KR101550269B1 - A method for direct dedifferentiation of hematopoietic stem cell from fibroblast cell - Google Patents

Abstract

The present invention relates to a method for inducing direct de-differentiation from fibroblasts to hematopoietic stem cells, and more particularly, to a method for inducing direct de-differentiation of fibroblasts from hematopoietic stem cells, Induced hematopoietic stem cells, and the differentiation-induced hematopoietic stem cells are differentiated into lymphoid cells and myeloid cells. Thus, the hematopoietic stem cell direct depletion induction method Can be usefully utilized in patient-specific cell therapy.

Description

[0001] The present invention relates to a direct method for direct dedifferentiation of hematopoietic stem cells from fibroblasts to hematopoietic stem cells,

The present invention relates to a method for directly inducing the differentiation of a hematopoietic stem cell into a hematopoietic stem cell through overexpression of a differentiation inducing gene in a fibroblast cell and a method for inducing the differentiation induced hematopoietic stem cell To a method of differentiating into an immune cell.

Based on scientific research, stem cells are increasingly recognized for their therapeutic potential in many areas, including metabolic, degenerative and inflammatory diseases, cancer, and repair of damaged tissues. In order to apply to various therapeutic application fields, attempts have been made to obtain stem cells from various human tissues and then to cultivate / differentiate / process the stem cells in various ways to finally apply them to patients.

Stem cells can be roughly classified into embryonic stem cells, adult stem cells, and recently reported degenerative stem cells. They are able to differentiate into cells that constitute the human body, and they are expected to play an important role as tools for treating incurable diseases. Embryonic stem cells are stem cells derived from the inner cell mass of a frozen embryo. It is extracted from frozen embryos to be discarded and has no legal problems. It is excellent in self-renewal ability and can supply a large amount of cells. However, since embryonic stem cells are extracted from embryos, they are subject to debate about bioethics in terms of destruction of life (Murry and Keller, 2008; Watt and Hogan, 2000). Adult stem cells exist in various organs or tissues of animals and are actually stem cells that can be separated from various tissues such as the adult bone, blood, placenta, and have no legal or ethical problems. In addition, extraction by taking into account tissue compatibility can eliminate the problem of immune rejection. However, adult stem cells have difficulty in securing enough cells because of inadequate in vitro proliferation, and have a problem in that they are less able to differentiate than embryonic stem cells (Watt and Hogan, 2000; Zaehres and Scholer, 2007). In contrast, the stem cell line is a stem cell made from adult cells using a de-differentiation factor (Lowry et al., 2008; Maherali et al., 2007; Nakagawa et al., 2008; Okita et al., 2007; 2006; Wernig et al., 2007; Yu et al., 2007). Degenerated stem cells are produced with differentiated adult cells, so there is no legal or ethical problem, and it has an advantage of being able to supply a large amount of cells because of its excellent self-reproduction ability. In addition, it is expected to be superior in differentiation ability similar to embryonic stem cells. Therefore, it is highly likely to be used for cell therapy, and since it is a cell extracted from itself, there is no problem about immune rejection.

Hematopoietic stem cells (HSCs) or hematopoietic stem cells (hematopoietic stem cells) are potential components of blood and are capable of differentiating themselves and have the ability to self-replicate and differentiate into all blood cells (Till, JE et al., 1961, Radiation research 14 , 213-222 .; Zon, LI, 2008, Nature 453 , 306-313). Hematopoietic stem cells or hematopoietic stem cells can be differentiated into cells (monocytes, macrophages, neutrophils, basophils, erythrocytes, platelets, etc.) and lymphatics (T cells, B cells, NK cells) produced in the bone marrow. However, hematopoietic stem cells have a small number of 0.05-0.1% of total bone marrow cells, and it is difficult to proliferate by in vitro culture (Spangrude, GJ et al., 1988, Science 241 , 58-62) , There are limitations in obtaining a large number of cells.

Recently, the development of induced pluripotent stem cell (iPS) technology developed by Yamanaka et al. Has enabled the development of degenerated stem cells to be applied in many disease treatments and regenerative medicine (Takahashi, K. et al., 2006, Cell 126 , 663-676). However, this stellate stem cell has a difficulty in practical use because it is actually induced by cancer (Ring, KL et al., 2012, Cell Stem Cell 11 , 100-109). On the other hand, making hematopoietic stem cells into de-differentiation technology can produce stem cells that do not have side effects such as cancer induction.

As a result, the inventors of the present invention have made efforts to find a method of producing hematopoietic stem cells capable of producing a safe, undifferentiated hematopoietic stem cell. As a result, it has been found that the overexpression of the gene for inducing differentiation induction from mouse embryo fibroblast cell (MEF) induced hematopoietic stem cell to differentiate into lymphoid cells and myeloid cells by inducing direct differentiation into hematopoietic stem cells having multipotency and inducing differentiation into hematopoietic stem cells from the fibroblasts, The present invention has been completed based on the finding that direct induction of cell differentiation into cells is useful for safe and rapid patient-specific cell therapy.

It is an object of the present invention to provide a direct induction method of dedifferentiation from a fibroblast cell to a hematopoietic stem cell (HSC).

Another object of the present invention is to provide a method of differentiating into natural killer (NK) cells in induced hematopoietic stem cell (iHSC) induced by differentiation according to the method of the present invention.

It is another object of the present invention to provide a method of differentiating hematopoietic stem cells into lymphoid cells or myeloid cells by the method according to the present invention.

In order to achieve the object of the present invention,

1) transforming a vector containing a dedifferentiation inducing gene into a fibroblast cell; And

2) a step of directly transforming fibroblasts into hematopoietic stem cells (HSCs), comprising culturing the transformant of step 1) and isolating the colonies.

In addition,

1) preparing an induced hematopoietic stem cell (iHSC) induced by differentiation according to the method of the present invention;

2) inducing differentiation into natural killer precursor cells by adding a natural killer (NK) cell precursor inducing agent to the hematopoietic stem cells derived in step 2); And

3) Differentiation The method of differentiation from the differentiation-induced hematopoietic stem cells into natural killer cells, including the step of treating natural killer precursor cells with IL-15 (interleukin-15) to differentiate into natural killer cells to provide.

In addition,

1) preparing hematopoietic stem cells which are induced to differentiate by the method according to the present invention; And

2) a step of differentiating the hematopoietic stem cells derived from the differentiation induced in step 1), thereby providing a method of differentiating hematopoietic stem cells into lymphoid cells or myeloid cells.

The present invention also relates to a method for producing an expression vector comprising an expression vector comprising any one or two or more degeneration inducing gene combinations selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, LOM2, c-Fos and c- And provides an agent for direct de-differentiation of fibroblasts into hematopoietic stem cells.

In addition, the present invention provides a cell therapy agent containing the differentiation-induced hematopoietic stem cells prepared by the method according to the present invention.

