WO2009035216A1

WO2009035216A1 - Cell delivery system for cell therapy comprising cells derived from embryonic stem cells

Info

Publication number: WO2009035216A1
Application number: PCT/KR2008/004722
Authority: WO
Inventors: Hyung-Min Chung; Sung-Hwan Moon; Ju-Mi Kim
Original assignee: Chabiotech Co., Ltd.
Priority date: 2007-09-12
Filing date: 2008-08-14
Publication date: 2009-03-19
Also published as: KR20090027376A; KR100984376B1

Abstract

The present invention provides a cell delivery system for cell therapy including cells derived from embryonic stem cells, which is formed by inserting the cells derived from embryonic stem cells into a carrier formed of matrigel. The cell delivery system is transplanted into the region of a living body, which does not directly in contact with a disease region; migration of the cells derived from embryonic stem cells from the transplanted region is inhibited by the carrier; and the cell delivery system is finally removed from the transplanted region. In the cell delivery system of the present invention, cell therapy is accomplished only by substances secreted from the cells derived from embryonic stem cells, such as cytokine, while the cells derived from embryonic stem cells are not directly in contact with a disease region. Therefore, the cell delivery system according to the present invention can avoid any formation of cancers and tumors, which may be caused from direct transplantation of cells derived from embryonic stem cells into a living body.

Description

CELL DELIVERY SYSTEM FOR CELL THERAPY COMPRISING CELLS DERIVED

FROM EMBRYONIC STEM CELLS

TECHNICAL FIELD

The present invention relates to a cell delivery system for cell therapy including cells derived from embryonic stem cells, and more specifically, to a cell delivery system for cell therapy including cells derived from embryonic stem cells, which is formed by inserting the cells derived from embryonic stem cells into a carrier formed of matrigel. The cell delivery system is transplanted into the region of a living body, which does not directly in contact with a disease region; migration of the cells derived from embryonic stem cells from the transplanted region is inhibited by the carrier; and the cell delivery system is finally removed from the transplanted region after cure of the disease.

BACKGROUND ART

Human embryonic stem cells retain infinite self-renewality and pluripotency which can be differentiated into three germ cell layers (endodermal, ectodermal, and mesodermal) which organize a human body (Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM, Embryonic stem cell lines derived from human blastocysts. Science (1998) 282:1145-1147). With recent successful studies on differentiation of human embryonic stem cells into neural cells, endothelial cells, muscle cells, pancreatic cells, or the like, the human embryonic stem cells are considered to play a critical role in studies of therapy for incurable diseases.

Thus, development of cell therapy, through methods for differentiating human embryonic stem cells into specific cells and methods for isolation of the differentiated cells, will offer critical techniques for incurable diseases and breakthrough and primitive therapy, as a novel therapy having excellent advantages compared with conventional treatments based on drugs and surgery. Cell therapy using vascular endothelial progenitor cells is considered to be directly or indirectly applied to rapidly increasing vascular diseases such as cardiovascular diseases, cerebrovascular diseases, and diabetic ulcer.

Meanwhile, angiogenesis is a process of growth of new blood vessels. That is, neo-vessels sprout from already existing blood vessels that is damaged, and theses sprouts are elongated, branched, and connected with other vessels to form a communicating vessel network. Treatments of neo-vascularization failure related diseases such as ischemic vascular diseases depend on rapid angiogenesis process. The neo-vascularization failure related diseases include diabetic ulcer; gangrene; wounds requiring angiogenesis; Burger's disease; hypertension; ischemic diseases such as cerebrovascular ischemia, renal ischemia, lung ischemia, joint ischemia, and ischemic myocardial infarction; obstructive vascular disease; and cardiovascular disease, but are not limited thereto.

