JP2009055817A - Method for analyzing cell fate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a method for analyzing fate determination and differentiation from blood vascular blast cells to a blood system cell line and an endothelial cell line, especially, a method for simultaneously analyzing the cell fate of the blood system cell line and the endothelial cell line. <P>SOLUTION: This method for analyzing the cell fate of stem cells comprises a process (first process) for preparing the single cells of stem cells and amplifying the obtained single cells, a process (second process) for culturing the amplified cells to produce colonies, and a process (third process) for dividing the single cells into groups on the basis of the forms of the produced colonies and measuring the number of the single cells divided into the same group. A graph for analyzing the cell fate of stem cells is obtained by developing the kinds of the obtained groups as cell fates in the X-axial direction and developing the number of the single cells classified in the same group as a frequency in the Y-axial direction. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell fate analysis method, and more particularly to a method for analyzing and evaluating stem cell fate.

  In the field of blood system and vascular system medicine, vascular endothelial progenitor cells (EPC) are found in CD34 positive cells, which are human blood stem cell-containing fractions (Non-patent Document 1), and CD34 positive cells and early CD133 positive cells, which are a blood stem cell fraction, have come to be considered as a hemangioblast (HB) -containing fraction. That is, it has been clarified that blood progenitor cells (hematopoietic stem / progenitor cells = HSPC) and EPCs differentiate from HB, which is a common pluripotent stem cell, in vascular islands in the embryonic stage. Even in adults, such evidence of undifferentiated HB has been confirmed (Non-Patent Documents 2 to 4), and EPC is fate of blood and vascular cell lineage from a common stem cell called HB together with HSPC in the bone marrow stem cell niche. It is presumed to be the result of a decision or differentiation.

In the blood system field, as a method for analyzing HSPC differentiation into blood cell lines based on morphological observation, a method using a non-adhesive culture dish and semi-solid medium methylcellulose was developed in the 1970s. The significance has been established (Non-Patent Document 5). That is, it is a method for evaluating the typing and number of colonies. However, as a matter of course, the analysis method is limited to blood cells, and cell biological analysis of HB is impossible.
Since the discovery of the adult EPC in 1997, two cell biological analysis methods have been used for the EPC differentiation mode evaluation system. One is that undifferentiated EPC-containing cells (bone marrow and peripheral blood CD34-positive and / or CD133-positive cells, mononuclear cells) are cultured in a liquid medium for culturing endothelial cells, and adherent cells are obtained. EPC culture assay method (Non-patent Documents 6 and 7) to be evaluated automatically, and the second is an EPC colony assay method using methylcellulose, which evaluates EPC colonies numerically (Non-patent Documents). 8). Either method is limited to numerical evaluation of EPC, and its differentiation process cannot be evaluated. Patent Documents 1 and 2 describe a method for analyzing the differentiation mode of EPC, but the analysis is limited to the analysis of endothelial cells, and the causal relationship with the differentiation into blood cell lineages is unknown.
T. Asahara, T. Murohara, A. Sullivan et al., Science 275: 964-967 (1997) E. Pelosi, M. Valtieri et al., Blood 100 (9): 3203-3208 (2002) S. Loges, B. Fehse et al., Stem Cells Dev. 13 (3): 229-242 (2004) UM. Gehling, S. Ergun et al., Blood 95 (10): 3106-3112 (2000) A. Hirao, Y. Takaue et al., Journal of Clinical Apheresis 10: 17-22 (1995) T. Asahara, T. Takahashi et al., EMBO J. 18 (14): 3964-3972 (1999) C. Kalka, H. Masuda et al., Circ Res. 86 (12): 1198-1202 (2000) B. Assmus, C. Urbich et al., Circ Res. 92 (9): 1049-1055 (2003) WO2006 / 090882 International Publication WO2006 / 090886 International Publication

  The present invention develops a method for analyzing cell fate of stem cells, particularly tissue stem cells, specifically, a method for analyzing fate determination and differentiation from blood hemangioblasts (HB) to blood cell lineages and vascular endothelial cell lines. In particular, it aims to develop a method for simultaneously analyzing the cell fate of blood cell lineage and vascular endothelial cell lineage.

  In view of the above problems, the present inventors first cultured a single cell isolated from HB, examined the mode of a colony produced by differentiation into a blood cell lineage or an endothelial cell lineage, Based on these patterns, cells were grouped to define a “cell commitment”. Then, for each obtained cell fate, its frequency (ie, the number of single cells that would present the relevant cell fate) was measured. By making the cell fate correspond to the frequency, the cell fate can be clarified and graphed, and can be quantified.

That is, the present invention is as follows:
[1] A step of preparing a single stem cell and amplifying the obtained single cell (first step),
A step of culturing the amplified cells to produce colonies (second step), and grouping the single cells based on the mode of the produced colonies, and measuring the number of single cells classified into the same group Process (3rd process)
A method for analyzing cell fate of a stem cell, comprising:
[2] The method described in [1] above, wherein the stem cells are blood hemangioblasts.
[3] The method according to [2] above, wherein the blood hemangioblasts are derived from bone marrow, umbilical cord blood or peripheral blood.
[4] The method according to [2] above, wherein the blood hemangioblasts are CD34 positive and / or CD133 positive.
[5] The method described in [2] above, wherein the produced colony is either a blood cell colony or a vascular endothelial cell colony.
[6] Blood system cell colonies are from the group consisting of erythroblast colonies (BFU-E), granulocyte / macrophage colonies (CFU-GM), macrophage colonies (CFU-M) and mixed colonies (CFU-Mix). The method of the above-mentioned [5], which is at least one selected.
[7] The vascular endothelial cell colony is at least one selected from the group consisting of an endothelial cell-like large cell colony (CFU-Large cell like EPC) and an endothelial cell-like small cell colony (CFU-small cell like EPC). The method according to [5] above.
[8] The method according to [1] above, wherein the first step is performed in the presence or absence of an additive factor.
[9] The method of the above-mentioned [8], wherein the additive factor is a growth factor.
[10] A screening method for a compound that affects the cell fate of a stem cell, comprising performing the method according to [1] above.
[11] A method for confirming the concentration of cells destined from a stem cell to a specific cell lineage, comprising performing the method according to [1] above.
[12] The method according to [11] above, wherein the stem cells are blood hemangioblasts and the cells destined for a specific cell lineage are vascular endothelial progenitor cells.
[13] A single stem cell is cultured, divided into groups based on the mode of colonies produced from the single cell, and the number of single cells classified into the same group is measured. The graph for analysis of the cell fate of a stem cell obtained by matching the number of single cells belonging to a group.
[14] Culturing single cells of stem cells, grouping them based on the mode of colonies produced from the single cells, expanding the types of the obtained groups as cell fate in the X-axis direction, The graph for the analysis of the cell fate of a stem cell obtained by expanding in the Y-axis direction using the number of single cells to be classified as a frequency.
[15] The graph according to [13] or [14] above, wherein the stem cells are blood hemangioblasts.
[16] The graph according to [15], wherein the colonies to be produced are either blood cell colonies or vascular endothelial cell colonies.
[17] The blood cell colony is from the group consisting of erythroblast colony (BFU-E), granulocyte / macrophage colony (CFU-GM), macrophage colony (CFU-M) and mixed colony (CFU-Mix). The graph according to [16], which is at least one selected.
[18] The vascular endothelial cell colony is at least one selected from the group consisting of an endothelial cell-like large cell colony (CFU-Large cell like EPC) and an endothelial cell-like small cell colony (CFU-small cell like EPC). The graph according to [16] above.