The direct induction of the differentiation of fibroblasts into hematopoietic stem cells of the present invention can be carried out by introducing genes Oct3 / 4, Sox2, Klf4 and c-Myc, which are important for pluripotency maintenance from mouse embryo fibroblast cells (MEF cells) Induced hematopoietic stem cells (HSCs) through overexpression of genes LMO2, c-Fos and c-Myb, which are important for the development of hematopoietic stem cells (HSCs) And can be usefully used for patient-specific cell therapy.

FIG. 1A shows a candidate gene selected to induce the differentiation of a hematopoietic stem cell (HSC) from a mouse embryo fibroblast cell (MEF cell).
Fig. 1B shows the expression of the decellularization candidate genes c-Myc, c-Jun, LMO2, c-Fos, c-Myb and Trim28 selected from hematopoietic stem cells.
HSC: hematopoietic stem cells;
pNK: premature natural killer cell;
mNK: mature natural killer cell;
MEF: mouse embryonic fibroblast; And
ES: Embryonic stem cells.
Fig. 1C is a graph showing the relationship between the differentiation candidate genes Oct3 / 4 (O), Sox2 (S), Klf4 (K), c-Myc (M), c- Jun (J), LMO2 Fig. 5 shows the number of colonies of induced hematopoietic stem cells (iHSC) derived from c-Myb (B) combination.
FIG. 2A shows the morphology of the induced differentiation-induced hematopoietic stem cell (iHSC)
pMXs: negative control;
iHSC: Reverse differentiation induced hematopoietic stem cells;
HSC: hematopoietic stem cells; And
iPS: induced pluripotent stem cells.
FIG. 2B is a graph showing the hematopoietic stem cell surface markers (CD34 ^- Flk2 ^- CD150 ⁺ CD48 ^- ) analysis results of the differentiation-induced hematopoietic stem cells.
FIG. 2C is a graph showing the hematopoietic stem cell surface marker analysis results of the differentiation-induced hematopoietic stem cell colonies # 106, # 13, # 110, # 103, # 19 and # 29.
FIG. 3A is a graph showing the effect of c-Kit and Sca-1 on hematopoietic stem cell colonies # 103, # 105, # 106, # 109 and # 110, hematopoietic stem cells and embryonic stem cells (ES) Fig. 3 is a diagram showing the expression pattern of the gene.
3B shows the gene expression patterns of c-Kit and Sca-1 in the differentiation-induced hematopoietic stem cell colonies # 101, # 116, # 117, # 118, # 120, # 121, # 103, # 104 and # 110 Fig.
FIG. 3c is a graph showing the protein expression patterns in the differentiation-induced hematopoietic stem cells, hematopoietic stem cells, and induced pluripotent stem cells (iPS).
FIG. 3D is a diagram showing the expression patterns of transcription factors in hematopoietic stem cell colonies # 103, # 104 and # 106 induced by differentiation, hematopoietic stem cells and embryonic stem cells.
FIG. 4A is a graph showing the ability of hematopoietic stem cells in vivo to differentiate into lymphoid cells.
4B is a graph showing the ability of hematopoietic stem cells derived from differentiation in vivo to differentiate into myeloid cells.
5A is a graph showing the ability of the hematopoietic stem cell colonies # 10, # 108, # 13, # 110 and # 106 induced in vitro to differentiate into lymphoid cells (CD3e ^- NK1.1 ⁺ NK cells) to be.
5B is a graph showing cytotoxicity of natural killer (NK) cells differentiated from the differentiation induced hematopoietic stem cell colonies # 543, # 554 and # 530.
5c is a graph showing the ability of the hematopoietic stem cell colonies # 600, # 602, and # 605 derived from in vitro differentiation to myeloid cells (Mac-1 ⁺ Gr-1 ⁺ ).
5D is a diagram showing the colony forming capacity of the differentiation-induced hematopoietic stem cell colonies # 106, # 110, # 114 and # 118:
CFU-G: granulocyte;
CFU-M: macrophage lineage; And
CFU-GM: granulocyte macrophage.
FIG. 6A is a graph showing gene expression patterns of the time-specific differentiation-activity-related factors in the differentiation-induced hematopoietic stem cells.
FIG. 6B is a graph showing gene expression patterns of markers of hematopoietic stem cells over time in the differentiation-induced hematopoietic stem cells.

Hereinafter, terms used in the present invention will be described.

In the present invention, the term "hematopoietic stem cell (HSC)" is a cell having the ability to produce fully differentiated hematopoietic cells and self-replicating ability. Typically, it does not produce progeny cells of other embryonic gonads when cultured as such in vitro, unless dedifferentiated or reprogrammed in some way.

In the present invention, the term " dedifferentiation "refers to a phenomenon in which the differentiation of cells is reversed, and mature cells resume dividing activity again. In other words, it refers to a case where the process of cell development from a stem cell to a general cell is reversed.

Hereinafter, the present invention will be described in detail.

The present invention

1) transforming a vector containing a dedifferentiation inducing gene into a fibroblast cell; And

2) a step of directly transforming fibroblasts into hematopoietic stem cells (HSCs), comprising culturing the transformant of step 1) and isolating the colonies.

In this method, the fibroblast of step 1) is preferably, but not limited to, a human or mouse embryo fibroblast cell (MEF).

In the above method, the vector of step 1) is preferably a recombinant viral vector, but is not limited thereto. In addition, the recombinant viral vector is preferably a retrovirus, an adenovirus, a herpes simplex virus and a lentivirus, more preferably a retrovirus, But is not limited thereto.

In this method, the reprogramming induction gene of step 1) is preferably any one or a combination of two or more selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, LOM2, c-Fos and c- But is not limited thereto.

In this method, the induced hematopoietic stem cell (iHSC) derived from step 2) is preferably Lineage (Lin) ^- c-Kit ⁺ Sca-1 ⁺ cells but not limited thereto. In addition, gene expression of the hematopoietic stem cells induced by differentiation is expressed in a manner similar to hematopoietic stem cells.

 In a specific embodiment of the present invention, the present inventors have conducted a serial analysis of gene expression (hereinafter, referred to as " gene expression analysis ") to select candidate genes for inducing regeneration from embryo fibroblast cells to hematopoietic stem cells (HSC) SAGE), and the expression of c-Myc, c-Jun, LOM2, c-Fos, c-Myb and Trim28 among the candidate genes selected in HSC was increased 1a and 1b). (L), c-Fos (F), and c (F), which are important for the development of hematopoietic stem cells, can be obtained by transforming retrovirus vectors containing the selected candidate genes into fibroblasts and confirming colony formation (O), Sox2 (S), Klf4 (K), and c-Myc (M), which play important roles in the maintenance of pluripotency of embryonic stem cells ) Gene, OSKMLFB, was found to be the optimal regeneration condition for hematopoietic stem cells (see Fig. 1C).

