JP2009148273A - Method for differentiating and proliferating endothelial cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for differentiating and proliferating an endothelial cell from an embryonic stem cell through a vessel precursor cell. <P>SOLUTION: The method for effectively differentiating and proliferating the endothelial cell, especially an arteria-based endothelial cell by increasing an intracellular cAMP concentration by using adrenomedullin, vascular endothelial growth factor (VEGF) or the like in a vascular endothelial growth factor receptor Flk-1 positive cell differentiated from the embryonic stem cell. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel method for differentiating and proliferating endothelial cells from vascular progenitor cells and subjecting them to cell transplantation. Specifically, the present invention relates to a novel method for effectively promoting the differentiation and proliferation of endothelial cells from vascular progenitor cells and obtaining cells for transplantation into a living body that is expected to have a therapeutic effect. That is, it teaches a method capable of securing a sufficient amount of embryonic stem cell-derived cells grown to a transplantable differentiation stage.

From mouse embryonic stem cells that can differentiate into both endothelial cells and mural cells (pericytes and vascular smooth muscle cells) that make up blood vessels and can construct blood vessels in vitro and in vivo by in vivo transplantation. Differentiated and proliferated “vascular progenitor cells (VPC)” were identified and reported (Non-patent Document 1). VPC is a positive cell for Flk-1 (VEGFR-2), which is a receptor for vascular endothelial growth factor (VEGF). The present inventors have reported that VPCs form blood vessels and increase blood flow when transplanted into adult mice (Non-patent Document 2).
In particular, the present invention aims to effectively induce differentiation from vascular progenitor cells obtained from embryonic stem cells to endothelial cells and promote proliferation, and to increase intracellular cAMP concentration, cAMP analogs and adrenomedullin And so on.

Adrenomedullin (AM) is a 52 amino acid peptide first reported in 1993 by Kitamura et al. AM is contained in many tissues such as normal adrenal medulla and heart, blood vessels (endothelial cells, vascular smooth muscle cells), lungs, brain and kidneys. AM is a complex of its receptor calcitonin-receptor-like receptor (CRLR) and receptor-activity-modyfying protein-2 (RAMP-2) It binds to the body and exerts its biological activity by increasing intracellular cAMP concentration (Non-patent Document 4). In AM knockout mice, embryonic lethality is caused by angiogenesis insufficiency, and AM has been shown to be an important physiological factor for the development and differentiation of blood vessels during development (Non-patent Document 5). It has been reported that AM is expressed in the developing cardiovascular tissue even in the fetal stage (Non-patent Document 6). In addition, according to reports of a plurality of laboratories including the present inventors, AM promotes the migration and proliferation of cultured endothelial cells (Non-patent Document 7), and further promotes the renewal of the blood vessel itself or its regeneration by administration to a living body. (Non-Patent Document 8).
However, to date, no effect of AM on embryonic stem cells has been reported. There is also no report on the effect of increasing intracellular cAMP concentration in VPC.

  The present inventors have discovered that an increase in intracellular cAMP concentration in an embryonic stem cell-derived VPC dramatically promotes differentiation into endothelial cells, and that the differentiated endothelial cells acquire properties as arterial endothelial cells. Furthermore, it has been found that administration of AM is effective as a method for increasing intracellular cAMP concentration of VPC. The expression of arterial endothelial cells is recognized at a neovascular site, and is considered to have an important significance during angiogenesis (Non-patent Document 9). However, regarding the endothelialization of endothelial cells, including the expression of EphrinB2, which is an arterial endothelial marker, there have been very few reports on the regulatory mechanism, and there have been no studies on embryonic stem cells.

  In order to differentiate from embryonic stem cells into fixed cells such as nerve cells, bone cells, immune cells and pancreatic β cells, cAMP has been described as a reagent to be added to the medium (Patent Document 1). However, there is no description regarding differentiation and proliferation induction into vascular endothelial cells. As a method for inducing vascular endothelial cells from embryonic stem cells, a method of culturing using a medium containing serum and vascular endothelial growth factor has been described (Patent Document 2). However, there is no description of adrenomedullin, prostanoic acid or intracellular cAMP here.