In order to treat neo-vascularization failure related diseases such as ischemic vascular diseases, there have been used drug therapy, percutaneous transluminal coronary angioplasty, artery bypass surgery, or the like. Recently, therapeutic angiogenesis for treating ischemic vascular diseases has been introduced, in which proteins or genes promoting formation of new blood vessels are administered into ischemic regions, thereby promoting the formation of collateral blood vessels. A vascular endothelial growth factor (VEGF) is commonly used in the gene therapy. In addition, it has been reported that an angiopoietin-1 variant, as a treatment by a protein, was used in treatments of cardiovascular diseases such as foot ulcer, heart diseases (myocardial infarction and cardiac ischemia), and stroke (Chung-Hyun Cho, Richard A. Kammerer, Hyuek jong Lee and Gou Young Koh, COMP-AngV. a designed angiopoietin-1 variant with nonleaky angiogenec activity, PNAS (2004) 101 5547-5552). In addition, fibroblast growth factor (FGF), transforming growth factor (TGF), and platelet-derived growth factor (PDGF) are known as an angiogenesis-related growth factor. However, since the angiogenesis-related growth factors are mainly obtained not from human cells or tissues, but cultured in E. coli or CHO cells, safety problems such as contamination with infectious substances which may occur during the culture need to be addressed. As cell therapy among methods of treating neo-vascularization failure related diseases such as ischemic vascular diseases, Asahara, et al. reported isolation of endothelial progenitor cells from peripheral blood and features thereof in 1997 (Takayuki Asahara, Toyoaki Murohara, Alison Sullivan, Jeffrey M. Isner, Isolation of Putative Progenitor Endothelial Cells for Angiogenesis, SCIENCE (1997), 275, 964-967), and research on treatment of neo-vascularization failure related diseases using endothelial progenitor cells has been conducted. For example, Hur, et al., reported that endothelial progenitor cells isolated by Asahara, et al. may be classified into early vascular endothelial progenitor cells and late vascular endothelial progenitor cells which have different features. According to Hur, et al, the early vascular endothelial progenitor cells secrete angiogenesis-related growth factors during the angiogenesis process, and the late vascular endothelial progenitor cells are directly related to formation of blood vessels during the angiogenesis process (Jin Hur, Chang-Hwan Yoon, Hyo-Soo Kim, Young-Bae Park, Characterization of Two Types of Endothelial Progenitor Cells and Their Different Contributions to Neovasculogenesis (ATVB) 2004, 24, 288). Research on possibility of vascular endothelial progenitor cells prepared by culturing not only peripheral blood but also cord-blood or mononuclear cells isolated bone marrow for cell therapy has been conducted. However, there is a need to secure the sufficient number of cells for administration in patients. Since human embryonic stem cells were cultured in 1998, there has been conducted research on methods for differentiation of vascular endothelial cells using human embryonic stem cells. According to studies conducted by Levenberg S, et al., embryoid bodies cultivated from human embryonic stem cells spontaneously differentiate into vascular endothelial cells, and the vascular endothelial cells are isolated from the differentiated embryoid bodies using a fluorescence activated cell sorter (FACS) (Levenberg S₁ Golub JS, Amit M, Itskovitz-Eldor J₁ Langer R. PNAS (2002) 99, 4391-4396). Kim, et al., reported a method for obtaining a number of vascular endothelial cells by specifically isolating vascular endothelial cells from embryoid bodies (Kim J, Moon SH, Lee SH, Lee DR, Koh GY, Chung HM. Stem Cells Dev. 2007 Apr; 16(2):269-80.). In addition, it has been reported that vascular endothelial cells differentiated from human embryonic stem cells can be used as cell therapy for vascular diseases (Lino S. Ferreira, Sharon Gerecht, Hester F. Shieh, Robert Langer, Vascular Progenitor Cells Isolated From Human Embryonic Stem Cells Give Rise to Endothelial and Smooth Muscle-Like Cells and From Vascular Networks In Vivo, Circulation Research, 2007 Aug 3; 101(3):286-94). However, in order to use cells differentiated from human embryonic stem cells for cell therapy, safety problems such as malignant tumors which might be caused from human embryonic stem cells and other unidentified side effects still need to be addressed.