In order to confirm the characteristics of the hematopoietic stem cell (iHSC) derived from the fibroblast-derived hematopoietic stem cell (iHSC) under the condition of the optimal regenerating gene combination, the inventors performed morphological analysis and fluorescent activity After performing a cell sorter (FACS) analysis, morphological a round of small shape indicates a shape similar to the hematopoietic stem ^{cells, Lineage (Lin) - c-} Kit + Sca-1 + (LKS) cell clusters, long-term cell clusters ( long-term population, LT-population, short-term population (ST-population) and multi-potent progenitor cells, the combination of the OSKMLFB gene from fibroblasts with hematopoietic stem cells (Figure 2A-2C). ≪ / RTI >

In addition, the present inventors have found that in the condition of the optimal regeneration gene combination, hematopoietic stem cells (iHSC), hematopoietic stem cells (HSC), embryonic stem cells (ES) and induced pluripotent stem Real-time PCR, Western blotting, immunostaining and RT-PCR were performed to compare the expression of genes and proteins between cells and iPS cells. As a result, The expression of c-Kit and Sca-1 gene, which are surface markers of hematopoietic stem cells, is increased in the induced hematopoietic stem cells (iHSC) and the expression of transcription factors specifically expressed in hematopoietic stem cells is increased, It was confirmed that the hematopoietic stem cells derived from the differentiation had molecular characteristics similar to those of the hematopoietic stem cells (see Figs. 3A to 3C).

In order to confirm the differentiation ability of hematopoietic stem cells derived from the fibroblasts in vivo, the present inventors used hematopoietic stem cells to inject hematopoietic stem cells and obtain bone marrow FCS analysis showed that LKS cells, natural killer (NK) cells, B cells and T cells and MAC-a ⁺ Gr-1 ⁺ cells, which are myeloid cells, The hematopoietic stem cells were able to regulate hematopoietic cell system (FIGS. 4A and 4B).

In order to confirm the differentiation ability of hematopoietic stem cells derived from the fibroblasts in vitro, the inventors of the present invention used a culture medium containing Flt3L, IL-7, stem cell factor (SCF) and IL-15 Induced differentiation of hematopoietic stem cells into NK cells, myeloid cells and hematopoietic progenitor cells was induced by the FACS analysis, cytotoxicity test, and colony forming ability assay. , Confirming that the de-differentiation-induced hematopoietic stem cells had a colony forming ability and a multipotent ability (Figs. 5A to 5D).

The present inventors also compared induction processes of hematopoietic stem cells and inducible pluripotent stem cells derived from the fibroblasts, and found that iHSC decreased the gene expression of pluripotency factors, and gene expression of HSC markers (FIG. 6A and FIG. 6B), it was confirmed that the induction of the differentiation-induced hematopoietic stem cells did not require induction of pluripotent stem cells.

Accordingly, the hematopoietic stem cell (iHSC) induced by the differentiation of the hematopoietic stem cell (hematopoietic stem cell) through direct over-differentiation of hematopoietic stem cells through overexpression of the differentiation inducing gene into the fibroblasts of the present invention has characteristics of hematopoietic stem cells, Cells and myeloid cells. Therefore, direct induction of the differentiation of the fibroblasts into hematopoietic stem cells can be advantageously used for the production of safe and efficient cell therapy agents.

In addition,

1) preparing hematopoietic stem cells (iHSCs) induced by differentiation according to the method of the present invention;

2) inducing differentiation into natural killer precursor cells by adding a natural killer (NK) cell precursor inducing agent to the hematopoietic stem cell induced by differentiation in step 2); And

3) a step of differentiating the natural killer precursor cells from the hematopoietic stem cells into natural killer cells, comprising the step of treating the natural killer precursor cells of step 2) with IL-15 (interleukin-15) to provide.

In this method, the hematopoietic stem cell derived from step 1) is preferably Lin ^- c-Kit ⁺ Sca-1 ⁺ cells, but is not limited thereto.

In this method, the natural killer cell precursor inducer of step 2) is preferably but not limited to SCF (stem cell factor), Flt3L (fms-like tyrosine kinase 3 ligand) or IL-7 (Interleukin-7).

The differentiated natural killer cells of step 3) are preferably CD3e ^- NK1.1 ⁺ NK cells, but are not limited thereto.

Since it was confirmed that differentiation induction from hematopoietic stem cells into natural killer cells was induced by the over-differentiation induction through overexpression of a dedifferentiation inducing gene in fibroblasts, the method of differentiation from the differentiation-induced hematopoietic stem cells into natural killer cells And can be usefully used for producing safe and efficient cell therapy products.

In addition,

1) preparing hematopoietic stem cells (iHSCs) induced by differentiation according to the method of the present invention; And

2) a step of differentiating the hematopoietic stem cells derived from the differentiation induced in step 1), thereby providing a method of differentiating hematopoietic stem cells into lymphoid cells or myeloid cells.

In this method, the hematopoietic stem cell derived from step 1) is preferably Lin ^- c-Kit ⁺ Sca-1 ⁺ cells, but is not limited thereto.

In this method, the differentiated lymphoid cells of step 2) are preferably but not limited to Lin ^- c-Kit ⁺ Sca-1 ⁺ cells, natural killer cells, B cells or T cells.

In this method, the differentiated myeloid cells of step 2) are preferably Mac-1 ⁺ Gr-1 ⁺ myeloid cells, but are not limited thereto.

The present invention has been confirmed to induce differentiation into hematopoietic stem cells and hematopoietic stem cells derived from hematopoietic stem cells induced by the over-differentiation induction of over-differentiation inducing genes in fibroblasts. Therefore, the hematopoietic stem cells from the differentiation- The cell differentiation method can be usefully used for the production of a safe and efficient cell therapy agent.

The present invention also relates to a method for producing an expression vector comprising an expression vector comprising any one or two or more degeneration inducing gene combinations selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, LOM2, c-Fos and c- And provides a direct direct differentiation inducing agent from fibroblasts to hematopoietic stem cells (HSCs).

The expression vector is preferably a linear DNA, a plasmid DNA, or a recombinant viral vector, more preferably a recombinant viral vector, but is not limited thereto.

The recombinant viral vector is preferably a retrovirus, an adenovirus, a herpes simplex virus and a lentivirus, more preferably a retrovirus, but is limited thereto It is not.

The present invention relates to a method for inducing differentiation into hematopoietic stem cells (HSC) through overexpression of a combination of Oct3 / 4, Sox2, Klf4, c-Myc, LOM2, c-Fos and c- By confirming that the differentiation-induced hematopoietic stem cell (iHSC) is induced to differentiate into lymphoid cells and myeloid cells, the direct reverse transcription from fibroblasts containing hematopoietic stem cells Differentiation inducers can be used to induce the differentiation of fibroblasts into hematopoietic stem cells.

In addition, the present invention provides a cell therapy agent containing hematopoietic stem cells (iHSCs) derived from differentiation into fibroblasts.