  It is possible to graph and quantify the cell fate of tissue stem cells, specifically the cell fate of blood and vascular endothelial cell lines from HB. In particular, by simultaneously graphing and quantifying the cell fate of the blood system and vascular endothelial cell lineage, it becomes possible to clarify and quantify the cell fate that could not be distinguished by the conventional single cell lineage analysis method. . Further, the method of the present invention is an evaluation with a single cell, and the cell lineage determination evaluation is accurate.

  The present invention relates to a method for analyzing the cell fate of a stem cell, for example, the determination of the fate from HB to a blood cell lineage and a vascular endothelial cell lineage, and the development of a method for analyzing differentiation. A method for simultaneously analyzing cell fate is provided. The analysis method of the present invention first includes a step of preparing and culturing a single cell of a stem cell for which analysis of the cell fate is desired.

  Stem cells are not particularly limited as long as they have a cell fate capable of differentiating into predetermined cells, but are preferably tissue stem cells, nervous stem cells, hematopoietic / vascular stem cells, mesenchymal stem cells, endoderm Examples include stem cells. Particularly preferred are hematopoietic and vascular stem cells whose phenotypes of each cell line are well studied, and the stem cells correspond to the above-described HB (blood hemangioblasts).

  The term “blood hemangioblast (HB)” used in the present invention is a progenitor cell of both blood cells and vascular endothelial cells, and blood cells (for example, erythrocytes, T-lymphocytes, B-lymphocytes, monocytes / macrophages, granulocytes, megakaryocytes), and especially undifferentiated cells that can become vascular endothelial cells via vascular endothelial progenitor cells (EPC). It is not limited. As HB, cells derived from bone marrow, umbilical cord blood or peripheral blood to be examined can be used. The HB can also be a mononuclear cell.

  The HB may further be CD34 and / or CD133 positive. Therefore, it is also preferable to select and use only CD34 positive and / or CD133 positive cells in advance as HB. For selection of CD34 positive and / or CD133 positive cells, cell sorting techniques commonly used in the art are used. Specifically, a substance having specific affinity for CD34 antigen and / or CD133 antigen is used. Magnetic cell separation method (MACS), fluorescent cell separation method (FACS) and the like. By this separation method, the stem cell can be made into a single cell.

  Examples of the substance having specific affinity for CD34 antigen or CD133 antigen include antibodies having specific affinity for these proteins or fragments thereof, and the specific affinity is defined by the antigen-antibody reaction. The ability to specifically recognize and bind. The antibody or a fragment thereof is not particularly limited as long as it can specifically bind to the protein, and may be a polyclonal antibody, a monoclonal antibody, or a functional fragment thereof. These antibodies or functional fragments thereof can be produced by methods generally performed in the art. For example, in the case of using a polyclonal antibody, there is a method in which the protein is injected subcutaneously into the back of the animal or into the abdominal cavity or vein of an animal such as a mouse or rabbit, and after waiting for the antibody titer to rise, antiserum is collected. In the case of using a monoclonal antibody, there is a method of preparing a hybridoma according to a conventional method and collecting the secreted solution. As a method for producing an antibody fragment, a method in which a cloned antibody gene fragment is expressed in a microorganism or the like is often used. The purity of the antibody, antibody fragment, etc. is not particularly limited as long as it retains specific affinity with the protein. These antibodies or fragments thereof may be labeled with a fluorescent substance or the like.

  HB can also be mobilized by a substance capable of mobilizing HB. Examples of substances capable of mobilizing HB include G-CSF, SDF-1, estrogen, VEGF, GM-CSF, angiopoietins 1 and 2, HGF, statins, and erythropoietin. CSF is preferred. In this case, HB may be provided in the form of a body fluid component (for example, peripheral blood) in which HB has been mobilized, which is obtained by administering a substance capable of mobilizing HB to a subject. The number of administrations of the substance capable of mobilizing HB to the test subject, the administration period, and the dosage vary depending on the type of substance used, and are not particularly limited as long as HB can be mobilized, but in the case of G-CSF, The administration frequency is, for example, usually 1 to 3 times a day, preferably 2 times. The administration period is, for example, about 3 to 7 days, preferably about 4 to 6 days, more preferably 5 days. The dosage is about 2-50 μg / kg / day, preferably about 5-20 μg / kg / day, more preferably about 10 μg / kg / day.

  The HB may be further collected from a test subject transplanted with HB mobilized by a substance capable of mobilizing HB.

  In the present invention, the test subject is an animal that is required to analyze the cell fate of a stem cell, and means a mammal in general including a human. The subject is preferably a human. In addition, use in mammals such as mice, rats, dogs, cats, rabbits, cows, pigs, goats, sheep, horses, monkeys and the like is also preferred.

  In the analysis method of the present invention, stem cells and single cells of CD34 and / or CD133 positive stem cells selected as necessary are cultured. Single cell culture can be performed by a conventional method or a method according to it, depending on the origin of the stem cell and the desired cell lineage. For example, if the stem cell is HB and the desired cell lineage is a vascular endothelial cell lineage, the methods disclosed in Patent Document 1 and Patent Document 2 are preferably used. If the stem cell is HB and the desired cell line is a blood cell line, a method using a non-adhesive culture dish and methylcellulose in a semi-solid medium (Non-Patent Document 5) is preferably used.

  In the method of the present invention, the culture of a single cell includes a step of preparing and amplifying a single cell of a tissue stem cell (first step) and a step of producing a colony from the amplified cell (second step). Usually, two steps are performed. The method of the first step is not particularly limited as long as the stem cells can be efficiently proliferated. However, when the tissue stem cells are HB, it is preferably performed by the method described in Patent Document 1. Specifically, it comprises incubating HB in a serum-free medium containing stem cell factor (SCF), interleukin 6 (IL-6), FMS-like tyrosine kinase 3 (Flt-3) and thrombopoietin (TPO).