Special Table 2002-504362 JP2003-9854

Yamasita J. et al., Nature. 2000, 408 (6808): 92-96 Yurugi-Kobayashi T et al., Blood. 2003, 101 (7): 2675-2678 Kitamura K. et al., Biochem. Biophys. Res. Commun (192: 553-560 (1993) McLatchie LM et al., Nature 393: 333-339 (1998) Shindo T et al. Circulation, 104: 1964-71 (2001) Montuenga LM, etc., Endocrinology 138: 440-451 (1997) Miyashita K et al., Hypertens Res 26: S93-98 (2003) Miyashita K et al., FEBS Lett. 2003, 544: 86-92 Galeet et al., Developmenta1 Biology 230: 151-160 (2001)

  Currently, 1 in 3 Japanese people die from stroke or ischemic heart disease due to arteriosclerosis, and these diseases are bedridden, dementia and other patients' quality of life (QOL) Is significantly reduced. Therefore, overcoming arteriosclerosis is one of the most important issues in modern medicine. As a fundamental treatment method for arteriosclerosis, a method of newly regenerating a blood vessel at a vascular occlusion site is most desirable. The present invention provides a method for the development of a new revascularization therapy by transplanting an embryonic stem cell-derived VPC and endothelial cells differentiated and proliferated from the VPC.

The present invention has many features and leads to the development of new revascularization medicine using embryonic stem cell-derived VPC and endothelial cells differentiated and proliferated from VPC.
In order to differentiate and proliferate endothelial cells in large quantities from embryonic stem cells, a method for revascularization medical treatment is provided which includes a step for increasing the cAMP concentration in a medium.
In order to increase cAMP concentration in a medium, a method of revascularization medicine comprising a step of using a cAMP analog and a phosphodiesterase inhibitor, or a prostaglandin I2 derivative beraprost or a salt thereof, which is a prostanoic acid derivative, is provided.
In addition, as another method for increasing the cAMP concentration in the culture medium, a method of revascularization medicine comprising the administration of AM and its derivatives or the administration of an expression plasmid incorporating the AM gene, and further the step of activating the AM receptor. I will provide a.

That is, the present invention
(1) It is characterized by differentiating and proliferating vascular progenitor cells, specifically vascular progenitor cells derived from embryonic stem cells, more specifically isolated vascular progenitor cells in the presence of a substance that increases intracellular cAMP concentration. A method of differentiating and proliferating endothelial cells, particularly arterial endothelial cells, from vascular progenitor cells;
(2) isolated endothelial cells prepared by the method of the present invention;
(3) A method for preparing vascular structural tissue in vitro, comprising culturing the isolated endothelial cells of the present invention in a three-dimensional manner;
(4) A method of increasing blood flow by forming blood vessels at the site of transplantation, wherein the isolated endothelial cells of the present invention are transplanted into a living body;
(5) A method for treating a vascular occlusive disease, which comprises transplanting the isolated endothelial cell of the present invention into a living body;
(6) A composition for differentiating and proliferating endothelial cells or arterial endothelial cells from vascular progenitor cells, comprising an intracellular cAMP concentration increasing substance;
(7) A composition for forming a vascular structural tissue, comprising a substance that increases intracellular cAMP concentration;
(8) A pharmaceutical composition for treating a vascular occlusive disease, comprising a substance that increases intracellular cAMP concentration;
(9) A composition for promoting differentiation and proliferation ability of an angiogenesis-inducing factor, comprising a substance that increases intracellular cAMP concentration.

The advantages of the present invention will become apparent from the following description of the invention.
By increasing the intracellular cAMP concentration, endothelial cells can be induced in a much larger amount than conventional endothelial cell induction methods.
Endothelial cells differentiated and proliferated by increasing intracellular cAMP concentration are mainly arterial endothelial cells having markers such as EphrinB2, which are differentiated and proliferated from embryonic stem cells by the method of the present invention. Means that it has properties as an arterial endothelial cell.
Endothelial cells that are differentiated and proliferated by increasing the intracellular cAMP concentration can be constructed in vitro in a three-dimensional manner, and the vascular structure can be constructed. Can increase blood flow.
According to the present invention, revascularization is possible, and vascular obstructive diseases, that is, atherosclerosis obliterans (ASO), ischemic heart disease, cerebrovascular disorders (including cerebral infarction, dementia), etc. , And the fundamental treatment of resuming and increasing blood flow is possible, leading to the development of revascularization medicine.