The hematopoietic stem cell (iHSC), which is induced by direct overexpression of hematopoietic stem cells into the fibroblasts of the present invention through overexpression of a differentiation inducing gene, has characteristics of hematopoietic stem cells, Cells, the direct induction of the differentiation of the fibroblasts into hematopoietic stem cells can be usefully used for the production of safe and efficient cell therapy agents.

< Example  1> Embryo fibroblast  And Of hematopoietic stem cells  detach

<1-1> Mouse embryonic fibroblast  detach

13.5-day-old experimental mouse (C57BL / 6J, Central Lab., Korea). The embryo's head and liver organs were separated, the tissue was finely ground, treated with 2.5 ml of trypsin, For 30 minutes. Then, DMEM + 10% medium was added, and the supernatant was removed by centrifugation, followed by filtration on a 40 μM mesh, followed by incubation at 37 ° C. in an incubator. The isolated mouse embryo fibroblast cells (MEF) were cultured in DMEM + 10% medium.

<1-2> Of hematopoietic stem cells  detach

Bone marrow was obtained from the femur and tibia of experimental mice and the lysis buffer was treated to remove red blood cells. Bone marrow cells were blocked for 15 minutes at 4 ° C and then incubated with biotinylated antibody cocktail for lineage containing B220, CD2, NK1.1, CD11b, Gr-1, and TER-119 antibodies. Was added and reacted at 4 ° C for 20 minutes. The cells are washed with FBS-added PBS, streptavidin-microbeads are added, and reacted again at 4 ° C for 20 minutes. A magnetic column was placed in Super MACS and the labeled beads were passed through the labeled beads. Then, the column was sufficiently washed with PBS, and the solution passed through the column was centrifuged to collect lineage negative cells (Lin ^- ). The Lin ^- addition of 20 ㎕ CD117 MACS microbeads (microbeads) in cell to cell and incubated at 4 ℃ 20 minutes. The cells were then passed through an MS column, the column was washed with PBS, and 1 ml PBS / FBS buffer was added to separate the hematopoietic stem cells (HSC) by positive CD117 ⁺ selection.

< Example  2> From embryonic fibroblasts  Into hematopoietic stem cells De-differentiation  Verify induction conditions

<2-1> From embryonic fibroblasts  Into hematopoietic stem cells De-differentiation  Candidate gene selection for induction

Serial analysis of gene expression (SAGE) was performed to select candidate genes to induce the differentiation of embryonic fibroblasts into hematopoietic stem cells.

Specifically, the gene expression sequencing method is a method for analyzing a gene highly expressed in a specific tissue or cell. The gene that is highly expressed in the hematopoietic stem cell can be analyzed and sorted, and the destiny of the hematopoietic stem cell is determined Genes could be selected based on genes that play an important role. In other words, after summarizing the degree of expression between hematopoietic stem cells and natural killer cells, SAGE data is used to determine the genes that are important in hematopoietic stem cell growth, development, differentiation, and self replication among genes expressed relatively high in hematopoietic stem cells Respectively.

As a result, as shown in FIG. 1A, compared with natural killer precursor cells (pNK) and mature natural killer cells (mNK), candidate genes overexpressed in hematopoietic stem cells (HSC) (Fig. 1A).

<2-2> From embryonic fibroblasts  Into hematopoietic stem cells De-differentiation  Identification of candidate gene expression for induction

When the candidate genes selected by the method described in Example <2-1> are compared with natural cells (NK), mouse embryonic fibroblasts (MEF) and embryonic stem cells (ES) Polymerase chain reaction (RT-PCR) was performed to confirm that it was highly expressed in stem cells (HSC).

Specifically, natural killer precursor cells (pNK), mNK cells, mouse embryonic fibroblasts (MEF), embryonic stem cells (ES) and hematopoietic stem cells obtained by the method described in the above Example <1-2> Cells (HSC) were recovered and total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, USA). 1-2 μg of RNA was incubated with 42 μl of a 2.5 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, Wis.) At 42 ° C. for 1 hour, at 95 ° C. The reaction was carried out for 5 minutes to synthesize cDNA. Using the above-mentioned genomic DNA as a template for PCR, polymerase chain reaction (PCR) was performed using the primer shown in Table 1 below to obtain c-Myc (Gene ID: Mm.2444), c MRNA (Gene ID: Mm.275071), LMO2 (Gene ID: Mm.29266), c-Fos (Gene ID: Mm.246513) Mm. 15701) gene.

primer The sequence (5 '- &gt; 3') c-Myc Forward CAG AGG AGG AAC GAG CTG AAG CGC (SEQ ID NO: 1) Reverse TTA TGC ACC AGT GTT TCG AAG CTG TTC G (SEQ ID NO: 2) c-Jun Forward TCC CCT ATC GAC ATG GAG TC (SEQ ID NO: 3) Reverse TGA GTT GC ACC CAC TGT TA (SEQ ID NO: 4) LMO2 Forward GGA TGG TCT CTG TGC ATC CT (SEQ ID NO: 5) Reverse TCC CAT TGA TCT TGG TCC AC (SEQ ID NO: 6) c-Fos Forward ATC CTT GGA GCC AGT CAA GA (SEQ ID NO: 7) Reverse GTA CAG GTG ACC ACG GGA GT (SEQ ID NO: 8) c-Myb Forward GCG TCA CTT GGG GAA AAC TA (SEQ ID NO: 9) Reverse CAA CGC TTC GGA CCA TAT TT (SEQ ID NO: 10) Trim28 Forward AAT GAT GCC CAG AAG GTG AC (SEQ ID NO: 11) Reverse ATC TCA CCA TGA GGC TCC AC (SEQ ID NO: 12) GAPDH Forward ATT GTC ATA CCA GGA AAT GAG CTT (SEQ ID NO: 13) Reverse GTG TTC CTA CCC CCA ATG TGT (SEQ ID NO: 14)

As a result, as shown in Fig. 1B, the expression of c-Myc, c-Jun, LMO2, c-Fos, c-Myb and Trim28 in hematopoietic stem cells compared with natural killer cells, embryonic fibroblasts and embryonic stem cells Respectively. In particular, it was confirmed that the expression of the six genes was significantly increased in hematopoietic stem cells as compared with embryonic fibroblasts (Fig. 1B).

<2-3> From embryonic fibroblasts  Into hematopoietic stem cells De-differentiation  Identification of induction best candidate gene combination

The dedifferentiation induction was induced through the combination of various candidate genes of the candidate gene selected by the methods described in the above Examples <2-1> and <2-2> including the pluripotency marker to determine the optimum conditions for induction of the differentiation Respectively.