  As the serum-free medium used in the first step, a medium usually used in the art can be used. For example, a serum-free medium known as a medium for the growth of hematopoietic stem cells can be used. Examples of the basal medium used as the serum-free medium include DMEM, MEM, IMDM and the like.

  Stem cell factor (SCF) that can be included in a serum-free medium is a glycoprotein consisting of 248 amino acids and having a molecular weight of about 30,000. There are a soluble type and a membrane-bound type by selective splicing, and the SCF used in the present invention may be any type of SCF as long as it is useful for culturing HB. Preferably it is a soluble type. The origin of SCF is not particularly limited, but a recombinant that can be stably supplied is preferable, and a human recombinant is particularly preferable. Those that are commercially available are known. The concentration of SCF in the serum-free medium varies depending on the type of SCF used and is not particularly limited as long as it is useful for HB culture. In the case of human recombinant SCF, for example, 10 to 1000 ng / mL, preferably 50-500 ng / mL, more preferably about 100 ng / mL.

  Interleukin 6 (IL-6) used in the present invention is a glycoprotein having a molecular weight of 210,000, isolated as a factor that induces terminal differentiation of B cells into antibody-producing cells. It is known to be involved in cell line proliferation and differentiation, acute phase reaction, and the like. IL-6 used in the present invention is appropriately selected. When used for culturing human HB, human IL-6 is preferable, and a recombinant that is expected to be stably supplied is particularly preferable. Those that are commercially available are known. The concentration of IL-6 in the serum-free medium varies depending on the type of IL-6 used, and is not particularly limited as long as it is useful for HB culture. In the case of human recombinant IL-6, for example, 1-6 500 ng / mL, preferably 5 to 100 ng / mL, more preferably about 20 ng / mL.

  FMS-like tyrosine kinase 3 (Flt-3) used in the present invention is known as a receptor tyrosine kinase that plays an important role in the control of early hematopoiesis. Several alternative splicing products are known but have been reported to stimulate hematopoietic stem cell proliferation. Flt-3 used in the present invention may be any type of Flt-3 as long as it is useful for HB culture. Those that are commercially available are known. The concentration of Flt-3 in the serum-free medium varies depending on the type of Flt-3 used, and is not particularly limited as long as it is useful for HB culture. In the case of human recombinant Flt-3 ligand, for example, 10 -1000 ng / mL, preferably 50-500 ng / mL, more preferably about 100 ng / mL.

  Thrombopoietin (TPO) used in the present invention is a kind of hematopoietic cytokine and is known to act specifically on the process of producing megakaryocytes from hematopoietic stem cells and promote the production of megakaryocytes. The origin of TPO used in the present invention is not particularly limited, but a recombinant that is expected to be stably supplied is preferable, and a human recombinant is particularly preferable. Those that are commercially available are known. The concentration of TPO in the serum-free medium varies depending on the type of TPO used and is not particularly limited as long as it is useful for HB culture. In the case of human recombinant TPO, for example, 1 to 500 ng / mL, preferably 5 to 100 ng / mL, more preferably about 20 ng / mL.

  In the serum-free medium used in the first step, the SCF, IL-6 are used from the viewpoint of more efficiently amplifying EPC or increasing the total number of amplified cells, as shown in Examples described later. In addition to Flt-3 and TPO, it may be preferable to further contain growth factors, especially vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) that can be used in the present invention is a growth factor that specifically acts on EPC, and is known to be produced mainly in cells surrounding blood vessels. Several types of VEGF proteins of different sizes are produced by alternative splicing, but VEGF used in the present invention may be any type of VEGF as long as EPC is amplified more efficiently or the total number of amplified cells is increased. . VEGF ₁₆₅ is preferred. The origin of VEGF and the like are not particularly limited, but a recombinant that can be stably supplied is preferable, and a human recombinant is particularly preferable. Those that are commercially available are known. The concentration of VEGF in the serum-free medium varies depending on the type of VEGF used and is not particularly limited as long as it is useful for HB culture. In the case of human recombinant VEGF ₁₆₅ , for example, about 5 to 500 ng / mL, Preferably it is about 5-200 ng / mL, More preferably, it is about 100 ng / mL.

  A preferred serum-free medium in the present invention is a serum-free medium containing about 100 ng / mL SCF, about 20 ng / mL IL-6, 100 ng / mL Flt-3, about 20 ng / mL TPO, about 100 ng Those containing / mL of VEGF are preferred.

  Each of the above components is dissolved in serum-free medium at a predetermined concentration, or a concentrated liquid (stock solution) of each component is prepared in advance and diluted to a predetermined concentration in serum-free medium to be used in the present invention. Serum-free medium for amplifying single cells can be prepared. For example, after dissolving components necessary for a commercially available serum-free medium to a predetermined concentration, sterilize by filtration sterilization or the like, or aseptically add a stock solution sterilized by filter sterilization to a commercially available serum-free medium, It can be prepared by diluting. Filtration sterilization can be performed according to a method commonly practiced in this field, for example, using a 0.22 μm or 0.45 μm Millipore filter.

The culture of HB in a serum-free medium containing the above-described factors is performed by seeding single cells of HB in a serum-free medium containing the above-mentioned factors. The culture conditions for HB are not particularly limited, and can be carried out under conditions that are usually performed in the art. For example, the cells are cultured for about 7 days at 37 ° C. in a 5% CO ₂ atmosphere.

  In the first step, “amplification” of a cell means increasing the number of cells while maintaining the undifferentiated state of the cell as much as possible. Amplification of HB in the first step can be achieved by culturing HB using, for example, the above-mentioned factors at the above-mentioned concentrations, and blood progenitor cells (HSPC) and vascular endothelial progenitor cells (EPC) are amplified. Preferably, this step (first step) is performed in an incubator suitable for single cell culture, such as a 96-well plate, due to the nature of single cell culture.