It is a photograph which shows the form of the cell which shows promotion of the concentration-dependent endothelial cell differentiation by 8-br-cAMP. It is a photograph which shows the form of the cell which shows promotion of endothelial cell differentiation by IBMX. It is a photograph which shows the form of the cell which shows concentration-dependent acceleration | stimulation of PECAM-1 positive endothelial cell differentiation by AM. It is a photograph which shows the form of the cell which shows inhibition of endothelial cell differentiation by AM derivative. It is the result of the flow cytometry which shows the induction effect of VE-cadherin / PECAM-1 positive endothelial cell by AM and cAMP. It is a photograph which shows the form of the cell which shows the property of the endothelial cell differentiated from embryonic stem cell origin VPC.

(1) A method for differentiating and proliferating endothelial cells from vascular progenitor cells, characterized by differentiating and proliferating vascular progenitor cells in the presence of an intracellular cAMP concentration-increasing substance. J. et al., Nature. 2000, 408 (6808): 92-96, can be differentiated from embryonic stem cells and obtained. Embryonic stem cells, also called embryonic stem cells or ES cells, transfer cells that constitute the inner cell mass, which is an undifferentiated stem cell present in the blastocyst, to the culture, and frequently It can be established by repeating dissociation and passage. These cells are known to maintain normal karyotypes and to proliferate and pass through indefinitely, maintaining the pluripotency that can differentiate into all types of cells, just like the inner cell mass. Yes.
In addition to embryonic stem cells, vascular progenitor cells can be prepared from other pluripotent stem cells capable of self-replication, such as fetal and bone marrow-derived cells. In the present invention, pluripotent stem cells derived from various animals such as humans, monkeys and mice can be used.

  In the present invention, “isolated vascular progenitor cells” can be used. “Isolated” refers to a state of being physically separated from a living body or being free from contamination with other types of cells. Vascular progenitor cells can be isolated using a cell sorter or the like as a marker of positive for Flk-1 (VEGFR-2), a receptor for vascular endothelial growth factor (VEGF).

In the present invention, “intracellular cAMP concentration increasing substance” means any substance that increases the cAMP concentration in vascular progenitor cells, including cAMP analogs, adrenomedullin and adrenomedullin derivatives, phosphodiesterase inhibitors, And prostanoic acid derivatives.
A “cAMP analog” is a compound having a structure similar to cAMP that increases intracellular cAMP concentration when contacted with a cell, such as 8-bromoadenosine-3 ′: 5′-cyclic monolin. Acid sodium salt (8-br-cAMP sodium salt), dibutyryl-cAMP.
`` Adrenomedullin '' or `` AM '' is a 52 amino acid peptide contained in many tissues such as normal adrenal medulla and heart, blood vessels (endothelial cells, vascular smooth muscle cells), lungs, brain, kidneys, etc. Kitamura K. et al., Biochem. Biophys. Res. Commun (192: 553-560 (1993)) AM can be easily prepared by the method described in JP-A-7-196693.
AM is its receptor calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein-2 (RAMP-2) or receptor It is known to bind to a complex with receptor-activity-modifying protein-3 (RAMP-3) and exert its biological activity by increasing intracellular cAMP concentration. Yes. Therefore, the term “adrenomedullin derivative” means any substance that activates the adrenomedullin receptor localized on embryonic stem cells. For example, one or a number of 52 amino acid residues constituting adrenomedullin Examples include peptides having an amino acid sequence in which one amino acid is deleted, substituted or added, and retaining the activity of adrenomedullin, such as AM (22-52) and CGRP (8-37). .

“Phosphodiesterase inhibitors” include, for example, 3-isobutylmethylxanthine (IBMX).
The “prostanoic acid derivative” used in the present invention means a number of unsaturated fatty acids having prostanoic acid as a basic skeleton and having a double bond, a hydroxy group or the like added thereto. Xanes and leukotrienes are included. In the present invention, a prostaglandin I ₂ derivative is particularly preferable, and beraprost or a salt thereof is particularly preferable. Here, “salt” means an ionic compound.

“Differentiating and proliferating” vascular progenitor cells in the presence of an intracellular cAMP concentration-increasing substance means culturing vascular progenitor cells with the intracellular cAMP concentration-increasing substance. The culture is carried out using a medium having a composition suitable for the purpose of cell maintenance or differentiation, adding the substance for increasing intracellular cAMP concentration of the present invention thereto, and under an environment known to those skilled in the art such as temperature. For example, as a medium to be used, a medium obtained by adding 5 × 10 ⁻⁵ M2-mercaptoethanol, 10% FCS or the like to a minimum essential medium for cell culture is usually used. Other substances useful for culture such as antibiotics can be added to each medium, and substitutes having equivalent functions may be used in place of the respective components. In addition, each component of the medium is used after being sterilized by a suitable method. In the present invention, angiogenesis-inducing factor, for example, vascular endothelial growth factor (VEGF) is added to the medium for culturing.

  In one embodiment of the invention, vascular progenitor cells are cultured in the presence of 10% FCS and VEGF in a culture vessel coated with collagen IV. When cultured under such conditions, the endothelial cells of the present invention are optimally obtained usually on the third day. Alternatively, it can be differentiated into endothelial cells to some extent even in a serum-free state.

  Endothelial cell isolation is directed against an appropriate label, an antibody to Flk-1 conjugated with allophycocyanin (APC), and an E-cadherin conjugated with an appropriate label, fluorescein isothiocyanate (FITC). The antibody can be used to collect a Flk-1-positive and E-cadherin-negative cell fraction using a cell sorter or the like. In addition, using an APC-conjugated VE-cadherin antibody, a FITC-conjugated PECAM-1 antibody, and a FITC-conjugated CD34 antibody, the differentiation of endothelial cells and the properties of the differentiated endothelial cells can be examined with a FACS cell sorter. In the present invention, the antibody used for cell isolation / purification may be either a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies are preferred when using FACS. Such antibodies can be easily prepared by those skilled in the art, or commercially available antibodies may be used.

  Many of the endothelial cells isolated in the present invention are arterial endothelial cells when they are Flk-1 positive, PECAM-1 positive, EphB4 binding molecule positive, and E-cadherin negative. Arterial endothelial cells mean endothelial cells that constitute arterial blood vessels. Endothelial cells grown in the presence of substances that increase intracellular cAMP concentration are not 100%, but many have the properties of arterial endothelial cells with increased expression of EphB4 binding molecule (EphrinB2), while intracellular cAMP Endothelial cells grown in the absence of a concentration-enhancing substance mainly have the properties of venous endothelial cells without EphB4 binding molecule expression (FIG. 6).

  In the present specification, “positive” for a certain molecule means that the cell expresses the molecule, and “negative” means not expressing the molecule. Whether a cell expresses a certain molecule can be determined by FACS or the like. The FACS usually includes a flow cytometer, a laser generator, an optical system, a data processing device, and a cell sorting device. By FACS, it is possible to measure scattered light (forward scattered light and side scattered light) and fluorescent signal information on cells fluorescently labeled with a specific substance, and to sort out cells that emit specific signal information.

(2) Isolated endothelial cell prepared by the method of the present invention As another aspect, the present invention provides an isolated endothelial cell prepared by the method of the present invention.
In the present invention, “isolated” means a state in which the substance is physically separated from a living body or a state in which there is no contamination of other types of cells.
Many of the endothelial cells isolated in the present invention are Flk-1 positive, PECAM-1 positive, EphB4 binding molecule positive and E-cadherin negative, as described above, and are arterial endothelial cells.

  The cells of the present invention preferably further express an adrenomedullin receptor. The adrenomedullin receptor can be confirmed using, for example, a mouse anti-AM monoclonal antibody (Michibata H et al., Kidney Int 53: 979-985 (1998)) in the case of a mouse.

(3) A method for preparing a vascular structure tissue in vitro, characterized by culturing the isolated arterial endothelial cells or endothelial cells of the present invention in a three-dimensional manner. For example, it means three-dimensional culture in which cells are embedded in a gel and cultured, and in the present invention, this can promote the construction of a vascular structure tissue. “Vascular structure tissue” means a tissue that constitutes a blood vessel such as an artery, vein, or lymphatic vessel.