Specifically, Oct3 / 4 (Gene ID: Mm.17031), Sox2 (Gene ID: Mm.65396), Klf4 (Gene ID: Mm.4325), and c- Myc and the candidate genes c-Jun, LMO2, c-Fos and c-Myb selected by the methods described in the above Examples <2-1> and <2-2> were subcloned. The pMXs retroviral vector was used as a negative control while confirming the transfection efficiency. Plat-E packing cells were cultured at 6 × 10 ⁵ cells / 6 wells for one day, and the subcloned gene was transformed with lipofectamine ^Plus . The medium is exchanged after 3-4 hours and the culture is recovered after 48 hours. The same amount of the virus cocktail was treated with mouse embryonic fibroblasts obtained by the method described in the above Example <1-1> together with 4 / / ml polybrene in several combinations, followed by culturing for 4 hours to one day And then replaced with fresh medium for infection for 3 days. The embryonic fibroblasts infected with various combinations were divided into mitomycin C-treated support cells, cultured in a hematopoietic stem cell culture medium, and the medium was changed every 2-3 days. After about 21 days, colonies were screened. For selection, the medium was removed, washed with PBS, treated with selective colony trypsin, and cultured in a hematopoietic stem cell culture medium.

As a result, as shown in Fig. 1C, the largest number of colonies were formed under the conditions of Oct3 / 4, Sox2, Klf4, c-Myc, Lmo2, c-Fos and c-Myb (OSKMLFB) It was confirmed that OSKMLFB among the candidate gene combinations for the reverse differentiation into hematopoietic stem cells was an optimal regeneration condition for hematopoietic stem cells (Fig. 1C).

< Example  3> derived hematopoietic stem cells De-differentiation  Confirm

 In order to determine whether the hematopoietic stem cells (iHSC) derived from fibroblasts and the hematopoietic stem cells have similar characteristics as the optimal regeneration conditions identified in Example 2-3, Fluorescence activated cell sorter (FACS) analysis was performed.

Specifically, the hematopoietic stem cells derived from mouse fibroblasts derived from differentiation conditions of Oct3 / 4, Sox2, Klf4, c-Myc, Lmo2, c-Fos and c- The morphology of stem cells and induced pluripotent stem cells (iPS) was observed using a microscope (Fig. 2a).

In addition, for analysis through a fluorescence activated cell sorter (FACS), hematopoietic stem cell (iHSC) colonies # 562 induced by the differentiation of the OSKMLFB gene combination conditions of Example <2-3> were cultured, (BD Bioscience) after reacting with c-Kit, Sca-1, CD135, and CD34, or CD48 and CD150, which had fluorescence bound thereto, at 4 ° C for 30 minutes, (Fig. 2B). As shown in Fig.

In addition, hematopoietic stem cell (iHSC) colonies # 106, # 13, # 110, # 103, # 19 and # 29 induced through the de-differentiation were divided into supporting cells treated with mitomycin C, After incubation with hematopoietic stem cell culture medium for 60 days, the surface markers of hematopoietic stem cells were confirmed using FACSCanto (BD Bioscience) after reacting with c-Kit and Sca-1 antibody conjugated with fluorescence as described above 2c).

As a result, it was confirmed that hematopoietic stem cells (iHSC) morphologically derived from fibroblasts showed a round and small shape similar to hematopoietic stem cells (Fig. 2a), as shown in Fig.

As shown in FIG. 2B and FIG. 2C, the LKS (Lin ^- c-Kit ⁺ Sca-1 ⁺ ) cell population, the long term population (Lin ^- c-Kit ⁺ Sca-1 ⁺ CD34 ^- CD135 ^- and the ^{Lin - c-Kit + Sca-} 1 + CD150 + CD48 - cells) and short cell clusters ^{(short term population, Lin - c} -Kit + Sca-1 + CD34 + CD135 - and the Lin ^- c-Kit ⁺ Sca-1 ⁺ CD150 ⁺ CD48 ⁺ cells) (Fig. 2b). Furthermore, it was confirmed that LKS cell clusters were still present in iHS cell clusters after a long time of culture (Fig. 2C). Therefore, it was confirmed that the differentiation of fibroblasts into hematopoietic stem cells was induced by the combination of OSKMLFB gene.

< Example  4> from fibroblasts De-differentiation  Identification of gene and protein expression of induced hematopoietic stem cells

(IHSCs), hematopoietic stem cells (HSC), embryonic stem cells (ES), and induced pluripotent stem cells (iPS) derived from fibroblasts (pMXs) Real-time PCR, western blotting, immunostaining, and RT-PCR were performed in order to perform PCR.

Specifically, real-time PCR was performed to confirm the expression of c-Kit and Sca-1 gene, which are surface markers of hematopoietic stem cells, in hematopoietic stem cells (iHSCs) derived from differentiation. (PMXs) obtained by the method described in the above Example <2-3>, the hematopoietic stem cell (iHSC) colonies # 103 and # 107 derived by the method described in Example <2-3> The hematopoietic stem cells (HSC) and the embryonic stem cells (ES) separated by the method described in the above Example < 1-2 > were recovered, The cDNA was synthesized as follows. Transcripts of the synthesized cDNAs were quantified using SYBR Premix Ex Taq (Takara Bio, Japan) and Real-time system TP 800 (Takara Bio, Japan), and GAPDH was used for standardization of the samples. Each primer was prepared using primer 3 software as shown in Table 2 (Fig. 3A).

primer The sequence (5 '- &gt; 3') c-Kit
Forward CCA TGT GGC TAA AGA TGA AC (SEQ ID NO: 15) Reverse CTG CTG GTG CTC GGG TTT G (SEQ ID NO: 16) Sca-1
Forward ATT ACC TGC CCC TAC CCT GA (SEQ ID NO: 17) Reverse CAT TGG GAA CTG CTA CAT TGC (SEQ ID NO: 18)

Western blotting was performed to confirm expression of c-Kit and Sca-1 protein, which are surface markers of hematopoietic stem cells, in the differentiation-induced hematopoietic stem cell (iHSC). (PMXs), and hematopoietic stem cell (iHSC) cell colonies # 101, # 116, # 117, # 118, # 120 and # 121, # 103, # 104 and # 110 were recovered and the cells were lysed using a lysis buffer. The dissolved cells were separated by SDS-PAGE and transferred to a nitrocellulose transfer membrane by terminating the reaction with about 10 μg of protein per sample in SDS sample buffer solution. Then, the primary antibody was incubated with anti-c-Kit (SANTA CRUZ BIOTECHNOLOGY, INC., USA) antibody, SANTA CRUZ BIOTECHNOLOGY, INC., USA antibody and SANTA CRUZ BIOTECHNOLOGY , INC., USA) antibody was reacted and HRP-conjugated secondary antibody was attached to the primary antibody attached to the membrane and confirmed by ECL (Amersham Biosciences, UK). Actin was used as a control protein (Figure 3b).