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM

The present inventors conducted extensive research to develop a cell delivery system for cell therapy using cells derived from embryonic stem cells (e.g., vascular endothelial progenitor cells or vascular endothelial cells), which can avoid safety problems such as malignant tumors which may be caused from human embryonic stem cells and other unidentified side effects. As a result, the present inventors have found that when cells such as vascular endothelial cells derived from embryonic stem cells are inserted into a specific carrier to form a cell delivery system and the cell delivery system is transplanted into a living body, migration of the cells from the transplanted region in the living body is inhibited by the carrier and desirable treatment effects can be obtained by cytokine, or the like secreted from the cells. Furthermore, since the transplanted cell delivery system is removed from the transplanted region by surgical operations, we also found that it is possible to avoid side effects such as malignant tumors that may be caused from cells derived from embryonic stem cells in the living body.

Therefore, the present invention provides a cell delivery system for cell therapy including cells derived from embryonic stem cells which can avoid side effects such as outbreak of malignant tumors.

TECHNICAL SOLUTION According to an aspect of the present invention, there is provided a cell delivery system for cell therapy comprising cells derived from embryonic stem cells, which is formed by inserting the cells derived from embryonic stem cells into a carrier formed of matrigel. The cell delivery system is transplanted into the region of a living body, which does not directly in contact with a disease region; migration of the cells derived from embryonic stem cells from the transplanted region is inhibited by the carrier; and the cell delivery system is finally removed from the transplanted region.

The inserting of the cells derived from embryonic stem cells into the carrier may be performed by inserting aggregated cell clusters of the cells derived from embryonic stem cells into the carrier. Preferably, the carrier is growth factor-free matrigel; the embryonic stem cells are human embryonic stem cells; and the cells derived from embryonic stem cells are labeled with a fluorescent material, more preferably, 1 , 1 '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate.

According to another aspect of the present invention, there is provided a cell delivery system for therapy of neo-vascularization failure related diseases comprising vascular endothelial cells differentiated from human embryonic stem cells, which is formed by inserting the differentiated vascular endothelial cells into a carrier formed of matrigel. The cell delivery system is transplanted into the region of a living body, which does not directly in contact with a disease region; migration of the differentiated vascular endothelial cells from the transplanted region is inhibited by the carrier; and the cell delivery system is finally removed from the transplanted region.

The inserting of the differentiated vascular endothelial cells into the carrier may be performed by inserting aggregated cell clusters of the cells derived from embryonic stem cells into the carrier. Preferably, the carrier is growth factor-free matrigel. The neo-vascularization failure related disease may be selected from the group consisting of diabetic ulcer; gangrene; wounds requiring angiogenesis; Burger's disease; hypertension; ischemic diseases comprising cerebrovascular ischemia, renal ischemia, lung ischemia, joint ischemia, and ischemic myocardial infarction; obstructive vascular disease; and cardiovascular disease. And also, the vascular endothelial cells differentiated from human embryonic stem cells may be labeled with a fluorescent material, more preferably, 1 , 1 '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate.

ADVANTAGEOUS EFFECTS

The cell delivery system according to the present invention is designed to envelop cells such as vascular endothelial cells derived from embryonic stem cells using a carrier such as matrigel, such that migration of the cells derived from embryonic stem cells out of the carrier can be inhibited and only substances secreted from the cells such as cytokine can be efficiently released. That is, in the cell delivery system of the present invention, cell therapy is accomplished only by substances secreted from the cells derived from embryonic stem cells, such as cytokine, while the cells derived from embryonic stem cells are. not directly in contact with a disease region. Therefore, the cell delivery system according to the present invention can avoid any formation of cancers and tumors, which may be caused from direct transplantation of cells derived from embryonic stem cells into a living body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F illustrate a process of preparing a cell delivery system according to the present invention. FIG. 1A is a image of vascular endothelial cells differentiated from human embryonic stem cells which are isolated and cultured, and FIG. 1 B is a image of the cultured vascular endothelial cells stained by DiI. FIGS. 1C to 1 E illustrate a cell delivery system prepared by inserting the vascular endothelial cells stained by DiI into growth factor-free matrigel and images of the cell delivery system after 72 hours, and FIG. 1 F is a image of a cell delivery system in which vascular endothelial cells stained by DiI are enveloped with matrigel containing growth factors after 24 hours. FIGS. 2A to 2D illustrate images of a mouse having hindlimb ischemia in which matrigel and a cell delivery system according to the present invention are respectively transplanted, obtained using an imaging fluorometer (Xenogen, U.S.A.) after 2 and 3 weeks of the transplantation. FIGS. 2A and 2B illustrate images of a mouse having hindlimb ischemia in which the matrigel and the cell delivery system according to the present invention are respectively transplanted, obtained after 2 weeks of the transplantation. FIGS. 2C and 2D illustrate images of a mouse having hindlimb ischemia in which the matrigel and the cell delivery system according to the present invention are respectively transplanted and removed after 3 weeks of the transplantation. FIG. 2E illustrates expression of angiogenic factors VEGF and Ang-1 in the matrigel and the cell delivery system isolated from the hindlimb mouse models, respectively, measured using reverse transcriptase-polymerase chain reaction (RT-PCR).

