WO2013108949A1 - Method for preparing human embryonic stem cell-derived perivascular progenitor cells, and composition for cell therapy containing same - Google Patents

Abstract

The present invention relates to a method for preparing perivascular progenitor cells from stem cell embryoid bodies, a cell therapy composition for preventing or treating cardiovascular diseases or retinal diseases containing the same as an active ingredient, and a method for treating cardiovascular diseases or retinal diseases using the composition, and more specifically, to a method for preparing perivascular progenitor cells from stem cells comprising the steps of: treating stem cell embryoid bodies with the BMP4 growth factor to induce the differentiation thereof into mesodermal cells; separating embryoid bodies containing the differentiated mesodermal cells into single cells; and culturing the same in a substrate-coated culture dish to proliferate only the adhered cells by natural selection, and to perivascular progenitor cells prepared by the method, a cell therapy composition for preventing or treating cardiovascular diseases or retinal diseases containing the same as an active ingredient, and a method for treating cardiovascular diseases or retinal diseases using the composition.

Description

Method for preparing human embryonic stem cell-derived perivascular progenitor cells and cell therapy composition comprising the same

The present invention is a method for producing perivascular progenitor cells from stem cells derived embryonic body, perivascular progenitor cells prepared by the above method, cardiovascular disease comprising the same as an active ingredient Or it relates to a cell therapy composition for the prevention or treatment of retinal disease, and a method for preventing or treating cardiovascular disease or retinal disease using the composition. More specifically, the stem cell-derived embryonic bodies are treated with BMP4 growth factors to induce differentiation into mesodermal cells, and after separating the embryoid bodies containing the differentiated mesodermal cells into single cells, the substrate is coated. Method for producing perivascular progenitor cells from stem cells that naturally grow only cells attached and cultured in a culture dish, perivascular progenitor cells prepared by the above method, prevention or treatment of cardiovascular disease or retinal disease comprising the same as an active ingredient And a method for treating cardiovascular disease or retinal disease using the composition.

In the vascular system, various types of cells come into contact with the microvascular basement membrane (BM), and these cells interact with vascular endothelial cells to participate in maintaining and determining the characteristics of the microvascular vessels. Among these cells, the cells that interact and specifically distribute between vascular endothelial cells and BM are perivascular progenitor cells. Perivascular progenitor cells are a kind of mesenchymal stem cells and are known to exist in capillaries, precapillaries, posterior capillaries, or collecting ducts of various human tissues such as the placenta, bone marrow, and adipose tissue. Although perivascular progenitor cells are similar to vascular smooth muscle cells, they are distinguished by markers, shape, and location in contact with the vascular endothelium. In the aorta or vein, vascular smooth muscle cells have a special structure and can be distinguished from perivascular cells because they are separated from BM where extracellular matrix is present. Perivascular progenitor cells, on the other hand, are present in the vascular endothelial basement membrane.

Perivascular progenitor cells are known to surround the outer portion of the vascular endothelial by about 10 to 50%. These differences indicate that the degree of enveloping blood vessels varies according to the type and proportion of perivascular progenitor cells and endothelial cells. In skeletal muscle, their ratio is about 1: 100 (perivascular progenitor cells: vascular endothelial cells) and in the retina, the ratio is 1: 1. Perivascular progenitor cells surround the blood vessels most often in the central nervous system microvascular. The reason why the distribution of perivascular progenitor cells in the central nervous system is higher than in other organs is not yet clear, but it is likely that it contributes to the formation and maintenance of the blood-brain barrier. In addition, perivascular progenitor cells show morphological diversity depending on the organs present. In the central nervous system, the shape of perivascular progenitor cells is flat or elongated. It appears in a variety of forms, from radioactive perivascular cells that are in contact with the outside of blood vessels to rounded or glomerular mesial cells, or to localized contact with BM.

Perivascular progenitor cells can differentiate into vascular smooth muscle cells during vascular growth or remodeling during development, whereas vascular smooth muscle cells can also differentiate into perivascular progenitor cells. Perivascular progenitor cells are also characterized by mesenchymal stem cells with the ability to differentiate into fibroblasts, osteoblasts, chondrocytes and adipocytes. Recently, it has been reported that perivascular progenitor cells identified in the human body have differentiation ability into cardiomyocytes.

Although the existence and role of perivascular progenitor cells have been underestimated for a long time, the perivascular progenitor cells have recently attracted attention as essential elements for the formation of microvascular system by acting as progenitor cells through various differentiation ability. , A key regulator in stabilization, maturation and remodeling. It is also considered an important target in understanding vascular physiology and studying the causes and treatment of vascular diseases. Peripheral progenitor cell studies recently identified in human tissues have identified their new function and emphasize the fact that perivascular progenitor cells have great biological significance. The demand for mass supply of perivascular progenitor cells is increasing. However, there is a lack of typical markers for perivascular progenitor cells, and it is practically impossible to identify enough cells to meet demand in human tissue. In addition, the amount of perivascular progenitor cells in limited tissues does not meet the demand for perivascular progenitor cells.

The present inventors have made diligent efforts to find a method for mass production of perivascular progenitor cells. As a result, the present inventors have developed a differentiation technique for obtaining perivascular progenitor cells by applying coating substrate-dependent single cell attachment technology from embryonic bodies derived from human embryonic stem cells. It was. In this process, it was found that high-purity perivascular progenitor cells can be isolated and obtained, thus completing the present invention.

An object of the present invention is to treat a BMP4 growth factor in an embryoid body derived from stem cells to differentiate into mesodermal cells, separating the differentiated mesodermal cells into single cells, and coating the substrate thereon. It is to provide a method for producing perivascular progenitor cells from stem cells, comprising the step of culturing in a cultured plate attached only to the cells attached naturally.

Another object of the present invention to provide a perivascular progenitor cell prepared by the above method.

Another object of the present invention is to provide a method for differentiating the perivascular progenitor cells into perivascular cells, smooth muscle cells, muscle cells, adipocytes or bone cells.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising the perivascular progenitor cells as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of retinal diseases, including the perivascular progenitor cells as an active ingredient.

It is another object of the present invention to provide a method for preventing or treating cardiovascular disease comprising administering the composition to an individual.

It is another object of the present invention to provide a method for preventing or treating a retinal disease comprising administering the composition to an individual.

By the method of producing perivascular progenitor cells derived from stem cells characterized by natural selection by substrate-specific adhesion culture of the present invention, the perivascular progenitor cells are purely separated without additional processes such as flow cytometry or magnetic cell sorting. It was confirmed that it can be grown by. In addition, it was confirmed that it has a characteristic multipotent ability of perivascular progenitor cells. Therefore, stem cell-derived perivascular progenitor cells prepared by the method of the present invention can be usefully used as a cell therapy composition for the treatment or prevention of cardiovascular diseases or retinal diseases.

1 is a diagram showing mesoderm specific induced differentiation of BMP4. (A) shows the induced differentiation of H9 hESC embryoid bodies by BMP4 added from the start of culture (EB day 0) to EB day 8. (B) shows the percentage of viable cells positive for KDR and PDGFRβ determined by flow cytometry on EB day 6. Bars represent standard error for the mean of three independent experiments compared to the NT group; ** p <0.01, *** p <0.001.

2 is a diagram showing substrate dependent adhesion of single cells derived from BMP4-EB. (A) single cells from undifferentiated H9 hESCs dispensed into cell culture dishes, (B) single cells from EB on day 6 of BMP4 induced differentiation dispensed into uncoated plastic Petri dishes, (C, D) coated with collagen Single cells from naturally-differentiated EBs dispensed into the dish, (E, F) single cells from EBs on day 6 of BMP4 induced differentiation dispensed into collagen coated dishes. Black scale bar, 20 μιη.

Figure 3 is a diagram showing substrate dependent natural selection of PDGFRβ-positive cell populations in BMP4-EB. (A) shows each cell dispensed in collagen coated dishes after separation of distinct PDGFRβ-positive and negative populations using FACS sorter, (B) shows single cell collagen matrix derived from BMP4-EB without separation Figure shows dependent attachment. Black scale bar, 20 μιη.

Figure 4 is a diagram showing the flow cytometry results of hESC-PVPC. (A) shows flow cytometry and (B) shows the overlap of each histogram for 8 independent hESC-PVPCs.

Figure 5 shows the generalized perivascular potential of hESC-PVPC. (A, B) are the results of microscopic analysis of long-term cultured H9 hESC-PVPC and human placenta-induced perivascular cells. Positive for blue DAPI, green NG2, negative for red SMA. (E) Introduce DiO-labeled hESC-PVPCs to the ablumenal surface of DiI-labeled HUVECs cell lines in vascular induction tunnels using EC-perivascular tube coassembly in three-dimensional collagen matrix. (F) Bilayer ellipsoid formation of DiO-labeled HUVECs (internal) and DiI-labeled hESC-PVPCs (outer). White scale bar, 20 mm.

6 is a diagram showing the characteristics of smooth muscle derived from hESC-PVPC. (A) RT-PCR analysis of αSMA expression in hESC, hESC-PVPC, PVPC-induced day 2-4 SMLC and mature SMLC, (B) is the real-time PCR analysis (n = 3). The significance level is * p <0.05, *** p <0.0001. Immunocytochemistry shows the expression of αSMA in hESC (C), SMLC (D) and contractile SMLC (E and F). αSMA (red), DAPI (blue), SM-MHC (green, E) and calponin (green, F). Scale bar is 100 μm.

7 is a diagram showing the characteristics of myocytes derived from hESC-PVPC. Morphological changes in hESC-PVPC under myocyte differentiation conditions. (A, D) is hESC-PVPC, (B, E) is myocyte differentiation medium and (C, D) is typical cell morphology in myocyte growth medium, respectively. Black scale bars in (A-C) show 20 μm (D-F) immunocytochemistry showing myocyte specific Desmin expression. Desmine is red and DAPI is blue.

8 shows the possibility of skeletal differentiation of hESC-PVPC. (A) and (B) confirmed the lipogenesis by microscopic analysis and oil red staining after differentiation under lipogenic conditions, and by (C) and (D) microscopic analysis and differentiation of alizarin red staining under bone formation conditions under (C) and (D) Formation was confirmed.

9 is a diagram showing the pigment transfer analysis between hESC-PVPC and other vascular systems. DiI-labeled hESC-PVPC (top panel: red) with calcine-labeled HUVEC (center panel, left: green), UASMC (center panel, center: green) or cardiomyocytes (center panel, right: green) Co-cultured for 1 hour in a 37 ° C. incubator of 5% CO 2. Calcine's green-fluorescence was detected in hESC-PVPC (bottom panel) with red-fluorescence of DiI (bottom panel: red green, asterisk).

10 is a diagram showing the improvement of ischemic limb rescue / blood flow in ischemic Fuji after hESC-PVPC transplantation. (A) shows image analysis showing a series of lower limb recovery on days 0 and 28 after treatment, and (B) shows blood perfusion rate of ischemic limbs measured by series of laser Doppler images on days 0 and 28 after treatment. Indicates.

FIG. 11 shows capillary density in ischemic myocardium increased by hESC-PVPC transplantation. (A to C) are representative photographs of the Masson tricolor-stained heart cross-section, (A) sham group injected with medium, (B) group injected with hESC-PVPC and (C) injected CB-EPC Photo for a group. (D) represents the fibrosis area of the infarct myocardium, (E) the scar length of the infarct myocardium, (F) the capillary density in the infarct myocardium, and (G) the infarct wall thickness of the infarct myocardium. (H, I) represents GS-lectin (green) staining capillaries of the cardiomyopathy region.

12 shows engraftment in the perivascular region of damaged blood vessels of hESC-PVPC transplanted in diabetic retinal disease model. DiI (red) was applied to the diabetic rats induced by streptozotocin (STZ). Labeled hESC-PVPC was injected into the retina to confirm engraftment after 4 weeks. The animal models used were mice with blood glucose levels of 250 mg / dl or more after the first 5 injections of diabetes (once a day), and were used after 8 weeks of diabetes in consideration of the time required to induce retinopathy. . For retinal blood vessel labeling, FITC-labeled dextran (dextran-FITC, green) was injected into the tail blood vessel before retinal extraction. It was confirmed that the transplanted hESC-PVPC is located in the retina, especially around the vessels of the damaged blood vessels.

Figure 13 is a diagram showing the transplantation of hESC-PVPC, human bone marrow-derived mesenchymal stem cells (BM-MSC) and human placenta-derived mesenchymal stem cells (placenta-MSC) in diabetic retinal disease model, and confirmed the engraftment for one month after transplantation to be. After 8 weeks of diabetes induced STZ, each cell labeled with DiI was transplanted into the retina of the mouse, and after 1, 2 and 4 weeks, the respective retinas were extracted and checked for engraftment. hESC-PVPC is engrafted in the perivascular area and does not cause additional retinal vascular damage.In the adult-derived mesenchymal stem cell transplant group, the transplanted cells do not engraft and retinal detachment and lens-retinal stenosis are induced. It was confirmed to lead to death.

As one aspect for achieving the above object, the present invention comprises the first step of differentiating into mesodermal cells by treating BMP4 growth factor in the stem cell-derived embryonic body (embryoid body); Separating a embryoid body comprising differentiated mesodermal cells into single cells; And a third step of culturing the isolated single cells in a substrate coated with a substrate, thereby proliferating only the attached cells by natural selection.

The first step is a step of differentiating mesodermal cells by treating BMP 4 growth factors in embryoid bodies derived from stem cells. The embryoid bodies derived from stem cells differentiated into mesodermal cells are cultured under appropriate differentiation conditions, thereby increasing the cell population in which the expression of mesodermal markers is increased by at least one or more. At this time, the expression of mesodermal markers can be detected by biochemical or immunochemical methods, and methods capable of detecting mesodermal markers can be used without limitation. For this purpose, specific polyclonal antibodies or monoclonal antibodies that bind to mesodermal markers can be used. Antibodies targeting individual specific markers can be used commercially and without limitation, those prepared by known methods. Representative mesodermal markers include, but are not limited to, KDR, PDGFRα / β CD29, CD31, CD34, CD73, CD90, and CD105. The first step may be performed for 6 to 8 days, preferably for 6 days.

According to one embodiment of the invention, the embryoid body formed by self-aggregation according to a method known in the art for 6 days in DMEM / F12 medium containing 10% SR containing BMP4 growth factor When cultured, it was confirmed by flow cytometry that KDR and PDGFRβ expression of mesodermal cell markers increased more than 30% and 50%, respectively, in the embryoid body (FIG. 1B).

As used herein, the term "stem cell" refers to a pluripotency capable of differentiating into cells derived from endoderm, mesoderm, and ectoderm of an animal, or a cell closely related to tissue or function. It means a cell having a multipotency that can be differentiated into. Means cells that have the ability to self-replicate and differentiate into two or more new cells, and include totipotent stem cells, pluripotent stem cells, and pluripotent stem cells ( multipotent stem cells).

As used herein, the term "embryonic stem cell" refers to an embryo that is capable of differentiating into all cells of an animal by extracting an inner cell mass from an blastocyst embryo immediately before implantation into an uterus of a mother. Means a cell having a pluripotency, but is not limited thereto, such as an embryonic body derived from embryonic stem cells or induced pluripotent stem cells (iPS cells) Similar stem cells are also included.

As used herein, the term "adult stem cell" refers to a cell having a multipotency by separating stem cells existing in each tissue and culturing in vitro, and including bone marrow stem cells, retinal stem cells, and M. glial cells in the retina. Cells, neural stem cells, and the like. The stem cells may be derived from mammals including humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs, mice, rats, and cats, preferably humans.

Stem cells of the present invention may be human embryonic stem cells (embryonic stem cells), induced pluripotent stem cells (iPSC) and somatic cell nuclear transfer cells (SCNT), preferably May be an embryonic stem cell, more preferably a human embryonic stem cell. The stem cells may be derived from mammals including humans, and most preferably may be human derived cells.

As used herein, the term "BMP4" refers to a growth factor as a protein encoded by the BMP4 gene in humans. Bond morphogenetic protein (BMP) is a signaling protein belonging to the transforming growth factor-β (TGF-β) superfamily that regulates early fetal differentiation, fetal tissue formation, and maintenance of adult tissue homeostasis. In particular, the difference in the concentration of BMP in the early gestational stage plays a decisive role in the formation of the axis during embryonic embryo formation. The extracellular secreted BMP binds to type I and type II serine / threonine kinase receptors on the cell membrane to initiate BMP signaling. The type 2 receptor phosphorylates the type 1 receptor, and the phosphorylated type 1 receptor phosphorylates the Smad protein, a substrate in the cell, for intracellular signaling. Smad proteins regulated by receptors are called receptor regulated Smads (R-Smads), and Smad-1, 2, 3, 5 and 8 belong to R-Smad. They bind to Smad-4, a common partner Smad (Co-Smad) in the cell, move into the cell nucleus and bind to transcription factors to regulate transcription of target genes (Yamamoto & Oelgeschlager, Naturwissenschaften, 91: 519-34, 2004 ).

The term "embryoid body" of the present invention refers to cell aggregates derived from embryonic stem cells. Cell aggregation occurs by drop culture, untreated cell culture dish culture or stirred culture, which prevents cells from adhering to the surface to form typical colony proliferation. Differentiation begins with aggregation, and cells begin to repeat a limited degree of embryogenicity. The goblet is derived from pluripotent cells and thus can consist of a wide variety of differentiated cell types. However, differentiation in embryoid bodies, despite occurring in a three-dimensional manner, is not very systematic compared to the case of carefully organized normal embryology. Embryos, however, are still good model systems for studying cellular and molecular interactions in the early stages of development, which are still difficult to study. Therefore, in the present invention, the differentiation was induced by treating BMP4 growth factor from the first day of the formation of embryoid bodies to differentiate them into mesodermal cells.

The term "mesodermal cell" of the present invention refers to cells capable of differentiating into muscle cells, bone cells, chondrocytes, adipocytes, reticular cells or other connective tissue cells, including blood cells, vascular endothelial cells, smooth muscle and myocardium, and the like. it means.

The second step is the step of separating the embryoid body containing the differentiated mesodermal cells into single cells, in the present invention, the enzyme was treated for single cell formation of the embryoid body. The enzyme may be used without limitation as long as it can inhibit intercellular binding by degrading cell surface proteins involved in intercellular binding. For example, trypsin, trypsin-EDTA, collagenase, etc. may be used, but is not limited thereto. Preferably it may be a hypoallergenic enzyme, more preferably TrypLE (Invitrogen).

In the third step, the isolated single cells are cultured in a substrate coated with a substrate, and only the attached cells are propagated by natural selection.

The term "natural selection" of the present invention does not use flow cell sorting or magnetic cell sorting techniques, which are used in the art to isolate highly differentiated specific cells. It means to separate naturally by using the inherent characteristics of the cell. In the case of the flow cytometry or magnetic cell sorting technology, there is an advantage in that purity can be confirmed through cell-specific markers. In addition, there is a problem in that the cost is increased by the use of a variety of reagents, machines, and the like. However, the method of the present invention does not require a machine or the like, and has the advantage of differentiating without the problem of cell deformation or high cost by using simple natural selection. In other words, the natural selection technique of the present invention focuses on the functional characteristics of the cells that can be distinguished, rather than focusing on cell specific markers. Examples are selective cultures due to media suitability or specific surface adhesion. The "substrate" of the present invention can be used without limitation so long as it can help the selective attachment of cells to the culture dish, for example collagen or laminin may be used, preferably collagen may be used. The third step may be performed for 16 to 24 hours, preferably 18 hours. The naturally selected high purity perivascular progenitor cells can continue to proliferate, differentiate into various tissue cells, or be recovered and cryopreserved.

In a specific example of the present invention, 5 mg / ml collagen was coated on a culture plate as a specific substrate. The BMP4 growth factor-induced mesodermal cell embryoid body was subjected to enzymatic treatment and single cell culture, followed by incubation for 18 hours in a collagen-coated culture dish. Thereafter, only the cells specifically attached to collagen were naturally selected through medium exchange.

Peripheral progenitor cells naturally selected in the third step show positive expression for platelet-derived growth factor receptor β (PDGFRβ). PDGFRβ is a protein that is encoded by the PDGFRB gene in humans. The gene encodes a cell surface tyrosine kinase receptor belonging to the PDGF family. The growth factor is one of specific markers of mesodermal cells as a proliferation agent for mesenchymal-derived cells.

According to a specific embodiment of the present invention, as a result of confirming by flow cytometry the cells before and after the culture by the natural selection technology, it is confirmed that the cells proliferated after the culture by the natural selection technology are cultured and grown in high purity only the cells positive for PDGFRβ. This showed similar levels of purity as the selective culture of only cells positive for PDGFRβ by flow cytometry (FIG. 3).

As another aspect, the present invention provides a perivascular progenitor cell prepared by the above method.

Peripheral progenitor cells prepared by the present invention showed positive expression patterns in the progenitor cell-specific PDGFR, CD44, CD146 and NG2 and mesenchymal stem cell-specific markers CD73 and CD90, etc. The markers CD31 and CD144 and the hematopoietic cell specific markers CD34 and CD45 and the like are not expressed (FIG. 4). In addition, the perivascular progenitor cells have differentiation ability into mesenchymal cell-derived differentiated bodies such as cartilage, osteoblasts and adipocytes, smooth muscle cells and myocytes (FIGS. 5 to 8), and are lost when applied as a cell therapy composition in a cardiovascular disease model. Has the potential to recover (FIGS. 10 and 11).

The term "progenitor cell" of the present invention refers to a cell capable of asymmetric division and is called a precursor, and because of asymmetric division, even if each cell has the same number of passages, Some are differentiated and some are multiplied, and may differ in age and nature.

In another aspect, the present invention, the perivascular progenitor cells produced by the above method is angiogenic growth factors (angiogenic growth factors), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), IGF (insulin-like) A method of differentiating perivascular progenitor cells into perivascular cells, which is characterized by culturing in EGM-2MV (SingleQuots, Cambrex Bio Science) medium containing growth factor (EGF), epidermal growth factor (EGF), ascorbic acid, and GA-1000. To provide. Preferably, 5% fetal bovine serum (FBS), 10 ng / ml angiogenic growth factor, 10 ng / ml FGF, 20 ng / ml VEGF, 10 ng / ml IGF, 10 ng / in EGM-2MV medium. It may be cultured in perivascular cell differentiation medium containing ml EGF, ascorbic acid, GA-1000, but is not limited thereto.

The term "pericyte" of the present invention refers to vascular wall cells buried in the basement membrane of the microvascular system, which forms a specific local contact with the vascular endothelium. Connective tissue cells that form around small vessels, also called Rouget cells, adventitial cells, or wall cells, are known to surround about 10-50% of the outer area of vascular endothelium. It is a long, narrow contractile cell that surrounds the precarotid artery outside the basement membrane. It is a relatively undifferentiated multipotent cell and functions to support blood vessels. If necessary, they can also be differentiated into fibroblasts, smooth muscle or macrophages. In addition to angiogenesis, it plays an important role in the stability of the blood-brain barrier. Adhesion to cells in the blood vessels has emerged as a blood flow regulator in the microvascular system, and in fact it is possible to regulate blood flow at the capillary level. Therefore, the study of perivascular cells is considered to be an important target for understanding vascular physiology and studying the causes and treatment of vascular diseases.

As another aspect, the present invention, the perivascular progenitor cells produced by the above method is a minimum essential medium non-essential amino acids (NEAA, Invitrogen), penicillin / streptomycin (penicillin / streptomycin; PS, Invitrogen), β-mercaptoethanol (Invitrogen) provides a method for differentiating perivascular progenitor cells into smooth muscle cells, characterized in that the culture medium. Preferably, it may be cultured in a smooth muscle cell differentiation medium containing 5% FBS, 1% NEAA, 1% PS, 0.1 mM β-mercaptoethanol in DMEM (Dulbecco's modified eagle medium) medium, but is not limited thereto.

The term " smooth muscle cell " of the present invention refers to a muscle without a horizontal pattern in muscle, and all internal organs other than the heart muscle of a vertebrate are smooth muscle. Although contraction rate is slow, it is involuntary muscle that does not feel fatigue easily. It is long and fusiform, rarely multinucleus, but usually has one elliptical nucleus in the center. It is found in various parts of the body, such as the walls of blood vessels and walls of hollow organs such as the stomach, intestines, and uterus. Because it plays a pivotal role in the regulation of various human functions, a number of human diseases are associated with the dysfunction of smooth muscle.

As another aspect, the present invention, the perivascular progenitor cells prepared by the above method is prepared by horse serum (HS, Invitrogen), chicken embryo extract (CEE, Sera Lab, Sussex, UK), PS It provides a method for differentiating perivascular progenitor cells into myocytes, characterized in that it is cultured in a culture medium comprising. Preferably, the medium may be cultured in myocyte differentiation medium containing 10% FBS, 10% HS, 1% CEE, 1% PS in DMEM high glucose (DMEM) medium, but is not limited thereto.

The term "myocyte" of the present invention, also known as muscle cells (muscle cells) arise from myoblasts (myoblast). Each myocyte contains a long myofibril, a long chain of sarcomere, and a contraction unit of the cell. Myocytes have a variety of differentiated forms of varying properties, including heart, skeleton, or smooth muscle cells.

As another aspect, the present invention, the perivascular progenitor cells prepared by the above method is insulin (Sigma-Aldrich), dexamethasone (Sigma-Aldrich), isobutylmethylxanthine (Sigma-Aldrich), India It provides a method for differentiating perivascular progenitor cells into adipocytes, which is characterized by culturing in a culture medium containing metasin (indomethacin, Sigma-Aldrich). Preferably in low glucose DMEM medium can be cultured in adipocyte differentiation medium containing 10% FBS, 5 μg / ml insulin, 1 μM dexamethasone, 0.5 mM isobutylmethyl xanthine, 60 μM indomethacin, but is not limited thereto. no.

The term "adipocyte" of the present invention refers to the major cells that make up adipose tissue that stores energy in the form of fat. It exists in two forms: white fat cells, which contain large fat droplets surrounded by a cytoplasmic layer, and polygonal brown fat cells, which contain a significant amount of cytoplasm, evenly dispersed fat droplets. White adipocytes secrete proteins that act as adipokine, such as resistin, adiponectin and leptin.

As another aspect, the present invention, the perivascular progenitor cells produced by the above method is cultured in a culture medium containing dexamethasone, β-glycerophosphate, ascorbic acid-2-phosphate It provides a method for differentiating into bone cells. Preferably, the present invention may be cultured in low concentration glucose DMEM medium in osteoblast differentiation medium containing 10% FBS, 1 μM dexamethasone, 10 mM β-glycerophosphate, 60 μM ascorbic acid-2-phosphate, but not limited thereto. .

The term "osteocyte" of the present invention is a star-shaped cell that is most abundant in dense bone tissue and includes a thin ring of nucleus and cytoplasm. Osteoblasts are trapped in the secreted matrix and form bone cells. Bone cells connect to each other through long cytoplasmic kidneys that fill small ducts called microtubules that are used to exchange nutrients and waste through gap junctions. On the other hand, osteocytes are reduced in their ability to synthesize, do not mitosis, are produced in the mesenchyme, and hydroxyapatite, calcium carbonate, and calcium phosphate accumulate around the cells.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising the prepared perivascular progenitor cells as an active ingredient.

The term "prevention" of the present invention means any action that inhibits or delays the onset of cardiovascular disease by the administration of the pharmaceutical composition according to the present invention, "treatment" refers to cardiovascular disease by the administration of the pharmaceutical composition It means any action that improves or beneficially changes the symptoms caused by it.

The composition of the present invention can be used without limitation in the prevention or treatment of cardiovascular diseases. The term "cardiovascular disease" of the present invention includes heart disease and vascular disease, and refers to a disease that occurs in the arteries and veins including the heart and affects the heart and blood vessels. Risk factors such as hyperlipidemia, diabetes and smoking are all causes of blood vessel damage and can lead to cardiovascular disease. However, there are usually no clear symptoms, usually called silent disease. Most of the conditions are already severe when symptoms begin to develop asymptomatically and worsen without awareness. Many of these cardiovascular diseases are also associated with adult diseases. Examples include hyperlipidemia, in which blood cholesterol and fat components are accumulated in blood vessels due to poor blood circulation, arteriosclerosis, which hardens as the arteries age, and high blood pressure caused by high pressure during the contraction and relaxation of the heart. Representatives include hypotension, angina caused by pericardial coronary artery disease, and myocardial infarction caused by heart muscle abnormalities.

Thus, the cardiovascular diseases treatable by the present invention include, for example, heart failure, hypertensive heart disease, arrhythmia, heart valve disease, ventricular septal defect, congenital heart disease, cardiomyopathy, pericardial disease, stroke, peripheral vascular disease, aneurysm, arteriosclerosis, Coronary artery disease or high blood pressure may be included, but is not limited thereto.

According to a specific embodiment of the present invention, it was confirmed that the perivascular progenitor cells of the present invention implanted in the mouse has an effect of improving ischemic limb relief / blood flow in ischemic Fuji (FIG. 10). It was also confirmed that there is an effect of increasing the capillary density in the ischemic myocardium (FIG. 11). Therefore, it was confirmed that perivascular progenitor cells derived from stem cells of the present invention have the potential to treat cardiovascular diseases as cell therapy compositions.

The term "pharmaceutical composition" of the present invention may be used interchangeably with "cell therapy composition", which is a cell and tissue prepared through isolation, culture and special manipulation from humans, which is used for the purpose of treatment, diagnosis and prophylaxis. U.S. FDA Regulations) treats, diagnoses, and treats live, autologous, or heterologous cells in vitro or otherwise alters the biological properties of cells to restore the function of cells or tissues. Means a drug used for the purpose of prevention.

The cell therapy composition of the present invention may further comprise a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the cells or humans exposed to the composition are not toxic. The carrier can be used without limitation so long as it is known in the art such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, preservatives and the like. The pharmaceutical compositions of the present invention can be prepared according to techniques commonly used in the form of various formulations. The cell therapy agent of the composition of the present invention can be administered by any route as long as it can induce migration to the disease site. In some cases, one may consider loading the vehicle with a means for directing stem cells to the lesion. Thus, the compositions of the present invention may be used topically (including buccal, sublingual, skin and intraocular administration), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and cerebrospinal fluid) or transdermal administration. It can be administered through several routes, including parenteral, most preferably directly to the affected area. In one embodiment, the stem cells can be administered to a subject by suspending it in a suitable diluent at a concentration of about 1 × 10 ³ to 5 × 10 ⁶ cells / ml, which dilution protects and maintains the cells and upon injection into the desired tissue. Used for ease of use. The diluent may include a saline solution, a phosphate buffer solution, a buffer solution such as HBSS, plasma, cerebrospinal fluid, or a blood component. In addition, the pharmaceutical composition can be administered by any device such that the active substance can migrate to the target cell. Preferred modes of administration and preparations are injections. Injections include aqueous solvents such as physiological saline solution, ring gel solution, Hank's solution or sterilized aqueous solution, vegetable oils such as olive oil, higher fatty acid esters such as ethyl oleic acid, and non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerin And non-invasive agents known in the art, suitable for the barrier to pass through, for mucosal permeation, and ascorbic acid, sodium hydrogen sulfite, BHA, tocopherol, EDTA as stabilizers for prevention of alteration. And the like, an emulsifier, a buffer for pH adjustment, phenyl mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like, and a pharmaceutical carrier such as a preservative for preventing the growth of microorganisms.

As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of retinal diseases, including the prepared perivascular progenitor cells as an active ingredient.

The term "prevention" of the present invention means any action that inhibits or delays the onset of retinal disease by the administration of the pharmaceutical composition according to the present invention, "treatment" refers to retinal disease by the administration of the pharmaceutical composition It means any action that improves or beneficially changes the symptoms caused by it.

In addition, the composition of the present invention can be used for the prevention or treatment of retinal diseases. As used herein, the term "retinal disease" refers to a disease caused by abnormal regeneration of vascular endothelial cells due to impaired pericyte or retinal degeneration due to leakage of blood or body fluids. Thus, it can be treated by implanting perivascular or differentiateable cells into the retina. Examples of retinal diseases treatable by the present invention include wet age related macular degeneration (neovascular or exudative AMD), glaucoma, diabetic retinopathy, wet age related retinopathy, Retinopathy of prematurity, but is not limited thereto.

According to a specific embodiment of the present invention, it is confirmed that the perivascular progenitor cells derived from the stem cells of the present invention transplanted into the retina of the diabetic retinal disease mouse model have an effect of engulfing the perivascular vessels of the damaged blood vessels to alleviate the damage. (FIG. 12). In addition, when compared with the transplantation of adult-derived stem cells, the transplanted adult-derived stem cells did not engraft in the desired location, it was confirmed that causing retinal detachment, lens-retinal stenosis excessive inflammatory reactions leading to death (Fig. 13). ). Therefore, it was confirmed that the perivascular progenitor cells derived from the stem cells of the present invention have the potential to treat retinal diseases as cell therapy compositions.

As another aspect, the present invention provides a method for preventing or treating a cardiovascular disease of a subject, comprising administering the pharmaceutical composition for preventing or treating the cardiovascular disease to a subject in need thereof.

The term "individual" of the present invention means any animal including a human having or may develop a cardiovascular disease as described above, and can effectively prevent or treat the diseases by administering the pharmaceutical composition of the present invention to an individual. The pharmaceutical composition of the present invention can be administered in parallel with existing therapeutic agents.

The term "administration" of the present invention means introducing a predetermined substance into a patient in an appropriate manner, and the route of administration of the composition may be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.

In addition, the pharmaceutical compositions of the present invention may be administered by any device in which the active substance may migrate to target cells. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, to prevent microbial growth Preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

As another aspect, the present invention provides a method for preventing or treating a cardiovascular disease of an individual, comprising administering the pharmaceutical composition for preventing or treating the retinal disease to an individual in need thereof.

The terms "individual" and "administration" of the present invention are the same as defined in the above text.

As another aspect, the present invention provides a use of the stem cell-derived perivascular progenitor cells for the prevention or treatment of cardiovascular diseases.

As another aspect, the present invention provides a use for the preparation of a cardiovascular disease therapeutic agent of the stem cell-derived perivascular progenitor cells.

As another aspect, the present invention provides a use for the prevention or treatment of retinal disease of the stem cell-derived perivascular progenitor cells.

As another aspect, the present invention provides a use for the manufacture of a therapeutic agent for retinal disease of the stem cell-derived perivascular progenitor cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example 1 Culture of Human Embryonic Stem Cells (hESCs)

In a culture dish (Nunc, Roskide, Denmark) coated with undifferentiated human embryonic stem cells (H9, CHA11-hESC) with 0.1% gelatin, mitomycin C (Sigma, St. Louis, MO, USA) was cocultured with mouse embryonic fibroblasts (MEFs) prepared by treatment at a concentration of 10 μg / ml for 90 minutes as support cells. At this time, hESC is DMEM / F12 (50:50, Invitrogen, Carlsbad, CA, USA), 20% (v / v) serum replacement (SR), 1% non-essential amino acid (Invitrogen), 1% penicillin strepto A culture consisting of mycin (Invitrogen), 0.1% beta-mercaptoethanol (Invitrogen), 4 ng / ml bFGF (basix fibroblast growth factor; Invitrogen) was used. The culture was replaced every day, subcultured to new support cells every 5-7 days, cell culture was incubated while maintaining 37 ℃, 5% carbon dioxide, 95% humidity.

Example 2 Formation of Embryoid Body

To form embryoid bodies for differentiation induction, human embryonic stem cells were separated from support cells with dispase, transferred to a new culture dish (Falcon / BD Biosciences, San Jose, CA, USA), washed once, and washed with DMEM / Aggregation was induced by suspension for 45 minutes in a culture medium in which 2% (v / v) serum replacement (SR; Invitrogen) was added to F12 (50:50, Invitrogen). Aggregated embryoid bodies were 10% (v / v) SR, 1 mM L-glutamine (Invitrogen), 1% nonessential amino acid (NEAA; Invitrogen), 1% penicillin streptomycin (Invitrogen), 0.1% beta Transfer to culture medium composed of mercaptoethanol (Invitrogen) was suspended in culture. Cultures were replaced every other day and maintained for 5-6 days.

Example 3: Increase of Mesodermal Cell Population in Embryos by BMP4 Growth Factor Treatment

DMEM with 1 mM L-glutamine, 1% non-essential amino acid, 0.1% beta-mercaptoethanol and 10% SR added on the first day of embryoid formation to differentiate embryoid bodies prepared by the above method into mesodermal cells The / F12 culture was treated with 20 ng / ml BMP4 growth factor (Peprotech Inc., Rocky Hill, NJ, USA), and was replaced with the cultured BMP4 growth factor once every two days from the next day.

As a result, it was confirmed that as the number of culture days treated with the BMP4 growth factor increased, the ratio of KDR and PDGFRβ, which are markers of mesodermal cells, increased in proportion to cells (FIGS. 1A and 1B). In addition, comparing the two markers KDR and PDGFRβ expression on the 6th day of culture with the control group (NT) not treated with BMP4 through flow cytometry, in particular, the expression rate of KDR was significantly improved compared to the control group in the experimental group treated with BMP4. (FIG. 1B).

Example 4: Single Cellization of Mesodermal Embryos by Hypoallergenic Enzyme Treatment

Mesoderm cell population-induced embryoid bodies treated with BMP4 growth factor were washed 1-2 times with phosphate buffer solution (Invitrogen 1X), and the embryonic bodies were placed in a 35 mm culture dish, Tryp-LE (Invitrogen) ) Was observed by observing with a microscope the release of intercellular binding of the embryoid body. The unbound cells were transferred to an Eppendorf tube, washed with phosphate buffer solution, and filtered using a 35 μm nylon mesh cell strainer to prepare single cells.

At this time, the average number of embryoid bodies obtained from human embryonic stem cells maintained in one 60 mm culture dish is 80 to 100, and one embryonic body is composed of 2,500 cells on average. Therefore, in one step of the present invention, embryonic bodies obtained from human embryonic stem cells of six 60 mm culture dishes on average are 500 to 600, and single cells obtained from them are 1.5 to 2 × 10 ⁶ .

The results are shown in FIG. FIG. 2A shows undifferentiated H9 hESC single cells dispensed in a cell culture dish, FIG. 2B shows spontaneous differentiation of single cells from EB on day 6 of BMP4 induction dispensed in an uncoated plastic Petri dish, 2C and 2D in collagen coated dishes Single cells from EBs, and 2E and 2F, are micrographs of single cells from EB on day 6 of BMP4 induction dispensed into collagen coated dishes. 2C and 2D show a non-uniform cell population in which epithelial cells and fibrous cells are observed together, while FIGS. 2E and 2F show a uniform cell population.

Example 5: Natural Selection of Perivascular Progenitor Cells through Coating Substrate Specific Adhesion Culture

Single cells prepared in Example 4 were incubated overnight in a cell culture apparatus at 37 ° C., 5% carbon dioxide conditions with EGM2-MV (Lonza) medium in a pre-prepared collagen coated culture dish. Single cells cultured in the above process is divided into floating and adherent population, the floating population was removed by natural selection through phosphate buffer solution and medium exchange process. In the process, cells attached through natural selection were set to passage 0. It is passaged every 2 to 3 days and propagates from passage 0 to passage 3 within 10 days of culture. In one process of the present invention, 1.5 to 2 × 10 ⁶ single cells are attached to 50 to 80% in a 35 mm collagen-coated culture dish, and in passage 3 to 3 to 5 × 10 ⁷ in five T-75 flasks. Multiplies.

Collagen used in the coating was a distilled water dilution solution of a collagen solution (Stem Cell Tech.) Of 3 mg / ml concentration. The collagen coating for passage 0 used a 5-fold diluted collagen solution, and subsequent passages used a 20-fold diluted collagen solution. The coating was performed by exposing the culture dish to the collagen solution of the corresponding concentration for at least 1 hour at a sterile work table at room temperature.

The results are shown in FIG. First, flow cytometry is shown in FIG. 3A for the types of cells cultured by categorizing positive and negative cell populations according to the expression level of PDGFRβ with a flow cytometer. In addition, cells cultured according to the natural selection method of the present invention is shown in Figure 3B. From the flow cytometry results after several days of culture, substrate specific natural selection, which is a characteristic step of the culturing method of the present invention, is capable of pure separation of perivascular progenitor cells without additional processes such as flow cytometry or magnetic cell sorting. Confirmed.

Example 6: Search for marker markers of perivascular progenitor cells

Cells prepared through the procedure of Examples 1 to 5 were analyzed by a flow cytometer (FACSCalibur flow cytometer; BD Bioscience, San Jose, CA, USA) using the markers of perivascular progenitor cells. Cells cultured in passage 3 were suspended using Tryp-LE, a hypoallergenic enzyme, transferred to an Eppendorf tube, washed with phosphate buffer solution and filtered using a 35 μm nylon mesh cell filter to 2% fetal bovine serum. Flow cytometry was performed after floating with phosphate buffer solution containing (Terracell, Webtec Inc., Charltte, NC, USA). Control markers include IgG _isotype (PE, APC; BD Biosciences), other PDGFRα (PE; R & D systems), PDGFRβ (PE; R & D systems), CD44 (PE, APC; R & D systems), KDR (PE; R & D systems) ), Tie-2 (PE; R & D systems), Flt-1 (PE; R & D systems), VE-CAD (PE; R & D systems), CD31 (PE; R & D systems), CD146 (PE; R & D systems), NG2 ( PE; R & D systems), CD34 (PE; R & D systems), CD45 (PE; R & D systems), CD105 (PE; R & D systems), CD90 (PE; R & D systems), CXCR4 (PE; R & D systems), SSEA4 (PE; R & D systems) and CD133 (APC; Miltenyi Biotec Inc.) were used.

The flow cytometry results are shown in FIG. 4. As a result, from Fig. 4a, the perivascular progenitor cells of the present invention are positive for PDGFRα, PDGFRβ, CD44, CD73, CD90, CD105, CD146, NG2, KDR, Tie2 and Flt1, and CD31, CD34, CD45, CD133, CD144 , CXCR4 and c-kit showed a negative immune phenotype.

Example 7: Differentiation of Perivascular Progenitor Cells

Peripheral progenitor cells identified by the method of Example 6 were continuously treated with 5% FBS, 10 ng / ml angiogenic growth factors, 10 ng / ml FGF, 20 ng / ml VEGF, 10 ng / ml IGF, 10 ng / ml When cultured in EGM-2MV (SingleQuots, Cambrex Bio Science) medium containing EGF, ascorbic acid and GA-1000, it showed similar characteristics to the perivascular cells derived from adults. This is shown in Figure 5 compared with human placental induced perivascular cells.

As a result, the similarity in cell morphology was observed in FIG. 5A (H9 hESC-PVPC) and FIG. 5B (human placenta-induced perivascular cells), and in FIG. 5C and FIG. 5D, NG2 (Green, a specific marker of perivascular cells). ), But the expression of adult tissue specific marker SMA (red) was not observed. Peripheral vascular progenitor cells formed by co-culture of vascular endothelial cells and human embryonic stem cell-derived vascular endothelial progenitor cells in the three-dimensional collagen gel of FIG. 5E and the low adhesion culture dish of FIG. 5F (FIG. In 5E, the vascular morphology and in FIG. 5F were located outside of the elliptic inner cell mass, that is, in the perivascular region.

Perivascular progenitor cells identified by the method of Example 6 were differentiated in DMEM medium containing smooth muscle cell differentiation medium, that is, 5% FBS, 1% NEAA, 1% PS, 0.1 mM β-mercaptoethanol, It was shown that the perivascular progenitor cells of the invention are capable of differentiating into smooth muscle cells. The expression of αSMA, a smooth muscle cell specific marker, was confirmed by RT-PCR, real-time PCR and immunocytochemistry by comparing with mature SMLC.

As a result, FIGS. 6A and 6B show that the smooth muscle differentiation medium gradually increases with the days of induction of differentiation. Immunofluorescence staining in perivascular progenitor cells prior to differentiation αSMA (red) was not expressed, but was expressed as it was differentiated into smooth muscle cells (FIGS. 6C and 6D). 6E and 6F showed that the mature smooth muscle cell specific markers SM-MHC (FIG. 6E, green) and calponin (FIG. 6F, green) were simultaneously expressed with αSMA.

Peripheral progenitor cells identified by the method of Example 6 were cultured in high glucose DMEM medium containing 10% FBS, 10% HS, 1% CEE and 1% PS to differentiate into myocytes. 7A and 7D show the perivascular progenitor cells prior to differentiation, FIGS. 7B and 7E show the cells that differentiate into myocytes in the myogenic differentiation medium, and FIGS. 7C and 7F finally show the morphology of muscle fibers under muscle growth medium. In order to confirm this, myocyte-specific marker desmine (red) was used.

Peripheral progenitor cells identified by the method of Example 6 were 10% FBS, 5 μg / ml insulin, 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, and 60 μM indomethacin and 10% of low glucose DMEM medium. Cultured in low glucose DMEM medium containing FBS, 1 μM dexamethasone, 10 mM β-glycerophosphate, and 60 μM ascorbic acid-2-phosphate to differentiate into adipocytes and osteoblasts, respectively, to determine the possibility of skeletal differentiation. Adipocytes generated by differentiation under the above conditions were identified by oil red staining, and bone cells by alizarin red staining, which are shown in FIG. 8.

Example 8: Intermaterial Exchange of Perivascular Progenitor Cells with Cardiovascular Cells

Interchangeability was confirmed through analysis of pigment transfer between human embryonic stem cell-derived vascular periphery progenitor cells (hESC-PVPC) and other cardiovascular cells of the present invention. Calcine is a fluorescence that is only transferred through gap-junction, which is part of cellular interactions. Thus, intercellular gap-junction formation is necessary for the transfer of calcine labeled in other cardiovascular cells to hESC-PVPC. DiI-labeled hESC-PVPC (FIG. 9, top panel, red) was calcine-labeled HUVEC (FIG. 9, middle panel left, green), UASMC (FIG. 9, middle panel center, green) or cardiomyocytes (FIG. 9, center). Right panel, green) and 5% CO 2 was incubated for 1 hour in a 37 ℃ incubator. Green fluorescence of calcine was detected with red fluorescence of DiI in hESC-PVPC (FIG. 9, bottom panel) (FIG. 9, bottom panel, red green, asterisk). From this, it was confirmed that hESC-PVPC forms a gap-junction with other cardiovascular cells, thereby allowing interactions such as mass transfer.

Example 9 Therapeutic Effect of Perivascular Progenitor Cells in an Ischemic Cardiovascular Disease Model

Human embryonic stem cell-derived perivascular progenitor cells (hESC-PVPC) of the present invention were transplanted into an ischemic disease animal model to confirm its applicability as a cell therapy. HESC-PVPC was transplanted into a mouse model of ischemic cardiovascular disease and the treatment effect was observed before and after transplantation. 10 shows ischemic limb rescue / blood flow improvement in ischemic Fuji after hESC-PVPC transplantation. Immediately after transplantation and at 28 days, a series of lower limb recovery was divided, and the images were analyzed and analyzed. The laser Doppler image was used to measure the recovery of blood perfusion of the ischemic limb. 11 shows increased capillary density in ischemic myocardium with hESC-PVPC transplantation. Heart sections of the Siamese group injected with media (FIG. 11A), the experimental group implanted with hESC-PVPC (FIG. 11B), and the comparative group injected with CB-EPC (FIG. 11C) were compared by Masson tricolor staining. 11D-11G show a decrease in infarct myocardial fibrosis area and infarct myocardial scar length and increase in capillary density and infarct wall thickness in infarct myocardium due to hESC-PVPC transplantation. 11H and 11I show staining of cardiomyopathy site capillary with GS-lectin (green). From the series of results, it was confirmed that the recovery effect of ischemic myocardial infarction through capillary regeneration in the hESC-PVPC transplantation experimental group.

These results suggest that the perivascular progenitor cells of the present invention can treat cardiovascular disease.

Example 10 Therapeutic Effect of Perivascular Progenitor Cells in Diabetic Retinopathy Disease Model.

As an animal model, hESC-PVPC was transplanted into mice induced with diabetic retinopathy to confirm the therapeutic effect. First, streptozotocin (streptozotocin, STZ) was administered to mice to induce diabetes. Diabetes-induced model was used to measure the blood glucose level after the first five times (once a day) STZ was used to select mice with a value of 250 mg / dl or more, in consideration of the time required to induce retinopathy Those after 8 weeks were used. For visualization, hESC-PVPC labeled with DiI (red) was used, and engraftment was confirmed 4 weeks after injection into the retina. For retinal blood vessel labeling, FITC-labeled dextran (dextran-FITC, green) was injected into the tail blood vessel before retinal extraction. As shown in FIG. 12, the transplanted hESC-PVPC was engrafted into the retina, and was found to be located particularly around the vessel of the damaged blood vessel.

In addition, adult-derived mesenchymal stem cells were transplanted and the results were compared. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) and human placenta-derived mesenchymal stem cells (placenta-MSCs) were transplanted in the same manner and transplanted for one month thereafter. Compared with. In the same manner as the hESC-PVPC, each cell was labeled with DiI and transplanted into the retina of STZ-induced diabetic 8-week-old mouse. hESC-PVPC is engrafted in the perivascular area and does not cause additional retinal vascular damage.In the adult-derived mesenchymal stem cell transplant group, the transplanted cells do not engraft and retinal detachment and lens-retinal stenosis are induced. It was confirmed to lead to death. This is shown in FIG. 13. The red label in the adult-derived mesenchymal stem cell transplant group in the figure is due to the autofluorescence of immune cells by inflammatory responses, not by DiI. It is clearly green-green luminescence that exists inside the retinal vessels regardless of cell transplantation pathway.

These results suggest that the perivascular progenitor cells of the present invention have excellent engraftment ability in the retina, and thus can treat retinal diseases that are not regenerated after perivascular cells are damaged.

Claims (18)

1) differentiating into mesodermal cells by treating bone morphogenetic protein 4 (BMP4) growth factor in the embryonic body derived from stem cells;

2) separating the embryoid body comprising mesodermal cells differentiated in step 1) into single cells; And

3) culturing the separated single cells of step 2) in a substrate coated with a substrate, and naturally only propagating the attached cells, a method for producing perivascular progenitor cells from stem cells.

The method of claim 1,

Stem cell of step 1) is from the group consisting of human embryonic stem cells (embryonic stem cells), induced pluripotent stem cells (iPSC) and somatic cell nuclear transfer cells (SCNT) Which is any one selected.

The method of claim 2,

The stem cell is a human-derived method.

The method of claim 1,

Cell separation of step 2) is made by treating trypsin enzyme.

The method of claim 1,

The substrate of step 3) is collagen or laminin.

The method of claim 1,

The naturally selected perivascular progenitor cells of step 3) are positive for platelet-derived growth factor receptor β (PDGFRβ).

Peripheral progenitor cells produced by the method of any one of claims 1 to 6.

Angiogenic growth factors (angiogenic growth factors), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), ascorb A method of differentiating perivascular progenitor cells into perivascular cells, wherein the perivascular progenitor cells are cultured in EGM-2MV medium containing acid and GA-1000.

Peripheral vascular progenitor cells are cultured in DMEM medium containing minimal essential medium non-essential amino acids (NEAA), penicillin / streptomycin (PS), and β-mercaptoethanol. How to differentiate into smooth muscle cells.

The method of differentiating perivascular progenitor cells into myocytes, wherein the perivascular progenitor cells of claim 7 are cultured in a high concentration glucose DMEM medium containing horse serum, cactus extract, and PS.

A method for differentiating perivascular progenitor cells into adipocytes, wherein the perivascular progenitor cells of claim 7 are cultured in a low glucose DMEM medium containing insulin, dexamethasone, isobutylmethylxanthine, and indomethacin.

The method of differentiating perivascular progenitor cells into bone cells, wherein the perivascular progenitor cells of claim 7 are cultured in a low concentration glucose DMEM medium containing dexamethasone, β-glycerophosphate, and ascorbic acid-2-phosphate.

The pharmaceutical composition for the prevention or treatment of cardiovascular diseases, including the perivascular progenitor cells of claim 7.

The method of claim 13,

Cardiovascular disease is selected from the group consisting of heart failure, hypertensive heart disease, arrhythmia, heart valve disease, ventricular septal defect, congenital heart disease, cardiomyopathy, pericardial disease, stroke, peripheral vascular disease, aneurysm, arteriosclerosis, coronary artery disease and hypertension Any one will be a pharmaceutical composition.

A pharmaceutical composition for preventing or treating retinal disease, comprising the perivascular progenitor cells of claim 7 as an active ingredient.

The method of claim 15,

The retinal disease is any one selected from the group consisting of wet macular degeneration, glaucoma, diabetic retinopathy, wet retinopathy and prematurity retinopathy.

Pharmaceutical for preventing or treating cardiovascular diseases according to claim 13 A method of preventing or treating cardiovascular disease in a subject, comprising administering the composition to a subject in need thereof.

Pharmaceutical for preventing or treating retinal diseases according to claim 15 A method of preventing or treating retinal disease in a subject, comprising administering the composition to a subject in need thereof.

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