Immunostaining was performed to confirm the expression of c-Kit and Sca-1 protein, which are surface markers of hematopoietic stem cells, in hematopoietic stem cells (iHSCs) derived from differentiation. The induced pluripotent stem cells (iPS), de-differentiation-induced hematopoietic stem cells (iHSC) and hematopoietic stem cells (HSC) in a circular glass coverslip (cover slip) in a 12-well plate containing 1 × 10 ⁵ cells / plate density For 24 h, then washed with cold 1 x PBS and fixed with 4% paraformaldhyde for 20 min at room temperature. The cells were then infiltrated with 0.2% Triton X-100 (Sigma, USA) for 15 min at room temperature and incubated with anti-SSEA (R & D systems, USA), anti- Antibody and DAP1 (Vector Labs, USA) and washed three times with PBS. Then, the cells were reacted with a secondary antibody (Invitrogen, USA) at room temperature for 2 hours, washed with PBS, and visualized using a LSM510 confocal microscope (Carl Zeiss, Fig. 3C).

RT-PCR was performed to confirm the expression of transcription factors of hematopoietic stem cells in the hematopoietic stem cells (iHSC) derived from differentiation. The mouse fibroblast negative control (pMXs) and the differentiation-induced hematopoietic stem cell (iHSC) cell colonies # 103, # 104 and # 106 obtained by the method described in Example 2-3 were recovered and hematopoietic stem cells And embryonic stem cells were subjected to RT-PCR as described in Example < 2-2 >. At this time, [Table 1] and [Table 3] were used as primers (Fig. 3d).

primer The sequence (5 '- &gt; 3') c-Kit
Forward CCA TGT GGC TAA AGA TGA AC (SEQ ID NO: 19) Reverse CTG CTG GTG CTC GGG TTT G (SEQ ID NO: 20) Sca-1
Forward ATT ACC TGC CCC TAC CCT GA (SEQ ID NO: 21) Reverse CAT TGG GAA CTG CTA CAT TGC (SEQ ID NO: 22) CD34
Forward CCA CAC ACT TTT TCA CAA CCA (SEQ ID NO: 23) Reverse CCA ACC TCA CTT CTC GGA TT (SEQ ID NO: 24) CD150
Forward TCT TGG GGA CTG GGA TAC AG (SEQ ID NO: 25) Reverse ATG AGG AGA GAC GGT GGA GA (SEQ ID NO: 26) Nanog
Forward ATG CGG ACT GTG TTC TCT CA (SEQ ID NO: 27) Reverse GCA ATG GAT GCT GGG ATA CT (SEQ ID NO: 28)

As a result, as shown in Fig. 3A, unlike fibroblasts and embryonic stem cells, which were negative control groups, the expression of c-Kit and Sca-1 gene, which are surface markers of hematopoietic stem cells, (Fig. 3A).

In addition, as shown in FIGS. 3B and 3C, the expression of c-Kit and Sca-1 protein was significantly increased in iHSC unlike fibroblasts (FIG. 3B). In addition, SSEA protein, which is a marker of embryonic stem cells, is expressed in inducible pluripotent stem cells. However, in the case of hematopoietic stem cells, the expression of c-Kit and Sca-1 protein is not expressed, (Fig. 3C).

In addition, as shown in FIG. 3D, it was confirmed that LMO2 protein, which is a transcription factor specifically expressed in hematopoietic stem cells as well as surface markers of hematopoietic stem cells, was highly expressed in hematopoietic stem cells derived from differentiation (FIG. 3D) . Therefore, it was confirmed that hematopoietic stem cells (iHSCs) derived from degeneration from fibroblasts have very similar molecular characters to hematopoietic stem cells.

< Example  5> in vivo in vivo ) Identification of differentiation ability

Hematopoietic stem cells play an important role in the reconstitution of blood when transplanted into recipient mice in which bone marrow has been removed. Therefore, FACS analysis was performed to confirm that the hematopoietic stem cell (iHSC) induced through de-differentiation from fibroblasts has the ability to regulate the hematopoietic cell system in vivo.

Specifically, after irradiation of 900 rad with 6-8 week old fed mice (CD45.1 ⁺ congenic mice) (C57BL / 6J, Central Lab., Korea), the irradiated mice were exposed to the tail vein 5 × 10 ⁸ mouse fibroblasts (pMXs) obtained by the method described in the above Example <2-3>, hematopoietic stem cells (iHSCs) induced through de-differentiation according to the method described in Example <2-3, Bone marrow cells prepared from the c-Kit ⁺ Sca-1 cells (CD45.2 ⁺⁾ conferred the mice (CD45.1 ⁺ congenic mice) ^(- one isolated from lin ^- c-Kit ⁺ Sca-1 cells, obtained from bone marrow or lin 1 x 10 &lt; ⁶ &gt;). After 16 weeks, the mice were sacrificed and bone marrow was obtained to express the markers of LKS, NK cells, B cells, T cells (Fig. 4A) and bone marrow cells (Fig. 4B) And analyzed by FACS.

As a result, as shown in Figs. 4A and 4B, the CD45.2 ⁺ lymphocytes LKS derived from hematopoietic stem cells (iHSC) derived from fibroblast differentiation from fibroblasts, (FIG. 4A). In the bone marrow of mice injected with the differentiation-induced hematopoietic stem cells, CD45.2 ⁺ Mac-a ⁺ Gr-1 ⁺ Cells were also present (Fig. 4B). Therefore, it can be seen that the hematopoietic stem cell (iHSC) induced through the de-differentiation of fibroblasts differentiates into a lymphoid lineage as well as a myeloid lineage in vivo.

< Example  6> in vitro in vitro ) Identification of differentiation ability

In order to confirm the multipotency of hematopoietic stem cells (iHSCs) induced by differentiation from fibroblasts through in vitro induction of differentiation into lymphatic and myeloid lineages, FACS analysis, cytotoxicity experiment And colonization ability analysis were performed.

Specifically, in order to induce the differentiation of hematopoietic stem cells (iHSCs) derived from the hematopoietic stem cells (iHSCs) obtained through the de-differentiation obtained by the method described in the above Example <2-3> into natural killer cells, a two-stage differentiation induction method was selected. (IHSC) colonies # 10, # 108, # 13, # 110 and # 106 obtained by the method described in the above Example 2-3 were inserted into a 24-well plate Becton and Dickinson Bioscience, USA) 1.5 x 10 ⁶ cells / well (cells / well) concentration of 10% in fetal calf serum (fetal bovine serum in the; FBS), 2 μg / ml indomethacin acid (indometacin) (Sigma-Aldrich , 50 ng / ml murine (Sigma-Aldrich USA), 30 ng / ml murine SCF (stem cell factor) (PeproTech, Rocky Hill, USA), 20 ug / ml gentamicin ) At 37 ° C, 5% CO ₂ for 7 days using RPMI 1640 medium containing Flt3L (PeproTech, USA), 0.5 ng / ml murine IL-7 (PeproTech, USA) Three days after the initial incubation, half of the culture supernatant was replaced with fresh medium containing the same composition of cytokine. For the differentiation into NK cells, the medium-stage precursor cells cultured for 7 days were collected and further cultured for 7 days in medium containing various concentrations of murine IL-15 (PeproTech, USA). At intervals of three days, half of the medium was replaced with fresh medium containing the same composition of cytokine. On day 14, the degree of differentiation of natural killer cells was analyzed by FACS as in Example 3 using anti-NK1.1 and anti-CD3e antibodies, which are antibodies against surface molecules of natural killer cells (NK cells) ).