FIGS. 3A and 3B illustrate images of a mouse having hindlimb ischemia in which matrigel and a cell delivery system according to the present invention are respectively transplanted and removed after 3 weeks of the transplantation, obtained using an imaging fluorometer after 12 weeks. FIGS. 3C and 3D illustrate images of dissected femoral artery in hindlimb of the mouse where necrosis does not occur (FIG. 3B).

FIG. 4 illustrates an image of a mouse having hindlimb ischemia in which a mixture of vascular endothelial cells and growth factor-free matrigel is transplanted into dorsal and hip regions of the mouse, obtained using an imaging fluorometer (Xenogen, U.S.A.).

FIGS. 5A and 5B illustrate images of a mouse having hindlimb ischemia in which VEGF, as an angiogenic factor, is injected in a tail vein every 2 days for 2 weeks and a mouse having hindlimb ischemia in which VEGF was injected in the ischemic regions every 2 days for 2 weeks.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a cell delivery system for cell therapy comprising cells derived from embryonic stem cells, which is formed by inserting the cells derived from embryonic stem cells into a carrier formed of matrigel, wherein the cell delivery system is transplanted into the region of a living body, which does not directly in contact with a disease region; migration of the cells derived from embryonic stem cells from the transplanted region is inhibited by the carrier; and the cell delivery system is finally removed from the transplanted region.

The embryonic stem cell may be any embryonic stem cell derived from a mammal, and preferably embryonic stem cells derived from human. The human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of human blastocysts. For example, the hESCs may be CHA-hES3 (Ahn SE, Kim S, Park KH, Moon SH, Lee HJ, Kim GJ, Lee YJ, Park KH1 Cha KY, Chung HM. "Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells", Biochem Biophys Res Commun. (2006) 10; 340(2): 403-408), but is not limited thereto. In addition, the hESCs may be easily established by those skilled in the art.

Cells derived from the embryonic stem cells, preferably human embryonic stem cells, may be immature or mature cells used for cell therapy, but are not limited thereto. The immature or mature cells may be prepared by partially or completely differentiating embryonic stem cells, preferably human embryonic stem cells, using a conventional method. The types of cells and methods of preparing the cells are not limited. For example, vascular endothelial cells differentiated from human embryonic stem cells may be prepared using a known method disclosed in e.g., Kim J, Moon SH, Lee SH, Lee DY, Koh GY and Chung HM, "Effective Isolation and Culture of Endothelial Cells in Embryoid Body Differentiated from Human Embryonic Stem Cells" Stem cell and Development (2007) 16:269.280.

