claim 1,
The human pluripotent stem cells are characterized in that the cells are differentiated into ectoderm, mesoderm, and endoderm. The method of claim 1,
Wherein said human pluripotent stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, embryonic germ cells, and embryonic tumor cells. The method of claim 1,
The mesoderm stem cells are characterized in that differentiation into one or more cells selected from the group consisting of blood progenitor cells, vascular progenitor cells, vascular endothelial cells and vascular smooth muscle cells. The method of claim 1,
The medium is characterized in that it further contains a human recombinant growth factor, cytokine, or serum. 6. The method of claim 5,
Wherein said human recombinant growth factor or cytokine is at least one selected from the group consisting of human recombinant BMP-4, human recombinant bFGF and human recombinant VEGF.

Images