  In the present invention, following the preparation and amplification (first step) of single cells of tissue stem cells, a step (second step) of producing colonies from the amplified cells is performed. The method of the second step is not particularly limited as long as a desired cell lineage colony is produced, but due to the property of producing colonies, an incubator having a larger culture area than the first step, for example, a 24-well plate It is preferable to carry out in an incubator. When the tissue stem cells are HB and the cell lineage is a blood cell lineage, a method using a methylcellulose medium for blood lineage that is a semi-solid medium is preferably used. By this culture, blood precursor cell colonies (HSPC colonies) are produced from HB. In the present invention, four types of HSPC colonies with different cell structures appear. Since such four types of HSPC colonies appear at the time when usually 10 days or more, for example, 12 days have elapsed since the HB suspension amplified in the first step was seeded on the semi-solid medium, In the present invention, HSPC colonies are formed, for example, by culturing for 12 days. The four types of colonies formed are erythroblast colonies (BFU-E), granulocyte / macrophage colonies (CFU-GM), macrophage colonies (CFU-M) and mixed colonies (CFU-Mix). The type of colony formed can be easily identified from its color and form. In addition, individual colonies can be collected and subjected to RT-PCR and confirmed at the gene expression level.

  The semi-solid medium used for HSPC colony production is not particularly limited as long as it enables HSPC colony formation. For example, the semi-solid medium known as a medium for proliferation of undifferentiated cells such as hematopoietic stem cells is known. Medium can be used. Examples of such a semi-solid medium include methyl cellulose medium, matrigel, collagen gel, meviol gel, and the like. When a methylcellulose medium is used as the semi-solid medium, the concentration of methylcellulose in the medium is not particularly limited, but is, for example, about 0.5 to 5%, preferably about 0.7 to 3%, more preferably about 1%. Used in

  In addition, when the tissue stem cell is an HB cell and the cell lineage is a vascular endothelial cell lineage, it can be suitably carried out according to the methods described in Patent Documents 1 and 2. Specifically, it includes culturing HB in a semi-solid medium containing vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). By such culture, vascular endothelial precursor cell colonies (EPC colonies) are produced from HB. In the present invention, two types of EPC colonies with different constituent cell sizes appear. Such EPC colonies of two sizes appear when the suspension of HB amplified in the first step is inoculated on a semi-solid medium and usually 10 days or more, for example, 14-18 days have passed. Therefore, in order to form EPC colonies in the present invention, the cells are cultured for 14 to 18 days, for example. Of the two types of colonies that are formed, colonies composed of large cells (endothelial cell-like large cell colonies; also referred to as CFU-Large cell like EPC, also referred to as large cell colonies) mainly contain cells having a major axis of about 50 to 200 μm. A colony composed of small cells (endothelial cell-like small cell colony; CFU-small cell like EPC, also referred to as small cell colony) mainly includes cells having a diameter of about 20 μm or less (for example, about 10 to 20 μm). Here, “mainly” means that about 30%, preferably about 50%, particularly preferably about 70% of the cell population constituting the colony is a cell having a major axis of about 50 to 200 μm (in the case of a large cell colony) or a diameter of 20 μm. It means the following cells (in the case of small cell colonies). When bone marrow fluid, umbilical cord blood, peripheral blood, etc. are collected over time from the test subject, and the EPC colony assay is performed on the collected sample, the time required for colony appearance after mobilization is large cell colony and small cell colony. Is known to be different. Large cell colonies appear slightly later than small cell colonies. That is, it is suggested that EPCs having different differentiation stages exist. Endothelial cell-like small cells that appear at an early stage can be said to be early differentiation stage EPCs, and endothelial cell-like large cells that appear at a later stage can be said to be late differentiation stage EPCs. In the analysis method of the present invention, EPCs having different degrees of differentiation can be distinguished based on the difference in cell size forming colonies as described above.

  The semi-solid medium used for EPC colony production is not particularly limited as long as it enables EPC colony formation. For example, a semi-solid medium known as a medium for proliferation of undifferentiated cells such as hematopoietic stem cells is known. Medium can be used. Examples of such a semi-solid medium include methyl cellulose medium, matrigel, collagen gel, meviol gel, and the like. When a methylcellulose medium is used as the semi-solid medium, the concentration of methylcellulose in the medium is not particularly limited, but is, for example, about 0.5 to 5%, preferably about 0.7 to 3%, more preferably about 1%. Used in

The semi-solid medium used for EPC colony production preferably contains vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Examples of VEGF used here are the same as those added to the serum-free medium in the first step. The concentration of VEGF in the semi-solid medium varies depending on the type of VEGF used and is not particularly limited as long as it is suitable for EPC colony production. In the case of human recombinant VEGF ₁₆₅ , for example, about 5 to 500 ng / mL, Preferably it is about 20-100 ng / mL, More preferably, it is about 50 ng / mL. The bFGF used here is a single-chain peptide of about 18 kDa and an isoelectric point of 9.0, and is known to have functions such as angiogenesis, various cell growth promotion, and differentiation induction. The origin of bFGF used in the present invention is not particularly limited, but a recombinant that is expected to be stably supplied is preferred, and a human recombinant is particularly preferred. Those that are commercially available are known. The concentration of bFGF in the semi-solid medium varies depending on the type of bFGF used, and is not particularly limited as long as it is suitable for EPC colony production. For example, when human recombinant bFGF is used, about 5 to 500 ng / mL, Preferably it is about 20-100 ng / mL, More preferably, it is about 50 ng / mL.

  In the second step, the semi-solid medium used for the production of EPC colonies further includes stem cell factor (SCF), interleukin, in addition to VEGF and / or bFGF, from the viewpoint of more efficiently forming EPC colonies. 3 (IL-3), insulin-like growth factor (IGF) and epithelial cell growth factor (EGF), one or more factors, preferably three or more factors, more preferably all factors It is preferable to contain.

  Examples of the stem cell factor (SCF) are the same as those added to the serum-free medium in the first step. The concentration of SCF in the semi-solid medium varies depending on the type of SCF used, and is not particularly limited as long as EPC colonies can be formed more efficiently. However, in the case of human recombinant SCF, 10 to 1000 ng / mL, preferably Is 50 to 500 ng / mL, more preferably about 100 ng / mL.

  Interleukin 3 (IL-3) is known as a cytokine that acts on hematopoietic stem cells and progenitor cells of various blood cell lineages and promotes their proliferation and differentiation. It consists of 152 amino acids in humans and 166 residues in mice, but has an apparent molecular weight of 28,000 due to sugar chain modification. IL-3 used in the present invention is appropriately selected according to the origin of EPC intended for colony production, but human IL-3 is preferable when used for colony production of human EPC, and stable supply is expected. Particularly preferred are recombinants. Those that are commercially available are known. The concentration of IL-3 in the semi-solid medium also varies depending on the type of IL-3 used, and is not particularly limited as long as EPC colonies can be produced more efficiently. If human recombinant IL-3 is used, About 1 to 500 ng / mL, preferably about 5 to 100 ng / mL, more preferably about 20 ng / mL.