In addition, the cytotoxicity of natural killer cells was measured using the Calcein-AM method. That is, target YAC-1 cells were reacted with 5 μg / ml calcein-AM for 1 hour at 37 ° C., and then the cells were reacted with the respective effector cells: target cells (E: T) Induced natural killer cells from the differentiation-induced hematopoietic stem cell (iHSC) colonies # 543, # 554 and # 530 obtained by the method described in the above Example <2-3>. The supernatant is collected and fluorescence is measured at a wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescent reader (FIG. 5B).

In order to induce differentiation into myeloid cells, the differentiation-induced hematopoietic stem cell (iHSC) colonies # 600, # 602 and # 605 obtained by the method described in Example 2-3 were cultured in DMEM containing 10 ng / Were cultured in RPMI 1640 medium containing 10 ng / ml GM-CSF, 10 ng / ml G-CSF, 50 ng / ml SCF, 20 ng / ml IL3 and 10 ng / ml BMP4 (PeproTech). On day 14, cells expressing the surface markers of bone marrow cells using anti-Gr-1 and anti-Mac-1 antibodies, which are antibodies to surface molecules of myeloid lineage, were treated with FACS (Fig. 5C).

In order to confirm the colony-forming capacity of the hematopoietic stem cell (iHSC) derived from the differentiation, the differentiation-induced hematopoietic stem cell (iHSC) obtained by the method described in Example <2-3> Colony formation assays were performed using methylcellulose-based medium (M3434, StemCell Technologies) in colonies # 106, # 110, # 114 and # 118. The cells were prepared at a ratio of 1:10 (v / v) with methocult media, and then 1 ml was added to a 35 mm culture plate. The cells were cultured at 37 ° C and 5% CO ₂ for 12 days. (Fig. 5D).

As a result, as shown in FIG. 5A, it was confirmed that natural killer cells were induced through differentiation induction in hematopoietic stem cells (iHSC) induced by de-differentiation similar to hematopoietic stem cells (HSC) (FIG.

In addition, as shown in FIG. 5B, it was confirmed that the cytotoxicity of the natural killer cells induced to differentiate from the fibroblasts was low and had the ability to kill tumor cells (FIG. 5B).

As shown in FIG. 5C, Mac-a ⁺ Gr-1 ⁺ myeloid cells differentiated from hematopoietic stem cells (iHSCs) induced by differentiation were increased, unlike fibroblasts. Especially, in # 600 iHSC, (Fig. 5C). As a result, it was confirmed that the differentiation-induced hematopoietic stem cells were differentiated into myeloid lineage (Fig. 5C).

5D, granulocytes (CFU-G) and macrophage lineage (CFU-M) are formed from hematopoietic stem cells (iHSCs) induced by de-differentiation. In particular, granulocytes It was confirmed that the macrophage (CFU-GM) was remarkably formed, and thus it was confirmed that the hematopoietic stem cells induced by the differentiation had characteristics of hematopoietic progenitor cells (FIG. 5d). Therefore, it was confirmed that the hematopoietic stem cells induced by the differentiation have the colonization ability and the multipotentialization ability through the above results.

< Example  7> Direct De-differentiation  Induced Hematopoietic stem cells (iHSC) and Induced pluripotent stem cells (iPS)  compare

In order to confirm the difference in the induction process of induced pluripotent stem cells (iPS) and the differentiation induction of hematopoietic stem cell (iHSC) induced by differentiation, the expression of markers of the differentiation potential markers and hematopoietic stem cells PCR.

Specifically, mouse fibroblasts (pMX), hematopoietic stem cells (iHSCs, 1, 2, 3, 4, 5, and 6 weeks) derived by the method described in Example 2-3, Stem cells (HSC) and embryonic stem cells (ES) were recovered and total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, USA). Then, a cDAN was synthesized as in the above Example <2-2>. Transcripts of the synthesized cDNAs were quantified using SYBR Premix Ex Taq (Takara Bio, Japan) and Real-time system TP 800 (Takara Bio, Japan), and GAPDH was used for standardization of the samples. The expression of Toct4, TSox2, TKlf4, Tc-Myc, Nanog and Eras mRNA was measured (Fig. 6a) in order to identify markers of pre-differentiation ability. To identify markers of hematopoietic stem cells, c- And CD150 mRNA expression was measured (Fig. 6B). Each primer was prepared using Primer3 software as shown in Table 4 below.

primer The sequence (5 '- &gt; 3') Oct4
Forward CTG AGG GCC AGG CAG GAG CAC GAG (SEQ ID NO: 29) Reverse CTG TAG GGA GGG CTT CGG GCA CTT (SEQ ID NO: 30) Sox2
Forward GGT TAC CTC TTC CTC CCA CTC CAG (SEQ ID NO: 31) Reverse TCA CAT GTG CGA CAG GGG CAG (SEQ ID NO: 32) c-Kit
Forward CCA TGT GGC TAA AGA TGA AC (SEQ ID NO: 33) Reverse CTG CTG GTG CTC GGG TTT G (SEQ ID NO: 34) Klf4
Forward CAC CAT GGA CCC GGG CGT GGC TGC CAG AAA (SEQ ID NO: 35) Reverse TTA GGC TGT TCT TTT CCG GGG CCA CGA (SEQ ID NO: 36) c-Myc
Forward CAG AGG AGG AAC GAG CTG AAG CGC (SEQ ID NO: 37) Reverse TTA TGC ACC AGT GTT TCG AAG CTG TTC G (SEQ ID NO: 38) Sca-1
Forward ATT ACC TGC CCC TAC CCT GA (SEQ ID NO: 39) Reverse CAT TGG GAA CTG CTA CAT TGC (SEQ ID NO: 40) Nanog
Forward ATG CGG ACT GTG TTC TCT CA (SEQ ID NO: 41) Reverse GCA ATG GAT GCT GGG ATA CT (SEQ ID NO: 42) E-Ras
Forward GGA ATG GCT TTG CCT ACA A (SEQ ID NO: 43) Reverse GAA TCT TGG ATA GTG GGG TCA (SEQ ID NO: 44) CD150
Forward TCT TGG GGA CTG GGA TAC AG (SEQ ID NO: 45) Reverse ATG AGG AGA GAC GGT GGA GA (SEQ ID NO: 46)

As a result, as shown in Figs. 6A and 6B, in the case of the hematopoietic stem cell (iHSC) derived from the differentiation, Oct4, Sox2, Klf4, and c-Myc, which are exogenously overexpressed factors, , Whereas Nanog and Eras are rarely expressed, whereas the markers of hematopoietic stem cells, c-Kit, Sca-1 and CD150, increase in time-dependent manner and show similar expression pattern to hematopoietic stem cells (HSC) Thus, it was confirmed that the generation of pluripotent stem cells was not necessary during the induction of the hematopoietic stem cell (iHSC) induced by the differentiation (FIG. 6).