(4) A method for forming blood vessels at a transplant site and increasing blood flow, characterized by transplanting the isolated arterial endothelial cells or endothelial cells of the present invention into a living body. And intravascular injection (intraarterial, intravenous).

(5) A method for treating a vascular occlusive disease, characterized by transplanting the isolated arterial endothelial cell or endothelial cell of the present invention into a living body. Arteriosclerosis, ischemic heart disease, cerebrovascular disorder such as cerebral infarction, dementia.
In this aspect, the present invention relates to the use of the isolated arterial endothelial cells or endothelial cells of the present invention for treating vascular occlusive disease.

(6) A composition for differentiating and proliferating endothelial cells or arterial endothelial cells from vascular progenitor cells, which contains a substance that increases intracellular cAMP concentration. The present invention relates to a composition containing a substance that increases intracellular cAMP concentration, which is used to differentiate and proliferate cells.
The “intracellular cAMP concentration increasing substance” is as described above. Such a composition is usually used for adding an intracellular cAMP concentration increasing substance to an embryonic stem cell-derived vascular progenitor cell for the purpose of increasing the intracellular cAMP concentration ex vivo.
In this aspect, the present invention relates to the use of an intracellular cAMP concentration increasing substance for preparing a composition for differentiating and proliferating endothelial cells or arterial endothelial cells from vascular progenitor cells.
The composition in this embodiment is typically a cell culture medium containing a substance that increases intracellular cAMP concentration.

(7) Composition for Forming Vascular Structure Tissue Containing Substance for Increasing Intracellular cAMP Concentration The invention of this aspect is used as a reagent for forming vascular structure tissue.

(8) A pharmaceutical composition for treating a vascular occlusive disease, comprising a substance that increases intracellular cAMP concentration. The invention of this embodiment is usually administered to a living body by injection. The administration site varies depending on the target disease and is usually intravenous administration. The dose is appropriately determined by a doctor and is usually 0.05 μg / min / kg body weight.

(9) A composition for promoting the differentiation and proliferation ability of an angiogenesis inducing factor, which contains a substance that increases intracellular cAMP concentration. As another aspect, the present invention promotes, increases and enhances angiogenesis of an angiogenesis inducing factor. The present invention relates to a composition containing a substance that increases intracellular cAMP concentration.
In the present invention, the angiogenesis-inducing factor has an activity of promoting the formation of new capillaries by accelerating various processes of vascular endothelial cell sprouting from the destruction site, migration / proliferation outside the blood vessel, and lumen formation. Means a factor. This includes, for example, vascular endothelial growth factor (VEGF), fibroblast growth factor-2, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and the like. The angiogenesis-inducing factor used in the present invention can be either naturally derived or prepared by genetic manipulation.

(10)
In another aspect, the present invention provides an embryonic stem cell according to the method of the present invention for preparing a composition containing an intracellular cAMP-concentrating substance, forming a vascular structural tissue, or treating a vascular occlusive disease. The use of an in vitro differentiation culture system from endothelial cells to arterial endothelial cells is provided. In addition, a method (for example, PNA microarray method) for comprehensively analyzing gene expression in cells undergoing differentiation in an in vitro differentiation culture system from embryonic stem cells to endothelial cells or arterial endothelial cells according to the method of the present invention. provide. Furthermore, the development of a gene expression database makes it possible to find new angiogenesis-inducing genes from the database.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.
Reference Example Differentiation and Proliferation from Embryonic Stem Cells to Flk-1 Positive Cells Differentiation and proliferation of Flk-1 positive cells by the method previously reported by the present inventors (Yamashita J et al., Nature 408: 92-96 (2000)) That is, mouse embryonic stem cells (provided by Dr. BM I Evans of Department of Molecular Embryology, Max-Planck-Institute fur Immunobiology, Freiburg, Germany) with 10% fetal calf serum (FCS) and 5 × 10 ⁻⁵ M 4-Mercaptoethanol-containing alpha minimum essential medium (differentiation medium) on a culture dish (Becton Dakinson) coated with type IV collagen for 4 days at 37 ° C. in an environment of 5% CO ₂ and 95% air Cultured.
Differentiated and proliferated Flk-1-positive cells were isolated with a purity of 97% or more by FACS Vantage (Becton Deakinson) according to a previously reported method (Nishikawa SI et al., Development 125: 1747-1457 (1998)). .

Example 1
Effect of various additives on differentiation and proliferation from Flk-1-positive cells to endothelial cells APC-conjugated anti-Flk-1 monoclonal antibody (AVAIS12 (Dev.Growth Differ. 39, 729-740 ( 1997))) and FITC-conjugated anti-E-cadherin monoclonal antibody (ECCD2 (Cell Struct. Funct. 11, 245-252 (1986))) was used to collect Flk-1-positive and E-cadherin-negative cell fractions. .
Endothelial cell differentiation and the properties of differentiated endothelial cells were investigated using APC-conjugated VE-cadherin antibody (VECD1 (Proc. Assoc. Am. Physicians 109, 362-371 (1997))), FITC-conjugated PECAM-1 antibody (Mec13 .3 (Pharmigen)), and FITC-conjugated CD34 antibody (RAM34 (Pharmigen)) using a FACS cell sorter.

Mouse embryonic stem cell-derived VPC, which is a purified Flk-1-positive cell isolated in the Reference Example, is present on a collagen IV-coated culture dish in the presence of 10% FCS and VEGF (10 ng / ml, R and D system). When cultured for 4 days, it differentiated into two types of cells: PECAM-1 and VE-cadherin positive endothelial cells and SMA positive wall cells. Under these conditions, the differentiation rate from VPC to endothelial cells was found to be about 30% of the total cells by FACS analysis.
Rat AM and AM derivative human AM- (22-52) -amide (both peptide studies) as an additive to differentiation medium containing 10% FCS and VEGF (50 ng / ml) (this culture medium is hereinafter referred to as medium A) ), 8-bromoadenosine-3 ': 5'-cyclic monophosphate sodium salt (8-br-cyclic AMP), IBMX, and except that beraprost, which is a prostaglandin I2 derivative, was added and cultured in the same manner. The effect of various additives on the differentiation and proliferation from Flk-1 positive cells to endothelial cells was examined.
8-Br-cAMP is added to medium A, and embryonic stem cell-derived VPCs are cultured on a collagen IV-coated culture dish for 4 days, and then the immunology by PECAM-1 which is an endothelial cell marker and SMA which is a wall cell marker Double staining was performed. The obtained results are shown in FIG. 1 (PECAM-1 (partially indicated by arrows in the figure), SMA (partly indicated by arrows in the figure), 40 times). FIG. 1 shows that the differentiation into endothelial cells is promoted in a concentration-dependent manner at a concentration of 8-br-cAMP of 0.1 mM to 0.5 mM. In addition, it was found that about 70% of the cells differentiated into endothelial cells by addition of 0.5 mM 8-br-cAMP. In the absence of VEGF, endothelial cell differentiation promoting action of 8-brc-AMP was not observed.

FIG. 2 shows the results obtained by adding IBMX (10 ⁻⁴ M) to medium A and culturing in the same manner. Compared with medium A alone, the addition of IBMX10 ^-4 M promoted the differentiation of PECAM-1-positive endothelial cells (blue) from embryonic stem cell-derived VPC (Fig. 2, PECAM-1 (partly with arrows) And SMA (partially indicated by arrows), 100 times).
The result of adding adrenomedullin (AM) to medium A and culturing in the same manner is shown in FIG. Addition of AM (10 ^-9 to 10 ^-6 M) promoted the differentiation and proliferation of PECAM-1-positive endothelial cells from embryonic stem cell-derived VPC in a concentration-dependent manner compared to culture with medium A alone (to endothelial cells) (FIG. 3, PECAM-1 (partly indicated by arrows), SMA (partly indicated by arrows), 200 times). Cells differentiated into endothelial cells by AM10 ^-6 M addition reached 50% of the total cells. The effect of promoting differentiation of PECAM-1-positive endothelial cells (blue) from VPCs derived from embryonic stem cells observed when AM (10 ⁻⁷ M) is added is the effect of AM (22-52) (10 ⁻⁴ M), which is an AM derivative. It was inhibited to the same extent as the medium A culture condition by addition (see FIG. 4, PECAM-1 (partly indicated by arrows), SMA (partly indicated by arrows), 100 Times)). From this, it was considered that the differentiation promoting effect of AM is an action through the AM receptor. A similar effect was observed when beraprost (10 nM), a prostaglandin I2 derivative, was added.

Furthermore, the effect of inducing VE-cadherin / PECAM-1-positive endothelial cells by AM and cAMP was confirmed by FACS analysis. The results are shown in FIG. According to FIG. 5, the induction of VE-cad / CD31-positive cells is 20% with Medium A alone, while the high induction effect is 37.9% when AM10 ^-6 M is added and 51.9% when 8-br-cAMP is added. Admitted.

Example 2
Characterization of endothelial cells differentiated from embryonic stem cells
It is known that EphrinB2 is expressed in arterial endothelial cells and its receptor EphB4 is expressed in venous endothelial cells (Adams, R et al Genes Dev. 13: 295-306 (1999)).
Therefore, after the cells that had been differentiated and proliferated in Example 1 were fixed with methanol by the immunohistochemical method (Hirashima et al., Blood 93: 1253-1263 (1999)) already reported, Mec 13.3 or anti-alpha smooth muscle actin (SMA) antibody (1A4 (SIGMA)) treated with alkaline phosphatase-conjugated anti-rat IgG antibody (EphB4-Fe chimera (ZYMED)) or horseradish peroxidase (HRP) -conjugated anti-mouse IgG antibody (ZYMED) Etc. were used for staining.
Furthermore, EphinB2 expression was examined by using EphB4 and human IgG chimeric protein to which EphrinB2 specifically binds, and perosidase-conjugated anti-human IgGFc goat IgG (ICN / CAPPEL). At this time, a Tyramid Signal Amplication Biotin system (Perkin Elmer Life Sciences) was used in order to increase the detection sensitivity of the binding between EphB4-human IgG chimeric protein and EphrinB2.

8-brcAMP (0.5 mM) was added to medium A, and the properties of endothelial cells differentiated from embryonic stem cells as described above were examined. The obtained result is shown in FIG. PECAM-1-positive endothelial cells differentiated from VPC under the culture conditions of medium A alone (FIG. 6a left, right) were EphrinB2-negative endothelial cells that did not bind to EphB4-Fc chimera (FIG. 6a). When AM10 ^-6 M and 8-br-cAMP 0.5 mM were added, most of the PECAM-1 positive endothelial cells had EphB4-Fc chimera binding properties (Fig. 6b, c middle, right) EphrinB2 positive endothelial cells ( FIG. 6, PECAM-1 (left), EphB4-Fc chimera (center, right), 200 times). FIG. 6 shows that when 8-brcAMP (0.5 mM) is added to medium A, a significant increase in the expression of an EphB4 binding molecule (considered EphrinB2) is observed compared to medium A alone (P <0.05). . A similar effect was observed when AM10 ^-6 M was added (P <0.05).
This indicates that AM or 8-br-cAMP differentiated arterial endothelial cells from vascular progenitor cells derived from embryonic stem cells.

Example 3
The expression of AM in mouse embryonic stem cells was performed using the mouse anti-AM monoclonal antibody (Michibata H et al., Kidney Int 53: 979-985 (1998)) already developed by the present inventors. (Naruko T et al., Circulation 94: 3103-3108 (1996)).
We examined the expression of AM and its receptor in vascular cells differentiated from embryonic stem cell-derived VPC, and we found AM expression in mural cells differentiated from embryonic stem cell-derived VPC, while AM receptor expression in endothelial cells Admitted.
This indicates that AM can act on endothelial cells derived from embryonic stem cells, and that AM secreted from simultaneously differentiated mural cells can act on endothelial cells.

The above results clearly show that by administering cAMP analog and IBMX, or prostaglandin I2 derivative beraprost or its salt or AM, or AM to embryonic stem cell-derived VPC, and increasing intracellular cAMP concentration, endothelium It shows that the differentiation of cells progresses dramatically.
The results further indicate that the above-mentioned additive promoted the differentiation and proliferation into endothelial cells of arterial system important in angiogenesis.
These effects of AM on embryonic stem cells are thought to be expressed through AM receptor mediation. Therefore, the present invention provides a new method for the development of revascularization medicine using embryonic stem cells. In other words, when embryonic stem cells are transplanted into the ischemic region to form blood vessels in the body and restore local blood flow, a new aim is to secure quantitative and adjust qualitative changes in embryonic stem cell-derived transplanted endothelial cells A cell processing method is provided.
In addition, a novel method for inducing revascularization is also a method for exhaustively analyzing gene expression of cells in the differentiation process in in vitro differentiation culture system from embryonic stem cells to endothelial cells or arterial endothelial cells using DNA microarray method etc. It is thought that it is useful for the search of genes and the development of revascularization drugs.

Claims (22)

  In vitro, vascular progenitor cells are differentiated and proliferated in the presence of a substance that increases intracellular cAMP concentration selected from the group consisting of adrenomedullin and adrenomedullin derivatives. To differentiate and proliferate.   The method according to claim 1, wherein the vascular progenitor cells are differentiated and proliferated from embryonic stem cells.   The method according to claim 1 or 2, wherein the isolated vascular progenitor cells are expanded.   The method according to any one of claims 1 to 3, wherein the adrenomedullin derivative activates an adrenomedullin receptor localized on embryonic stem cells.   The method according to any one of claims 1 to 4, wherein arterial endothelial cells are differentiated and proliferated from vascular progenitor cells.   The method according to claim 5, wherein the arterial endothelial cells are Flk-1 positive, PECAM-1 positive, EphB4 binding molecule positive, and E-cadherin negative.   The method according to claim 6, wherein the arterial endothelial cells further express an adrenomedullin receptor.   The method according to any one of claims 1 to 7, wherein the growth is performed in the presence of an angiogenic factor.   Claims for proliferating endothelial cells from vascular progenitor cells, wherein the cells are proliferated in the presence of a substance that increases intracellular cAMP concentration selected from the group consisting of vascular endothelial growth factor (VEGF) and adrenomedullin and adrenomedullin derivatives. 8. The method according to 8.   An isolated endothelial cell prepared by the method of any one of claims 1-9.   A method for preparing a vascular structural tissue in vitro, comprising culturing isolated endothelial cells prepared by the method according to any one of claims 1 to 9 in a three-dimensional manner.     The pharmaceutical composition containing endothelial cells according to claim 10 for forming blood vessels at a transplant site and increasing blood flow.     The pharmaceutical composition containing the endothelial cell according to claim 10 for treating a vascular occlusive disease.   The pharmaceutical composition according to claim 13, wherein the vascular occlusive disease is obstructive arteriosclerosis, ischemic heart disease, or cerebrovascular disorder.   A composition for differentiating and proliferating endothelial cells from vascular progenitor cells, comprising an intracellular cAMP concentration increasing substance selected from the group consisting of adrenomedullin and adrenomedullin derivatives.   A composition for differentiating and proliferating arterial endothelial cells from vascular progenitor cells, comprising an intracellular cAMP concentration increasing substance selected from the group consisting of adrenomedullin and adrenomedullin derivatives.   The composition according to claim 15 or 16, for differentiating and proliferating endothelial cells from vascular progenitor cells derived from embryonic stem cells.   The composition according to any one of claims 15 to 17, further comprising an angiogenic factor.   19. The composition of claim 18, comprising a vascular endothelial growth factor (VEGF) and a substance that increases intracellular cAMP concentration selected from the group consisting of adrenomedullin and adrenomedullin derivatives.   The composition according to any one of claims 15 to 19, which is a cell culture medium.   A composition for forming a vascular structural tissue, comprising an intracellular cAMP concentration increasing substance selected from the group consisting of adrenomedullin and an adrenomedullin derivative.   A composition for promoting differentiation and proliferation ability in an angiogenesis-inducing factor, comprising an intracellular cAMP concentration increasing substance selected from the group consisting of adrenomedullin and an adrenomedullin derivative.

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