The cell delivery system according to the present invention includes a carrier formed of matrigel. The carrier prevents cells derived from embryonic stem cells inserted thereinto from migrating from the transplanted region to another region in the living body and efficiently releases a variety of substances (such as cytokine) secreted from the inserted cells. Thus, the use of the carrier makes it possible for the cell delivery system to be transplanted into a living body which does not directly in contact with a disease region. The carrier is formed of matrigel. The "matrigel", one of extracellular matrix materials derived from tumor cell lines, is a matrix extracted from Engelbreth-Holm-Swarm (EHS) sarcoma. The matrigel includes laminin, type IV collagen, heparin, and various unknown growth factors. Since the matrigel provides a substrate necessary for cell growth (e.g., Vukicevic et al., Exp. Cell Res, 202(1), 1-8(1992)), it is used as a substrate for culture or proliferation of cells. In addition, the matrigel remained in gel state at room temperature, in liquid state at 4⁰C , and in solid state at -2O⁰C . When vascular endothelial cells or vascular endothelial progenitor cells are cultured in matrigel including various growth factors, vascular cord or vascular tube are formed since the various growth factors function as substrates for angiogenesis. The growth factor includes TGF-beta secreted from Engelbreth-Holm-Swarm sarcoma, fibroblast growth factor, or the like. Growth factor-free matrigel (e.g., growth factor-free matrigel purchased from BD Bioscience (U.S.A.)) may be preferably used in the cell delivery system according to the present invention.

The cell delivery system may be formed by inserting cells derived from embryonic stem cells into a carrier formed of matrigel. The inserting of the cells derived from embryonic stem cells into the carrier may be performed by inserting aggregated cell clusters of the cells derived from embryonic stem cells into the carrier so that migration of the cells derived from embryonic stem cells may be efficiently inhibited.

The number of cells derived from embryonic stem cells and the amount of the carrier may be adjusted for cell therapy by those of ordinary skill in the art. For example, when the cell delivery system may be used for cell therapy of ischemic blood vessel, 9-11 cell clusters, each including 5.0 X 10⁶ vascular endothelial cells, may be inserted into matrigel having a volume of about 400-500 μl to form a cell delivery system. The number the cells and the amount of the carrier may vary as desired. The cells derived from embryonic stem cells contained in the cell delivery system for cell therapy may be labeled with a fluorescent material. The fluorescent material may be DiA (4-(4-ditetradecylaminostyry!)-N-methylpyridinium iodide), DiD (DiICI 8(5) or i .i'-dioctadecyl-S.S.S'.S'-tetramethylindodicarbocyanine,

4-chlorobenzenesulfonate salt), DiI (DiICI 8(3) or 1 ,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiO (DiOC18(3) or 3,3-dioctadecyloxacarbocyanine perchlorate), DiO-C14(3) (S.S'-dihexadecyloxacarbocyanine, hydroxyethanesulfonate), DiR (DiICI 8(7) or I .i'-dioctadecyltetramethyl indotricarbocyanine Iodide), or the like. Among them, the fluorescent material may be DiI having a wavelength of about 533 nm or DiO having a wavelength of about 488 nm, and preferably DiI having a wavelength of about 533 nm. The cell delivery system may be monitored in the living body in real time using the fluorescent material. For example, the position of cells transplanted into the living body may be monitored in real time using an imaging fluorometer (Xenogen), or the like.

The transplanted cell delivery system may be removed from the transplanted region by surgical operations after the treatment is completed. Thus, risks of outbreak of cancers, tumors, or the like which may be caused from the transplanted cells can be completely removed.

In accordance with one embodiment, there is provided a cell delivery system for therapy of neo-vascularization failure related diseases comprising vascular endothelial cells differentiated from human embryonic stem cells, which is formed by inserting the differentiated vascular endothelial cells into a carrier formed of matrigel, wherein the cell delivery system is transplanted into the region of a living body, which does not directly in contact with a disease region; migration of the differentiated vascular endothelial cells from the transplanted region is inhibited by the carrier; and the cell delivery system is finally removed from the transplanted region. The vascular endothelial cells differentiated from the human embryonic stem cells may be prepared using a conventional method, such as a method disclosed in Kim J, Moon SH, Lee SH, Lee DY, Koh GY and Chung HM, "Effective Isolation and Culture of Endothelial Cells in Embryoid Body Differentiated from Human Embryonic Stem Cells" Stem cell and Development (2007) 16:269.280. The vascular endothelial cells differentiated from embryoid bodies positive for a vascular endothelial cell marker such as PECAM, CD34, KDR/Flk-1 , CD133, Tie-2, VE-cadherin, and vWF may be isolated using a fluorescence activated cell sorter (FACS).