  Insulin-like growth factor (IGF) is a polypeptide with a primary structure similar to proinsulin, also called somatomedin, having a molecular weight of about 7000, and it is known that there are two types, IGF-I and IGF-II, which are similar to each other. It has been. Both have similar actions and are known to promote the growth of various cells in vitro. The IGF used in the present invention may be any type of IGF as long as it enables EPC colony production. IGF-I is preferred. The origin of IGF is not particularly limited, but a recombinant that is expected to be stably supplied is preferred, and a human recombinant is particularly preferred. Those that are commercially available are known. The concentration of IGF in the semi-solid medium varies depending on the type of IGF used, and is not particularly limited as long as EPC colonies can be produced more efficiently. However, when human recombinant IGF-I is used, about 5 to 500 ng is used. / ML, preferably about 20-100 ng / mL, more preferably about 50 ng / mL.

  Epidermal growth factor (EGF) is a 53-amino acid protein having an action of promoting differentiation and proliferation of epithelial cells, and is known to have no sugar chain bound thereto. It has 3 disulfide bonds at about 6 kDa. The origin of EGF used in the present invention is not particularly limited, but a recombinant that is expected to be stably supplied is preferred, and a human recombinant is particularly preferred. Those that are commercially available are known. The concentration of EGF in the semi-solid medium varies depending on the type of EGF to be used, and is not particularly limited as long as EPC colonies can be produced more efficiently. When human recombinant EGF is used, about 5 to 500 ng / mL. , Preferably about 20-100 ng / mL, more preferably about 50 ng / mL.

  The semi-solid medium used for EPC colony production is selected from the group consisting of VEGF and bFGF, and SCF, IL-3, IGF and EGF, from the viewpoint of more efficiently forming EPC colonies. It is preferable to further contain serum and / or heparin in addition to two or more factors.

  When serum is used, its origin and the like are not particularly limited, but it is expected to be used in a relatively large amount because it is added to the medium. Accordingly, commercially available serum from bovine, horse, human, etc. (eg fetal serum) is used. More preferred is fetal calf serum (FCS). The serum is preferably used after inactivation. The concentration of serum in the semi-solid medium varies depending on the type of serum used, and is not particularly limited as long as EPC colonies can be produced more efficiently. In the case of FCS, about 10 to 50%, preferably about 15 to 40%, more preferably about 30%.

  Heparin is a glucosaminoglycan having a repeating structure of a disaccharide of a uronic acid residue containing either D-glucuronic acid or L-iduronic acid and D-glucosamine as a skeleton. Heparin is abundant in the small intestine and lungs of mammals, and many commercial products are extracts derived from porcine intestine, and the molecular weight is about 7,000 to 25,000. The heparin used in the present invention may be derived from animal tissue, or may be chemically and physically degraded low molecular weight heparin as long as it enables EPC colony production. For example, porcine intestinal heparin is used. Those that are commercially available are known. The concentration of heparin in the semi-solid medium varies depending on the type of heparin used, and is not particularly limited as long as EPC colonies can be produced more efficiently. In the case of porcine intestinal heparin, about 0.2 to 10 U / ML, preferably about 1 to 5 U / mL, more preferably about 2 U / mL.

  Each physiologically active substance described above is dissolved in a predetermined concentration in a semi-solid medium, or a concentrated liquid (stock solution) of each physiologically active substance is prepared in advance and diluted to a predetermined concentration in a semi-solid medium. A semi-solid medium for producing EPC colonies can be prepared. For example, a physiologically active substance necessary for a commercially available semi-solid medium is dissolved to a predetermined concentration and then sterilized by filtration sterilization or the like. Alternatively, a stock solution sterilized by filter sterilization or the like is aseptically added to a commercially available semi-solid medium. The bioactive substance-containing semi-solid medium used in the present invention can be prepared by adding and diluting. Filtration sterilization can be performed according to a method commonly practiced in this field, for example, using a 0.22 μm or 0.45 μm Millipore filter.

  Although the production of EPC colonies can be confirmed visually, whether or not the obtained colonies are actually composed of EPC is, for example, the ability to take up acetylated LDL (acLDL) or the ability to bind to UEA-1 lectin, It can be carried out by confirming the expression of VE-cadherin, KDR (also referred to as VEGFR-2), vWF (for example, by RT-PCR or fluorescent immunohistochemical method) and the like. For example, when colonies are double-stained with DiI-labeled acetylated LDL (acLDL-DiI) and FITC-labeled UEA-1 lectin (UEA-1 lectin-FITC), EPC is stained together.

  The present invention includes a step of culturing single cells of stem cells and grouping them based on the mode of colonies produced from the single cells. For example, when HB is used as a stem cell, CFU-Large cell like EPC or CFU-small cell like EPC is formed if it is an EPC colony, and BFU-E, CFU-GM, CFU-M, or CFU is formed if it is an HSPC colony. -Mix is produced. Each single cell is grouped by examining what colonies it produces.

  For example, in the case of a vascular endothelial cell line, a cell that produces CFU-Large cell like EPC but does not produce CFU-small cell like EPC (corresponding to the LVZ region in the examples described later), does not produce CFU-Large cell like EPC. Can be divided into three groups: cells producing CFU-small cell like EPC (corresponding to the EVZ region in the examples described later), and cells producing both (corresponding to the IVZ region in the examples described later). Table 1 below shows an example of the mode of colonies produced for each blood cell lineage. In this way, the characteristics of individual cells that can be divided into groups based on each mode are referred to as “cell fate”, and the present invention analyzes such “cell fate”. Enable and provide its applications.

  In the present invention, the number of single cells having the same cell fate is measured with respect to the “cell fate” grouped based on the mode of the produced colonies (third step). For example, a single cell that produces colonies of CFU-Mix, BFU-E, CFU-GM, and CFU-M after amplification and differentiation (first step) has cell fate 1. Yes (see Table 1 above). A single cell that will produce all four colonies has a cell fate of 1, and the number is measured. For example, when HB is seeded in a 96-well plate in the first step, one HB exists in each well. Since the cell fate of the HB single cell is determined by evaluating what kind of colonies derived from HB existing in each well, the single cell fate frequency is the same as the cell fate frequency. It can be easily known by measuring the number of cells, that is, the number of wells.

  In the present invention, EPC colonies and HSPC colonies produced from tissue stem cells, particularly HB single cells, can be assayed at the same time, and the present inventors have referred to such assays as HELIC (Hematopoietic and Endothelial Lineage Commitment) assays. Named. The HELIC assay can simultaneously analyze the cell fate of the blood cell lineage and the cell fate of the vascular endothelial cell lineage.

  In the analysis method of the present invention, in addition to the above-described factors necessary for culturing single cells, the purpose is to examine whether or not the determination of cell fate is affected, or the determination of cell fate is already affected. Alternatively, a known factor may be added (hereinafter also referred to as an added factor). Examples of the additive factor used in the present invention include clinically applied drugs or drugs scheduled for clinical application, including cytokines, growth factors, sex hormones, antineoplastic agents, antihyperlipidemic agents, antihypertensive agents and the like. Examples of cytokines include stromal derived factor-1 (SDF-1) having an angiogenesis promoting action. Examples of growth factors include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), angiopoietin-1 and the like. Examples of sex hormones include estrogen and progesterone having an angiogenesis promoting action. As clinically applied drugs, reductions in vascular endothelial progenitor cell activity have been reported, particularly in lifestyle-related diseases such as hyperlipidemia and hypertension. Thus, in particular, it is possible to analyze the influence of these therapeutic agents on the fate determination of vascular endothelial cells from HB. Conversely, it is also possible to analyze the influence of an antineoplastic drug on the fate determination from HB to vascular endothelial cells in various malignant tumors with high angiogenic ability.

The present invention also provides a method of screening for compounds that affect cell fate using the analysis method of the present invention.
Specifically, the following method is exemplified.
(1) A single stem cell is prepared.
Similar to the analysis method of the present invention described above, when single cell selection is required, it can be performed at this stage (for example, selection of CD133 positive cells).
(2) The single cell obtained in (1) above is amplified and cultured to produce colonies.
First step: The culture conditions are appropriately set according to the origin of the stem cells. The two groups are cultured in the presence of the test compound and in the absence of the test compound. Except for the presence or absence of the test compound, this step is carried out according to the analysis method of the present invention described above.
Second step: Colonies are produced from the stem cells amplified in the presence or absence of the test compound. This step is performed according to the analysis method of the present invention described above.
(3) Group (cell fate) based on the mode of colonies produced, and measure the number (frequency) of single cells having the same cell fate. This step is performed according to the analysis method of the present invention described above.
(4) By examining the relationship between the cell fate and its frequency in the presence and absence of the test compound, it is determined whether or not the test compound affects the cell fate. For example, if the cell fate in the presence and absence of the test compound is graphed as described below, and the shape of the graph changes significantly, it is determined that the test compound has an effect on the cell fate. Can do.

  The amount of the test compound to be present is set so as not to let the cell have a lethal effect, and screening is preferably performed by changing the dose stepwise. In the above example, the test compound is used at the stage of stem cell amplification (first step). However, the test compound can be used at the stage of colony production (second step) depending on the purpose.

The analysis method of the present invention cultivates single cells of stem cells, groups them based on the mode of colonies produced from the single cells, and develops the types of the obtained groups in the X-axis direction as cell fate. A graph can be obtained by expanding the number of cells having the same cell fate as the frequency in the Y-axis direction. By graphing, cell fate can be quantified, and the influence of the test compound described above can be quantified. The type of graph is not particularly limited as long as the cell fate is associated with the frequency, but a line graph obtained by expanding the cell fate on the X axis and the frequency on the Y axis, and statistically processing it. The bar graph obtained by the above is preferable.
The analysis method of the present invention may be performed for one cell line derived from a stem cell, but is preferably performed simultaneously for two or more cell lines. When two cell lineages are performed simultaneously, the causal relationship between both cell lines can be known by superimposing the graphs of both cell lines.

  The graph for cell fate analysis of the present invention includes any type of graph obtained by classifying cell fate and measuring the frequency in addition to the above-described line graph and bar graph.

  The present invention also provides a method for confirming the enrichment of cells destined for a specific cell lineage by applying the analysis method of the present invention. For example, when the stem cell is HB, the stem cell is induced to differentiate into a blood cell lineage or a vascular endothelial cell line in the future. While analyzing which cell line is destined by the analysis method of the present invention, when a cell destined to a specific cell line is amplified, its concentration (purity) can also be confirmed. Here, “amplify and concentrate cells destined to a specific cell lineage” means that the purity of the target cell lineage is increased by amplification culture that is destined to the target cell line from a pluripotent stem cell. Raise (concentrate) and increase (amplify) the number of cells in the cell lineage. As a method for amplifying and concentrating cells destined to a specific cell line, for example, a vascular endothelial cell line, a method of incubating in a serum-free medium containing stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 and thrombopoietin (Patent Document 1). When an attempt is made to amplify and concentrate vascular endothelial progenitor cells (EPC) from HB according to the method described in Patent Document 1, the purity or concentration can be examined by this method. A vascular endothelial cell lineage colony is produced from the EPC-containing cell population obtained through amplification and concentration, and the frequency thereof is examined. If amplification and concentration are appropriately performed, the frequency of cells that produce EPC colonies increases. By making a graph, it is possible to judge more easily and to quantify.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the examples are described for explaining the present invention, and do not limit the present invention in any way.

Example 1: HELIC Assay The experimental protocol is shown in FIG.
(1) Isolation and amplification of CD133 positive cells HB was used as stem cells. As the cell suspension containing HB, a cell suspension containing mononuclear cells derived from cord blood (CB) was used. First, the collected blood was layered on Histopaque-1077, and mononuclear cells were separated by density gradient centrifugation. The separated mononuclear cells were washed with PBS-EDTA, and the mononuclear cells after removing the platelets were collected and suspended in a buffer solution to prepare a cell suspension.
Next, the cell suspension was subjected to MACS using an anti-CD133 antibody to recover CD133 positive cells. In detail, it carried out according to the protocol of a package insert using the CD133 positive cell isolation kit (The product made by Militenyi Biotec, catalog number 130-050-801).
60 plates (60 wells) were seeded on 1 plate, one CD133 positive cell per well (single cell) per well by BD FACS Vantage system (FIG. 2a-1). Similarly, 4 plates were prepared. The plate was cultured in a CO ₂ incubator at 37 ° C. in the presence of 5% CO ₂ for 7 days (FIG. 2a-2). The plate used was a 96-well Primaria plate (BD Labware). A culture solution with 100 μL of VEGF (100 ng / mL) added or not added was used per well. The culture solution used was aseptically added the components shown in Table 2 below to a Stem span serum-free medium.

In Table 2, “h” indicates that it is derived from human, and “r” indicates that it is a recombinant produced by genetic engineering. Other abbreviations are as described above.
Seven days later, the number of amplified cells in the well in which the cells were amplified was counted.

(2) Colony Production 300 μL per well of methylcellulose medium (H4435, Stem Cell Tec.) For blood cell lineage colony production was placed in a 24-well suspension culture dish (Greiner).
Moreover, the methylcellulose culture medium for vascular endothelial cell lineage colony production was produced based on the composition shown in Table 3 using the methylcellulose culture medium (H4236, Stem Cell Tec.). That is, each component shown in Table 3 was aseptically added to the methylcellulose medium so as to have a predetermined concentration. 300 μl per well was placed in a 24-well Primalia dish (BD Labware).

In Table 3, “h” indicates that it is derived from human, and “r” indicates that it is a recombinant produced by genetic engineering. Other abbreviations are as described above.
For each well in which single cells were amplified, half of the cell culture solution (50 μL each) was added as it was to the previously prepared methylcellulose medium for blood cell lineage and vascular endothelial cell lineage.
HSPC colony production ability and EPC colony production ability were evaluated after 12 days and 14-18 days, respectively.
HSPC colonies were determined as CFU-Mix, CFU-GM, CFU-M, and BFU-E (FIGS. 2a-5, 6, 7, and 8), and EPC colonies were endothelial cell-like small cell colonies (CFU-small cell). like EPC) and endothelial cell-like large cell colonies (CFU-large cell like EPC) were determined (FIGS. 2a-3, 4).
As is clear from FIG. 2a, some of the single CD133-positive cells were found to have blood HSPC colonies and vascular endothelial EPC colonies, and were confirmed to be HB-containing fractions. .

In addition, RNA was collected from CFU-small cell like EPC and CFU-large cell like EPC produced from a single cell, and vascular endothelial growth factor was confirmed by RT-PCR. Expression of receptor-2 (vascular endothelial groth factor receptor-2; VEGFR-2), vascular endothelial cadherin (VE cadherin), endothelial NO synthase (endothelial nitric oxide syntase; eNOS) It was confirmed that these colonies were vascular endothelial EPC colonies (FIG. 2b).
The procedure for RT-PCT is shown below.
Total RNA was extracted using RNeasy micro (Mini) kit (QIAGEN). Reverse transcription was performed using superscript (invitrogen) to prepare cDNA. PCR reaction was performed using Ex Taq kit (TaKaRa). The primers used for the PCR reaction are as follows.
eNOS:
forward: 5'-AACCACATCAAGTATGCCACCAACC-3 '(SEQ ID NO: 1)
reverse: 5'-CGTGCCGATCTCAGTGCTCA-3 '(SEQ ID NO: 2)
VEGFR-2:
forward: 5'-TGTGATATGTCTGAGACTGAATGCG-3 '(SEQ ID NO: 3)
reverse: 5'-CCCAGCATTTCACACTATGGTACC-3 '(SEQ ID NO: 4)
VE cadherin:
forward: 5'-ACACCAAGCTCACCCTTCGTC-3 '(SEQ ID NO: 5)
reverse: 5'-CTTGTCATGCACCAGTTTGGC-3 '(SEQ ID NO: 6)

Furthermore, it was cultured in a methylcellulose medium for vascular endothelial cell lineage colony production using a 35 mm Primalia dish in which a sterilized 24 mm x 24 mm glass plate (Matsunami Glass Industry) with 1000 CD133 positive cells was placed, and the CFU-small that appeared Cell-like EPC and CFU-large cell-like EPC colonies were immunostained with VEGFR-2, eNOS, and VE cadherin antibodies and observed with a fluorescence microscope. All colonies were stained and vascular endothelial EPC colonies. This was confirmed (FIG. 3).
The procedure for immunostaining is shown below.
After removing methylcellulose by adding 1 mL of ice-cooled PBS, 1 mL of 4% paraformaldehyde was added, and incubation was performed at 4 ° C. for 3 hours. Thereafter, washing was carried out twice by incubation with PBS for 5 minutes. Blocking was performed with 5% goat serum-PBS-0.1% Triton-X100 for 30 minutes. Each primary antibody mouse anti-human VEGFR-2 antibody (V9134, Sigma), mouse anti-human eNOS (NOS Type III 610296, BD Pharmingen) was used in 5% goat serum- at dilutions of 1:50 and 1: 500, respectively. Dilute with PBS-0.1% Triton-X100. Mouse anti-human VE cadherin antibody (HM2032, Hycult) was diluted with 5 mM goat serum-PBS-0.1% Triton-X100 containing 2 mM CaCl ₂ at a magnification of 1:10. Diluted primary antibody was added and incubated at 4 ° C. for 12 hours. Each wash was washed 3 times with 5 min PBS incubation. Alexa488-goat anti-mouse IgG (H + L) (Molecular Probe) was added after dilution with 5% goat serum-PBS-0.1% Triton-X100 at a magnification of 1: 500, and incubated at room temperature for 1 hour. Washing was performed 3 times by incubation with PBS for 5 minutes. A colony glass stained with VECTASHIELD HardSet Mounting Medium with DAPI (H-1500, Vector Lab) was embedded in a fluorescent microscope slide glass. The images were observed and photographed with confocal microscopy (FluoView ^™ FV1000, Olympus).

(3) Graph preparation and analysis The colony produced by said (2) was analyzed. In the single evaluation of HSPC colonies, it is confirmed that there is a total of 10 cell fates in the absence of VEGF (VEGF-) and in the presence of VEGF (VEGF +) from the mode of production colonies from a single cell. Okay (Fig. 4a, X-axis direction). Cell fate was developed on the X axis, and the number (frequency) of cells having such cell fate was plotted on the Y axis. The frequency of single cells determined for any cell fate is not significantly different in the two groups of VEGF− and VEGF + (60 single cells × 4 samples = 240 single cells / group). Not (FIG. 4b).
In single evaluation of EPC colonies, there were a total of three cell fates in VEGF- and VEGF + from the mode of production colonies from single cells (Figure 4c, X-axis direction). That is, the cells were classified into three types of determined cell fate areas: single cells producing only small cell EPC colonies, single cells producing only large cell EPC colonies alone, and single cells producing both colonies. From this, the degree of differentiation of single cells destined to EPC is as follows: CFU-small cell like EPC alone early basal zone = EVZ, both intermediate basal zone = IVZ, CFU-large cell like EPC It was found that classification was possible in the form of late basal zone = LVZ. Based on this, when the frequency of fate determination to EPC of single HB cells was evaluated, no change was observed from EVZ to LVZ in the VEGF− group, but about doubling EVZ in LVZ in the VEGF + group, VEGF-group. The frequency was about twice that of LVZ, and it was found that VEGF promotes the differentiation of CD133-positive cells into the endothelial system. In addition, the frequency of determining HB single cells to EPC is about 1.5 times higher in the VEGF + group than in the VEGF− group, and it has become clear that VEGF has the ability to determine the cell lineage to EPC for CD133-positive cells. (FIGS. 4c and d).

  Furthermore, both cell lines were analyzed simultaneously by overlapping cell lineage determination methods in the blood system and vascular endothelial system. By assessing blood and vascular endothelial fate at the same time, it was found that there were a total of 34 cell fate in the VEGF- and VEGF + groups. Speaking only of the vascular endothelial cell lineage, the cells were classified into four types of determined cell fate regions of EVZ, IVZ, LVZ, and hematopoietic zone = HZ having no ability to produce EPC colonies but only blood colony producing ability. VEGF was found to significantly reduce the cell fate frequency of HZ to about 40% (FIGS. 4e and f). That is, it is considered that differentiation from HB to blood cell lineage is suppressed and differentiation into vascular endothelial cell lineage is promoted.

  The HELIC assay showed that VEGF promotes the cell lineage fate determination and differentiation of CD133 positive cells into the vascular endothelial system and suppresses cell fate determination into the blood system alone. As described above, the HELIC assay is a cell fate determination that cannot be analyzed by single evaluation of the blood system or vascular endothelial system by analyzing the determination of the blood system or vascular endothelial system of the single cell of the cell fraction containing HB. It was revealed that can be accurately, quantitatively and easily evaluated. The development of this assay will be groundbreaking in hematology and vascular biology stem cell biology.

  The analysis method of the present invention makes it possible to graph and quantify the fate of blood system and vascular endothelial cell lineage, particularly by simultaneously graphing and quantifying the fate of blood system and vascular endothelial cell lineage. Thus, it becomes possible to clarify and quantify the cell fate, which could not be discriminated by the conventional single cell lineage analysis method.

SEQ ID NO: 1 RT-PCR primer for eNOS detection (forward)
SEQ ID NO: 2 Primer for RT-PCR for detecting eNOS (reverse)
SEQ ID NO: 3 Primer for RT-PCR for detection of VEGFR-2 (forward)
SEQ ID NO: 4 Primer for RT-PCR for detecting VEGFR-2 (reverse)
SEQ ID NO: 5 RT-PCR primer for VE cadherin detection (forward)
SEQ ID NO: 6 Primer for RT-PCR for detecting VE Cadherin (reverse)

FIG. 1 is a diagram showing the procedure of the analysis method of the present invention. FIG. 2 is a diagram showing that CD133-positive cells in which blood HSPC colonies and vascular endothelial EPC colonies are produced are present and are HB-containing fractions. FIG. 2a shows a phase contrast image, and FIG. 2b is a diagram in which production of EPC colonies was confirmed by RT-PCR. FIG. 2a1 shows the state of the cells 12 hours after seeding with the CD133-positive single cells, and FIG. 2a2 shows the state of the cells after the single cells were amplified and cultured for 7 days. 2a3, 4 show the state of EPC colony production, and FIGS. 2a5, 6, 7, 8 show the state of HSPC colony production. FIG. 3 is an image showing the result of fluorescent staining indicating that the produced colonies are EPC colonies. FIG. 4 is a graph showing the relationship between cell fate and its frequency. FIG. 4a is a graph showing the relationship between the cell fate of a blood cell lineage and the frequency of cells having that fate. There are 10 types of cell fate. FIG. 4 b is a graph showing the results of examining the effect of VEGF on the fate determination of blood cell lineages. FIG. 4c is a graph showing the relationship between the cell fate of the vascular endothelial cell lineage and the frequency of cells having that fate. There are three types of cell fate. FIG. 4d is a graph showing the results of examining the effect of VEGF on the fate determination of vascular endothelial cell lineage. FIG. 4e is a graph in which the relationship between the cell fates of the blood cell lineage and the vascular endothelial cell lineage and the frequency of the cells having the fate are overlapped and divided into 34 types of cell fates. FIG. 4f is a graph showing the results of examining the effect of VEGF on the fate determination of blood cell lineages and vascular endothelial cell lineages.

Claims (18)

Preparing a single stem cell and amplifying the resulting single cell (first step);
A step of culturing the amplified cells to produce colonies (second step), and grouping the single cells based on the mode of the produced colonies, and measuring the number of single cells classified into the same group Process (3rd process)
A method for analyzing cell fate of a stem cell, comprising: The method according to claim 1, wherein the stem cells are blood hemangioblasts. The method according to claim 2, wherein the blood hemangioblasts are derived from bone marrow, umbilical cord blood or peripheral blood. The method according to claim 2, wherein the blood hemangioblasts are CD34 positive and / or CD133 positive. The method according to claim 2, wherein the produced colony is either a blood cell colony or a vascular endothelial cell colony. The blood cell colony is at least selected from the group consisting of erythroblast colony (BFU-E), granulocyte / macrophage colony (CFU-GM), macrophage colony (CFU-M) and mixed colony (CFU-Mix). 6. The method of claim 5, wherein the method is one. The vascular endothelial cell colony is at least one selected from the group consisting of an endothelial cell-like large cell colony (CFU-Large cell like EPC) and an endothelial cell-like small cell colony (CFU-small cell like EPC). 5. The method according to 5. The method according to claim 1, wherein the first step is performed in the presence or absence of an additive factor. 9. The method of claim 8, wherein the additive factor is a growth factor. A method for screening a compound that affects the cell fate of a stem cell, comprising performing the method according to claim 1. A method for confirming the enrichment of cells destined from a stem cell to a specific cell line, comprising performing the method according to claim 1. The method according to claim 11, wherein the stem cells are blood hemangioblasts, and the cells destined for a specific cell lineage are vascular endothelial progenitor cells. A single stem cell is cultured, grouped based on the mode of colonies produced from the single cell, the number of single cells classified into the same group is measured, and each group belongs to that group Graph for analyzing cell fate of stem cells, obtained by matching the number of single cells. A single cell of stem cells is cultured and grouped based on the mode of colonies produced from the single cell, and the type of the obtained group is expanded in the X-axis direction as a cell fate and classified into the same group The graph for analysis of the cell fate of a stem cell obtained by expanding in the Y-axis direction with the number of single cells as a frequency. The graph according to claim 13 or 14, wherein the stem cells are blood hemangioblasts. The graph according to claim 15, wherein the produced colony is either a blood cell colony or a vascular endothelial cell colony. The blood cell colony is at least selected from the group consisting of erythroblast colony (BFU-E), granulocyte / macrophage colony (CFU-GM), macrophage colony (CFU-M) and mixed colony (CFU-Mix). The graph according to claim 16, which is one type. The vascular endothelial cell colony is at least one selected from the group consisting of an endothelial cell-like large cell colony (CFU-Large cell like EPC) and an endothelial cell-like small cell colony (CFU-small cell like EPC). 16. The graph according to 16.