<110> Korea Research Institute of Bioscience and Biotechnology <120> A method for direct dedifferentiation of hematopoietic stem cell          from fibroblast cell <130> 13p-11-62 <160> 46 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> c-Myc forward primer <400> 1 cagaggagga acgagctgaa gcgc 24 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> c-Myc reverse primer <400> 2 ttatgcacca gagtttcgaa gctgttcg 28 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Jun forward primer <400> 3 tcccctatcg acatggagtc 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> c-Jun reverse primer <400> 4 tgagttgcac ccactgtta 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMO2 forward primer <400> 5 ggatggtctc tgtgcatcct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LMO2 reverse primer <400> 6 tcccattgat cttggtccac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Fos forward primer <400> 7 atccttggag ccagtcaaga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Fos reverse primer <400> 8 gtacaggtga ccacgggagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Myb forward primer <400> 9 gcgtcacttg gggaaaacta 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Myb reverse primer <400> 10 caacgcttcg gaccatattt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Trim28 forward primer <400> 11 aatgatgccc agaaggtgac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Trim28 reverse primer <400> 12 atctcaccat gaggctccac 20 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 13 attgtcatac caggaaatga gctt 24 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 14 gtgttcctac ccccaatgtg t 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Kit forward primer <400> 15 ccatgtggct aaagatgaac 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> c-Kit reverse primer <400> 16 ctgctggtgc tcgggtttg 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sca-1 forward primer <400> 17 attacctgcc cctaccctga 20 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sca-1 reverse primer <400> 18 cattgggaac tgctacattg c 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Kit forward RT-PCR primer <400> 19 ccatgtggct aaagatgaac 20 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> C-Kit reverse RT-PCR primer <400> 20 ctgctggtgc tcgggtttg 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sca-1 forward RT-PCR primer <400> 21 attacctgcc cctaccctga 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sca-1 reverse RT-PCR primer <400> 22 cattgggaac tgctacattg c 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD34 forward primer <400> 23 ccacacactt tttcacaacc a 21 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD34 reverse primer <400> 24 ccaacctcac ttctcggatt 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD150 forward primer <400> 25 tcttggggac tgggatacag 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD150 reverse primer <400> 26 atgaggagag acggtggaga 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog forward primer <400> 27 atgcggactg tgttctctca 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog reverse primer <400> 28 gcaatggatg ctgggatact 20 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oct4 forward primer <400> 29 ctgagggcca ggcaggagca cgag 24 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oct4 reverse primer <400> 30 ctgtagggag ggcttcgggc actt 24 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Sox2 forward primer <400> 31 ggttacctct tcctcccact ccag 24 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sox2 reverse primer <400> 32 tcacatgtgc gacaggggca g 21 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Kit forward real-time PCR primer <400> 33 ccatgtggct aaagatgaac 20 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> c-Kit reverse real-time PCR primer <400> 34 ctgctggtgc tcgggtttg 19 <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Klf4 forward primer <400> 35 caccatggac ccgggcgtgg ctgccagaaa 30 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Klf4 reverse primer <400> 36 ttaggctgtt cttttccggg gccacga 27 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> c-Myc forward real-time PCR primer <400> 37 cagaggagga acgagctgaa gcgc 24 <210> 38 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> c-Myc reverse real-time PCR primer <400> 38 ttatgcacca gagtttcgaa gctgttcg 28 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sca-1 forward real-time PCR primer <400> 39 attacctgcc cctaccctga 20 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sca-1 reverse real-time PCR primer <400> 40 cattgggaac tgctacattg c 21 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog forward real-time PCR primer <400> 41 atgcggactg tgttctctca 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nanog reverse real-time PCR primer <400> 42 gcaatggatg ctgggatact 20 <210> 43 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> E-Ras forward real-time PCR primer <400> 43 ggaatggctt tgcctacaa 19 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> E-Ras reverse real-time PCR primer <400> 44 gaatcttgga tagtggggtc a 21 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD150 forward real-time PCR primer <400> 45 tcttggggac tgggatacag 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> CD150 reverse real-time PCR primer <400> 46 atgaggagag acggtggaga 20

Claims (12)

1) transforming a vector containing both Oct3 / 4, Sox2, Klf4, c-Myc, LOM2, c-Fos and c-Myb as a dedifferentiation inducer into fibroblast cells; And
2) direct transformation of fibroblasts into hematopoietic stem cells, comprising culturing the transformant of step 1) and isolating the colonies.
The method according to claim 1, wherein the fibroblasts in step 1) are human or mouse embryo fibroblast cells.
delete 2. The method of claim 1, wherein step 2) de-differentiation-induced hematopoietic stem cells for the ^{Lineage (Lin) - c-Kit} + Sca-1 + induced differentiation directly reverse to the hematopoietic stem cells to cells of at fibroblasts, characterized Way.
1. A method for producing hematopoietic stem cells comprising the steps of:
2) Stem Cell Factor (SCF), Flt3L (fms-like tyrosine kinase 3 ligand) or IL-7 (Interleukin-3) as a natural killer cell precursor inducer in the differentiation-induced hematopoietic stem cells of step 1) 7) to induce differentiation into natural killer precursor cells; And
3) Differentiation of NK precursor cells of step 2) into differentiation-induced hematopoietic stem cells into natural killer cells, comprising treating IL-15 (interleukin-15) with natural killer cells.
delete [Claim 5] The method according to claim 5, wherein the differentiated natural killer cells of step 3) are CD3e ^- NK1.1 ⁺ NK cells.
1. A method for producing hematopoietic stem cells comprising the steps of: And
2) differentiating the differentiated hematopoietic stem cells of step 1) into a lymphatic cell or a myeloid cell.
The stem cell according to claim 8, wherein the differentiated lymphoid cells of step 2) are Lin ^- c-Kit ⁺ Sca-1 ⁺ cells, natural killer cells, B cells or T cells. Lt; RTI ID = 0.0 &gt; lymphocytic &lt; / RTI &gt; cells or myeloid cells.
[Claim 10] The method according to claim 8, wherein the differentiated myeloid cells of step 2) are Mac-1 ⁺ Gr-1 ⁺ myeloid cells, wherein the differentiated hematopoietic stem cells are transformed into lymphoid cells or myeloid cells Lt; / RTI &gt;
Direct induction of direct dedifferentiation into hematopoietic stem cells in fibroblasts containing an expression vector comprising a reprogramming induction gene combination of both Oct3 / 4, Sox2, Klf4, c-Myc, LOM2, c-Fos and c-Myb.
A cell therapy agent containing the differentiation-induced hematopoietic stem cells prepared by the method of claim 1 .

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