The cell delivery system for therapy of neo-vascularization failure related diseases include a carrier formed of matrigel. The carrier prevents the differentiated vascular endothelial cells inserted thereinto from migrating from the transplanted region to another region in the living body and efficiently releases a variety of substances (such as cytokine) secreted from the inserted cells. Thus, the use of the carrier makes it possible for the cell delivery system to be transplanted into a living body which does not directly in contact with a disease region. More preferably, growth factor-free matrigel (e.g., BD Bioscience (U.S.A.)) may be used in the cell delivery system according to the present invention.

The cell delivery system may be formed by inserting differentiated vascular endothelial cells into a carrier formed of matrigel. The inserting of differentiated vascular endothelial cells into the carrier may be performed by inserting aggregated cell clusters of the differentiated vascular endothelial cells into the carrier so that migration of the cells derived from embryonic stem cells may be efficiently inhibited. For example, 9-11 cell clusters, each including 5.0 X 10⁶ vascular endothelial cells, may be inserted into matrigel having a volume of about 400-500 μl to form a cell delivery system for therapy of neo-vascularization failure related diseases. The number the differentiated vascular endothelial cells and the amount of the carrier may vary as desired.

The neo-vascularization failure related diseases may be: diabetic ulcer; gangrene; wounds requiring angiogenesis; Burger's disease; hypertension; ischemic diseases comprising cerebrovascular ischemia, renal ischemia, lung ischemia, joint ischemia, and ischemic myocardial infarction; obstructive vascular disease; and cardiovascular disease.

The differentiated vascular endothelial cells contained in the cell delivery system for cell therapy may be labeled with a fluorescent material. The fluorescent material may be DiA (4-(4-ditetradecylaminostyryl)-N-methylpyridinium iodide), DiD (DiICI 8(5) or 1 ,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt), DiI (DiICI 8(3) or 1 ,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiO (DiOCI 8(3) or 3,3-dioctadecyloxacarbocyanine perchlorate), DiO-C14(3) (3,3'-dihexadecyloxacarbocyanine, hydroxyethanesulfonate), DiR (DiICI 8(7) or 1 ,1 '-dioctadecyltetramethyl indotricarbocyanine Iodide), or the like. Among them, the fluorescent material may be DiI having a wavelength of about 533 nm or DiO having a wavelength of about 488 nm, and preferably DiI having a wavelength of about 533 nm. The cell delivery system may be monitored in the living body in real time using the fluorescent material. For example, the position of cells transplanted into the living body may be monitored in real time using an imaging fluorometer (Xenogen), or the like.

Hereinafter, the present invention will be described more specifically with reference to the following examples. The following examples are only for illustrative purposes and are not intended to limit the scope of the invention.

Example 1 : Vascular endothelial cells stained by 1.1'-dioctadecyl-β.S.S'.S'-tetramethylindocarbocyanine perchlorate (DiI)

Vascular endothelial cells were naturally differentiated from human embryonic stem cells CHA-hES3 (Ahn SE, Kim S, Park KH, Moon SH, Lee HJ, Kim GJ, Lee YJ, Park KH, Cha KY, Chung HM. Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells. Biochem

Biophys Res Commun. 2006 10; 340(2):403-408) using a method introduced by Kim, et al. (Kim J, Moon SH, Lee SH, Lee DY, Koh GY and Chung HM, "Effective Isolation and

Culture of Endothelial Cells in Embryoid Body Differentiated from Human Embryonic Stem Cells" Stem cell and Development (2007) 16:269.280). Among theses cells, cells positive for vWF were isolated using a FACS.

A cell-labeling solution was prepared by diluting 10 μl of 1 ,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) solution in a serum-free DMEM (high glucose) at a dilution ratio of 1:200, and mixed with the isolated vascular endothelial cells. Then, the mixture was cultured in an incubator at 37⁰C for about 15-20 minutes. The culture medium was removed, and the resultant was washed three times with phosphate buffered saline (PBS). Then, stain of DiI was identified using a fluorescence microscope.

Example 2: Preparation of cell delivery system using matrigel 5 X 10⁵ of vascular endothelial cells stained by DiI prepared in Example 1 were seeded on an EGM-2/MV culture medium (Cambrex, U.S.A.) such that drops are formed on the bottom surface of a culture dish. The culture dish was turned over and cultured in an incubator for 24 hours so as to obtain aggregated cells formed in a ball shape (sorted cells or aggregated cell clusters). Growth factor-free matrigel (BD Bioscience, U.S.A.) in gel state was cut into cylindrical pieces having a diameter of 50-70 mm and a height of 50-70 mm, and the aggregated cell clusters were inserted in the cylindrical matrigel. Then, the resultant was cultured in an incubator at 37 °C for 30 minutes to obtain a cell delivery system in which cells were enveloped by the matrigel (FIGS. 1 C to 1 E).

A cell delivery system was prepared in the same manner as described above using matrigel containing growth factors (BD Bioscience, U.S.A.). However, cells grew from aggregated cell clusters and came out of the cell delivery system within a short period of time (24 hours) (FIG. 1 F). Thus, the cell delivery system cannot appropriately inhibit migration of cells in a living body, when using matrigel containing growth factors.

Experimental Example 1

(1) Preparation of mouse model having hindlimb ischemia 50 μl of an anesthetic prepared by mixing ketamine and Rumpun in a ratio of 4:1 was injected into each of 6-7 week-old Balb/C Nude (Charles River Laboratories) via intraperitoneal injection. The anesthetized mouse was fixed, and 1 cm skin incision was vertically made in a boundary between abdomen and femoral regions to remove fat in the boundary between the abdomen and the femoral regions. Common iliac artery in the abdomen region was ligated twice using 4-0 black silk suture along external iliac artery. An upper region of blood vessel of ankle area divided into two branches along the external iliac artery was tied. Blood vessel from the node of blood vessel in ankle area to the node in femoral region was isolated such that muscles were not damaged. Then, the avulsed skin was sutured.

(2) Evaluation of angiogenesis by administration of cell delivery system The mice having hindlimb ischemia prepared in step (1) described above were classified into two groups (n=5). In the first group, growth factor-free matrigel was transplanted into a dorsal region of the mouse. In the second groups, a cell delivery system prepared in Example 2 (cell delivery system prepared using growth factor-free matrigel) was transplanted into a dorsal region of a mouse. The transplantation of the matrigel or the cell delivery system was performed as follows. The dorsal skin of the mouse having hindlimb ischemia was incised for about 1 cm, the matrigel or the cell delivery system was transplanted into the incised region using a spoon such that the shapes of the matrigel and the cell delivery system were not deformed, and the skin was sutured. The transplanted matrigel or cell delivery system was removed with peripheral region thereof using operating scissors, and the skin was sutured.

FIGS. 2A and 2B illustrate images of a mouse having hindlimb ischemia in which matrigel and a cell delivery system according to the present invention were respectively transplanted, obtained using an imaging fluorometer (Xenogen, U.S.A.) after 2 weeks of the transplantation. In addition, FIGS. 2C and 2D illustrate images of a mouse having hindlimb ischemia in which matrigel and a cell delivery system according to the present invention were respectively transplanted and removed after 3 weeks of the transplantation, obtained using an imaging fluorometer (Xenogen, U.S.A.). As shown in FIG. 2B, when the cell delivery system prepared according to the present invention was transplanted, necrosis did not occur due to efficiently performed antiogenesis, and the cell delivery system stayed at the transplanted region. In addition, expression of angiogenic factors VEGF and Ang-1 in the matrigel and the cell delivery system isolated from the hindlimb mouse models, respectively, was measured using RT-PCR. As a result, VEGF and Ang-1 were specifically expressed in the cell delivery system (FIG. 2E).

FIGS. 3A and 3B illustrate images of a mouse having hindlimb ischemia in which matrigel and a cell delivery system according to the present invention were respectively transplanted and removed after 3 weeks of the transplantation, obtained using an imaging fluorometer after 12 weeks. FIGS. 3C and 3D illustrate images of dissected hindlimb of the mouse in which necrosis did not occur (FIG. 3B). As shown in FIG. 3B, therapeutic effects can be maintained even though the transplanted cells are removed if the treatment is conducted for a predetermined period of time. Furthermore, it can be seen that a neo-vessel was formed in the region marked by a blue arrow in which blood vessel was removed marked by a black arrow in FIG. 3D.

Experimental Example 2: Transplantation of mixture of matrigel and vascular endothelial cells

Aggregated cell clusters of vascular endothelial cells obtained in the same manner as in Example 2 were mixed with about 450 g of growth factor-free matrigel, and the mixture was transplanted into dorsal and hip regions of a mouse having hindlimb ischemia. FIG. 4A illustrates an image of the mouse after 1 week using an imaging fluorometer. As shown in FIG. 4A, when the cells were not enveloped by matrigel but simply mixed with the matrigel, they migrated to peripheral regions, and thus side effects such as outbreak of tumors may be caused by the transplanted cells.

Experimental Example 3: Comparison with direct administration of angiogenic factor

100 ng VEGF, as an angiogenic factor, was injected into a tail vein of a mouse having hindlimb ischemia every 2 days for 2 weeks. 100 ng of VEGF was injected into ischemic regions of a mouse having hindlimb ischemia every 2 days for 2 weeks. The results are shown in FIGS. 5A and 5B. Necrosis occurred in FIG. 5A (injected into the tail vein) and FIG. 5B (injected into the ischemic regions). Therefore, it can be seen that necrosis does not occur when the cell delivery system according to the present invention is transplanted into a living body due to an angiogenic factor secreted from differentiated vascular endothelial cells inserted into the cell delivery system.

Claims

1. A cell delivery system for cell therapy comprising cells derived from embryonic stem cells, which is formed by inserting the cells derived from embryonic stem cells into a carrier formed of matrigel.

2. The cell delivery system of claim 1 , wherein the inserting of the cells derived from embryonic stem cells into the carrier is performed by inserting aggregated cell clusters of the cells derived from embryonic stem cells into the carrier.

3. The cell delivery system of claim 1 , wherein the carrier is growth factor-free matrigel.

4. The cell delivery system of any one of claims 1 to 3, wherein the embryonic stem cells are human embryonic stem cells.

5. The cell delivery system of any one of claims 1 to 3, wherein the cells derived from embryonic stem cells are labeled with a fluorescent material.

6. The cell delivery system of claim 5, wherein the fluorescent material is 1 ,1'-dioctadecyl-3,3,3\3'-tetramethylindocarbocyanine perchlorate.

7. A cell delivery system for therapy of neo-vascularization failure related diseases comprising vascular endothelial cells differentiated from human embryonic stem cells, which is formed by inserting the differentiated vascular endothelial cells into a carrier formed of matrigel.

8. The cell delivery system of claim 7, wherein the inserting of the differentiated vascular endothelial cells into the carrier is performed by inserting aggregated cell clusters of the vascular endothelial cells differentiated from human embryonic stem cells into the carrier.

9. The cell delivery system of claim 7, wherein the carrier is growth factor-free matrigel.

10. The cell delivery system of any one of claims 7 to 9, wherein the neo-vascularization failure related disease is selected from the group consisting of diabetic ulcer; gangrene; wounds requiring angiogenesis; Burger's disease; hypertension; ischemic diseases comprising cerebrovascular ischemia, renal ischemia, lung ischemia, joint ischemia, and ischemic myocardial infarction; obstructive vascular disease; and cardiovascular disease.

11. The cell delivery system of any one of claims 7 to 9, wherein the vascular endothelial cells differentiated from human embryonic stem cells are labeled with a fluorescent material.

12. The cell delivery system of claim 11 , wherein the fluorescent material is 1 , 1 '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate.