JP6188075B2 - Immortalized cell lines of peripheral tissue capillaries - Google Patents

Description

  The present invention relates to an immortalized vascular endothelial cell line derived from capillaries of a non-human mammal, an immortalized pericyte cell line, an immortalized mesenchymal stem cell-like pericyte cell line, and a method for preparing the same.

In conventional angiogenesis research, there are many restrictions on observation of new blood vessels and capillaries because the target is minute and the distribution is irregular (Non-Patent Document 1), and the following problems (i) to (iii) There is a point.
(I) In the histological technique, immunostaining is indispensable in order to identify capillaries in tissue sections, and it is difficult to objectively evaluate the overall degree of angiogenesis.
(Ii) Use limited organs (conjunctiva, retina, cerebral dura mater) and animals other than mammals (eg chicken egg amniotic membrane, zebrafish) that can be observed in a two-dimensional distribution or permeable tissue. There are few ideal experimental systems that are versatile in medical research.
(Iii) As an in vitro angiogenesis assay, tube formation using endothelial cells such as human-derived large blood vessel endothelial cells and angiogenesis from tissue sections using large blood vessel tissue sections under three-dimensional gel culture Although the observation method is frequently used, since most of the observation is to the level of immature endothelial cell tube formation, it cannot be said that the original intended angiogenesis is evaluated.

The in vitro angiogenesis assay method using vascular cells has a high degree of freedom in setting the conditions, and enables detailed observation such as changes over time. There are problems (i) to (ii).
(I) Tissues of relatively large blood vessels that can prepare endothelial cells in large quantities have been frequently used so far. However, large blood vessel-derived endothelial cells are different from endothelial cells of new blood vessels, and there are problems of bias due to individual differences and cell separation preparation, so they are not ideal sources for observing angiogenesis.
(Ii) It is ideal to separate and prepare endothelial cells and pericytes derived from capillaries rather than large blood vessels. However, the capillaries that exist in the tissue are micro-tissues that are "dissolved" in various other cells, and there is no specific separation method, and in the process of increasing a small number of cells, There is also a problem that the characteristics of the cell itself change, such as cell aging, and it is difficult to prepare capillary-derived cells efficiently and stably.

  In response to such problems, many immortal cell lines including capillary cells have been established using SV40 temperature-sensitive mutant tsA58T antigen gene expressing animals. The vascular endothelial cells and pericytes reported so far are limited to those derived from the brain or retina (all derived from rats) (Non-patent Documents 2 to 5), and many organ diseases derived from other than the brain eyes There are no peripheral capillary-derived cell lines involved. This is because it is difficult to purify the capillaries into pure frames from tissues other than special organs such as the brain and retina where the connective tissue is rough or the capillaries are dense.

  So far, bone marrow endothelial progenitor cells have been isolated from bone marrow of SV40 temperature-sensitive mutant tsA58T antigen gene expressing rats, and the cells have been subcultured to produce an immortalized vascular endothelial cell line (Patent Document 1). However, there is a debate as to how much fully differentiated “endothelial cells” are immortalized vascular endothelial cell lines obtained by differentiating from bone marrow endothelial progenitor cells, and true endothelial cells. In addition, an immortalized vascular pericyte cell line derived from brain capillaries obtained by homogenizing cerebral tissue of SV40 temperature-sensitive mutant tsA58T antigen gene expressing rat has been reported (Patent Document 2). However, it is unclear whether the cells obtained by a simple method of homogenizing and centrifuging cerebral tissue are derived from brain capillaries.

JP 2001-231549 A JP 2001-224367 A

Int. J. et al. Exp. Path 90, 195-221, 2009 Eur. J. et al. Cell Biol. 81, 145-152, 2002. Mol. Pharmacol. 61, 1289-1296, 2002. Exp. Eye Res. 72,163-172,2001. Cell Tissue Res. 326, 749-758, 2006. Nature reviews 8,726-736,2008 Cell Stem Cell, 3, 301-313, 2008

  An object of the present invention is to provide an immortalized cell line of capillary constituent cells derived from peripheral capillaries involved in many organ diseases.

  In order to achieve the above object, the inventors prepared a tissue containing capillary blood vessels, separated cells from the tissue with a collagenase enzyme solution, and said that there is some specificity for endothelial cells and pericytes. The cells were established by sorting and passaging endothelial cells and pericytes using antibodies against other markers (eg, CD146 and NG2, respectively).

  That is, the present invention relates to a method for preparing an immortalized pericyte cell line derived from capillaries of a non-human mammal and an immortalized mesenchymal stem cell-like pericyte cell line, and immortalization derived from capillaries of a non-human mammal. The present invention relates to a method for preparing a vascular endothelial cell line.

In a first embodiment, the present invention provides a method of preparing an immortalized pericyte cell line and an immortalized mesenchymal stem cell-like pericyte line derived from a capillary tube of a non-human mammal, comprising the following steps:
(A) a step of separating cells from a tissue densely containing capillaries prepared in the body of a non-human mammal introduced with the SV40 temperature-sensitive mutant tsA58T antigen gene;
(B) selecting a cell expressing at least one selected from NG2, PDGFRb, and NGFR, subculturing the cell to obtain an immortal cell line, and (c) from among the immortal cell lines Select cell lines that do not have the ability to differentiate into mesenchymal cells as immortalized pericytes, and select cell lines that have the ability to differentiate into mesenchymal cells as immortalized mesenchymal stem cell-like pericytes. Process.

The above method may further comprise the following steps as required:
(D) A step of selecting a cell line expressing a pericyte marker from an immortalized cell line.

  Examples of pericyte markers include PDGFRb, aSMA, myocardin, angiopoietin-1, calponin, caldesmon, ang-like4, and NGF.

  For a tissue containing capillary blood vessels, for example, a carrier containing an extracellular matrix component is placed around a damaged blood vessel of a non-human mammal into which an SV40 temperature-sensitive mutant tsA58T antigen gene has been introduced, and the capillary blood vessels are developed there. Can be prepared.

  The carrier is, for example, one or more extracellular matrix components selected from collagen, fibronectin, vitronectin, proteoglycan, laminin, tenascin, entactin, elastin, fibrin, hyaluronic acid, or a reconstituted base such as Matrigel (registered trademark). Contains a membrane matrix.

  In one embodiment, the carrier is collagen or collagen gel.

  The present invention provides an isolated capillary-derived immortalized pericyte cell line that expresses NG2 and does not have the ability to differentiate into mesenchymal cells, obtained by the preparation method described above.

  The present invention also provides an isolated capillary-derived immortalized mesenchymal stem cell-like pericyte cell line that expresses NG2 and has the ability to differentiate into mesenchymal cells obtained by the preparation method described above.

  The immortalized mesenchymal stem cell-like pericellular line of the present invention is further positive for at least one mesenchymal stem cell marker selected from Sca1, CD29, CD44, CD105, and CD106, and is aSMA positive, CD11b and Characterized by being CD45 negative.

  The immortalized mesenchymal stem cell-like pericytes of the present invention can be differentiated into endothelial cells, skeletal muscle cells, osteoblasts, and adipocytes.

In a second embodiment, the present invention provides a method of preparing an immortalized vascular endothelial cell line derived from a non-human mammal capillary, comprising the following steps:
(A) separating cells from a tissue densely containing capillaries prepared in the body of a non-human mammal introduced with the SV40 temperature sensitive mutant tsA58T antigen gene, and (b) from CD146, rWF, and CD31 A step of selecting cells expressing at least one selected and subculturing the cells to obtain an immortalized cell line.

The above method may further comprise the following steps as required:
(C) A step of selecting a cell line expressing a marker for vascular endothelial cells from an immortalized cell line.

  Examples of vascular endothelial cell markers include VEGFR2 (Flk1), vWF, VEGFR1 (Flt1), PECAM1 (CD31), VE-cadherin, TIE1, and TIE2.

  For a tissue containing capillary blood vessels, for example, a carrier containing an extracellular matrix component is placed around a damaged blood vessel of a non-human mammal into which an SV40 temperature-sensitive mutant tsA58T antigen gene has been introduced, and the capillary blood vessels are developed there. Can be prepared.

  The carrier is, for example, one or more extracellular matrix components selected from collagen, fibronectin, vitronectin, proteoglycan, laminin, tenascin, entactin, elastin, fibrin, hyaluronic acid, or a reconstituted base such as Matrigel (registered trademark). Contains a membrane matrix.

  In one embodiment, the carrier is collagen or collagen gel.

  The present invention provides a capillary-derived immortalized vascular endothelial cell line that expresses CD146, rWF, and CD31 obtained by the preparation method described above.

  In a third embodiment, the present invention provides a method for preparing a capillary-like structure. The method is characterized in that a capillary-like structure is prepared by three-dimensionally coculturing the dead vascular endothelial cell line of the present invention and the immortalized pericyte cell line of the present invention.

  Alternatively, a capillary-like structure can be prepared by three-dimensionally culturing the immortalized mesenchymal stem cell-like pericytes of the present invention using a medium containing VEGF. In this case, after three-dimensional culture using a medium containing VEGF, the medium may be replaced with a medium that does not contain VEGF but contains TGF for three-dimensional culture.

  When the present invention is used, cell preparation is simplified and cell conditions (single gene background, no individual differences) are made uniform, so that it can be expected that variation in experimental results is minimized. In addition, since conventional blood vessel research has used various animals in many cases, it is beneficial to use an in vitro assay method using appropriate passage cells from the viewpoint of animal welfare and research management efficiency. It is.

  It is clear that capillary dysplasia is deeply involved not only in tumors and retinopathy but also in the pathology of important intractable diseases that threaten the health of Japanese people, such as arteriosclerosis, heart failure, and insulin resistance. It has become. In addition, expectations for regenerative medicine have been increasing recently, and there is a high need for research on “angiogenesis” as there is an intravascular tissue regeneration construction as a universally indispensable issue for organ regeneration. Compared to the conventional experimental system for mere “endothelial cells”, this cell line, which is a capillary constituent cell, and the assay method using these cell lines maintain normal blood capillaries and maintain more mature blood vessels. It is very useful for elucidating the mechanism for regeneration.

  Furthermore, pluripotent mesenchymal stem cells (MSCs) present in many tissues are expected for their role in various disease states and application to regenerative medicine (Nature reviews 8, 726-736, 2008). ). Recently, it has become clear that (at least) a part of tissue MSC whose localization in the tissue is unknown as well as a mechanism of undifferentiated maintenance or differentiation control as stem cells exist as capillary pericytes. (Cell Stem Cell, 3, 301-313, 2008). This cell line (MSC-PC) and the assay method using this cell line have led to the elucidation of the above-mentioned questions related to tissue-derived MSC, and are not only angiogenesis but also an intractable disease that is a problem in an aging society Can be expected to be useful as a useful tool in a wide range of medical and medical fields, such as elucidation of pathological conditions (remodeling of fat and bone tissue (metabolic syndrome, osteoporosis), fibrosis), and development of regenerative medicine (preparation of regenerative cell tools) .

FIG. 1 is a diagram showing a capillary tissue formed around a damaged blood vessel. FIG. 2 is a diagram showing monolayer growth (A), RT-PCR result (B), and lumen (C) obtained by three-dimensional culture of a vascular endothelial cell line (c-EC). FIG. 3 is a diagram showing multilayer growth (A), RT-PCR results (B), and capillary-like structures (C) obtained by three-dimensional culture of pericytes (c-PC). FIG. 4 is a diagram showing the formation of a capillary-like structure by co-culture of a vascular endothelial cell line (c-EC) and a pericyte line (c-PC). FIG. 5 is a diagram showing that the mesenchymal stem cell-like pericytes (MSC-PC) have multipotency. FIG. 6 is a diagram showing the formation of capillary-like structures by mesenchymal stem cell-like pericytes (MSC-PC). FIG. 7 is a diagram showing the ability to attach cells to the surface of a capillary endothelial cell line (c-EC). FIG. 8 is a diagram showing pluripotency in a living body of a mesenchymal stem cell-like pericyte cell line (MSC-PC). (A) Differentiation into skeletal muscle cells by introduction into cardiotoxin-damaged skeletal muscle tissue, (B) Differentiation into capillaries by introduction into subcutaneous adipose tissue at the lower limb ischemia site (B1: differentiation into pericytes, B2 : Differentiation into endothelial cells), (C) differentiation into adipocytes by introduction into subcutaneous adipose tissue. FIG. 9 is a diagram showing a surface marker profile of a mesenchymal stem cell-like pericyte cell line (MSC-PC).

  This specification includes the contents described in the specification of Japanese Patent Application No. 2012-24854, which is the basis of the priority of the present application.

  The present invention relates to an immortalized vascular endothelial cell line, an immortalized pericyte cell line, and an immortalized mesenchymal stem cell-like pericyte cell line derived from capillaries of non-human mammals. Specifically, immortalized vascular endothelial cell lines, immortalized pericytes, and immortalized mesenchymal stem cell-like pericytes, methods for preparing these cell lines, and capillary-like structures reconstructed by these cell lines Concerning structure.

1. Method for preparing immortalized vascular endothelial cell line (c-EC), immortalized pericyte cell line (c-PC), and immortalized mesenchymal stem cell-like pericyte cell line (MSC-PC) Endothelial cell (EC) ) Are thin and flat cells constituting the inner surface of the blood vessel. In the present invention, “immortalized vascular endothelial cell line” or “c-EC” refers to an immortalized vascular endothelial cell clone that retains the function and characteristics as a vascular endothelial cell.

  A pericyte (also referred to as a pericyte) is a cell that exists so as to surround a capillary wall, and is a mesodermal cell that is also called a Rouget cell. At present, it is presumed that pericytes present in a living body are not composed of a single type of cell, but the type cannot be identified by a known marker. The inventors further identified a cell line having a mesenchymal stem cell (MSC) -like differentiation ability from a plurality of pericyte lines, and designated it as a “mesenchymal stem cell-like pericyte cell line”. . Therefore, in the present invention, the “immortalized pericellular line” or “c-PC” is an immortalized pericellular clone that retains the function and characteristics as a pericyte and has the ability to differentiate into mesenchymal cells. Refers to a cell clone that does not have an "immortalized mesenchymal stem cell-like pericyte line" or "MSC-PC" is an immortalized pericyte that retains function and properties as a pericyte and is an osteoblast A cell clone having the ability to differentiate into cells belonging to the mesenchymal system such as adipocytes, muscle cells, chondrocytes, endothelial cells, stem cells, or nerve cells.

1.1 Preparation of Tissue Containing Capillaries In order to prepare an immortalized vascular endothelial cell line, first, tissue containing capillaries is prepared in vivo in a non-human mammal.

  In the large blood vessels, capillaries (vasa vasorum: vascular blood vessels) that feed the blood vessels are distributed from the outer membrane of the blood vessel wall to the media, and when vascular injury causes plaque formation and thickening of the blood vessel wall, vasa around the blood vessel of the same site Vasorum angiogenesis is known to be enhanced (see Am J Physiol 298, H295-305, 2010. Cardiovascu Res 75, 649-658, 2007). Therefore, in the present invention, a carrier containing an extracellular matrix component serving as a place for angiogenesis is placed and introduced around a damaged blood vessel of a transgenic animal into which the SV40 temperature-sensitive mutant tsA58T antigen gene has been introduced, and here a capillary vessel is introduced. By expanding, a tissue containing capillary blood vessels is created.

  As an extracellular matrix component, use an extracellular matrix component such as collagen, fibronectin, vitronectin, proteoglycan, laminin, tenascin, entactin, elastin, fibrin, hyaluronic acid, or a reconstituted basement membrane matrix such as Matrigel (registered trademark). Can do. Collagen may be derivatized (acylated or esterified), and may be modified collagen such as gelatin, collagen gel, hydrogel and the like.

  The carrier containing the extracellular matrix component only needs to be in a form that can be removed as a tissue containing newly-developed capillaries, and may be a gel or sponge composed of the extracellular matrix component, The matrix may be coated on a suitable support.

  Specifically, for example, by placing a collagen-coated carrier (for example, a tube or sheet) or a collagen gel or the like around the damaged blood vessel of the animal, a capillary vessel is developed on the surface of the carrier or the collagen gel. . Next, by peeling off the collagen film or collagen gel surface on the surface of the carrier, a thin film tissue densely containing capillaries can be obtained.

  In the present invention, “comprising capillaries“ closely ”” means a state in which connective tissues are rough and capillaries are densely distributed, such as those present in the brain and retina.

  In the present invention, rodent animals such as mice and rats can be used as non-human mammals. In addition, a transgenic animal into which an oncogene such as ras gene or c-myc gene, adenovirus E1A gene, SV40 virus large T antigen gene, or human papillomavirus HPV16 gene can be used. In particular, transgenic animals into which the SV40 temperature sensitive mutant tsA58T antigen gene has been introduced are preferably used. The cells derived from such transgenic animals are isolated from living tissues and cultured at a low temperature (32 to 34 ° C.), so that intracellular T antigen is stably expressed. The cells can be immortalized while maintaining the characteristics of the cells, and the T antigen is invalidated by culturing at a normal culture temperature (36 to 38 ° C.), and the cells are used for experiments in a form that excludes the influence of the T antigen as much as possible. can do.

1.2 Separation of cells Cells are separated from a thin film tissue containing capillary blood vessels. Separation of cells can be carried out according to a method well known to those skilled in the art, for example, by separation using an enzyme such as collagenase, trypsin, dispase, elastase, chymotrypsin, and pronase.

1.3 Selection by marker and immortalization (1) Immortalized vascular endothelial cell line (c-EC)
From the separated cells, cells expressing vascular endothelial cell marker are selected. As a marker for selection, any of cell surface markers CD146, rWF, and CD31 is preferable from the viewpoint of ease of selection, and CD146 or rWF is particularly preferable from the viewpoint of expression level, and CD146 is most preferable. Selection of cells can be performed according to methods well known to those skilled in the art, for example, after labeling cells with an antibody against the above marker, according to fluorescence activated cell sorting (FACS), magnetic cell separation, etc. Cells can be separated and selected.

  The separated cells are subcultured at a low temperature using an endothelial cell medium to be immortalized. As the medium, for example, a commercially available medium for endothelial cells such as EGM-2 medium and EBM-2 medium can be used. The subculture is performed at a low temperature in order to stably express the intracellular T antigen and to exert its function. Here, low temperature is 32-34 degreeC, Preferably it is 32.5-33.5 degreeC, More preferably, it is 33 degreeC.

  Vascular endothelial cells are adherent cells that have the property of growing by attaching to the extracellular matrix, and the cell lines of the present invention have similar properties. Therefore, it is preferable to use an appropriate scaffold in the culture. The scaffold is not particularly limited as long as it is a matrix, substrate or carrier that can adhere and divide and proliferate, for example, fibronectin, vitronectin, collagen, proteoglycan, laminin, tenascin, entactin, elastin, fibrin, hyaluronic acid, gelatin, Examples thereof include poly-L-lysine and poly-D-lysine. In particular, it is desirable to use fibronectin as a matrix.

(2) Immortalized pericyte cell line (c-PC) and immortalized mesenchymal stem cell-like pericyte line (MSC-PC)
A cell expressing a pericyte marker is selected from the separated cells. As the marker for selection, NG2, PDGFRb, and NGFR, which are cell surface markers, are preferable, and NG2 is particularly preferable from the viewpoint of easy selection and distinction from vascular endothelial cells. Selection of cells can be performed according to methods well known to those skilled in the art, for example, after labeling cells with an antibody against the above marker, according to fluorescence activated cell sorting (FACS), magnetic cell separation, etc. Cells can be separated and selected.

  The isolated cells expressing NG2 are subcultured at a low temperature using a basic medium to be immortalized. As the basic medium, MEM medium, BME medium, DME medium, α-MEM medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI medium, StemSpan medium, StemPro medium and these Mixtures can be used.

  The subculture is performed at a low temperature in order to stably express the intracellular T antigen and to exert its function. Here, low temperature is 32-34 degreeC, Preferably it is 32.5-33.5 degreeC, More preferably, it is 33 degreeC.

  Pericytes and mesenchymal stem cells are adherent cells that have the property of growing by attaching to the extracellular matrix, and the cell lines of the present invention have similar properties. Therefore, it is preferable to use an appropriate scaffold in the culture. The scaffold is not particularly limited as long as it is a matrix, substrate or carrier that can adhere and divide and proliferate, for example, fibronectin, vitronectin, collagen, proteoglycan, laminin, tenascin, entactin, elastin, fibrin, hyaluronic acid, gelatin, Examples thereof include poly-L-lysine and poly-D-lysine. In particular, it is desirable to use collagen as a matrix.

  Next, whether the obtained immortalized cell line has the ability to differentiate into mesenchymal cells (for example, osteoblasts, adipocytes, muscle cells, chondrocytes, endothelial cells, stem cells, nerve cells, etc.) The cell line that does not have differentiation potential is selected as an immortalized pericyte cell line, and the cell line that has differentiation ability is selected as an immortalized mesenchymal stem cell-like pericyte cell line. Specifically, the cell line is cultured at a normal culture temperature using a differentiation medium for mesenchymal cells well known to those skilled in the art, and the expression of each mesenchymal cell marker is examined according to a method well known to those skilled in the art. Alternatively, the cells are stained to determine whether they have differentiated. Here, the normal culture temperature is 36 to 38 ° C, preferably 37 ° C.

  In one aspect of the present invention, the obtained immortalized cell line is cultured at 37 ° C. using an adipocyte differentiation medium, and is immortalized by confirming oil red staining and FABP4 expression. Pericytes and immortalized mesenchymal stem cell-like pericytes may be selected. As the medium, a commercially available adipocyte differentiation medium such as DMEM medium containing isobutylmethylxanthin (3IBMX), dexamethasone, insulin and the like can be used. If it is stained with oil red or positive for FABP, it can be said that it has differentiated into adipocytes, so the cell line is an immortalized mesenchymal stem cell-like pericyte cell line and is not stained with oil red or is negative for FABP Since it can be said that it did not differentiate into adipocytes, such a cell line is an immortalized pericyte cell line.

  In another embodiment of the present invention, the obtained immortalized cell line is cultured at 37 ° C. using an osteoblast differentiation medium, and alizarin red staining or osteopontin expression is performed. By confirming, an immortalized pericyte cell line and an immortalized mesenchymal stem cell-like pericyte cell line may be selected. As the medium, a commercially available osteoblast differentiation medium such as an MEM medium containing ascorbic acid, hydrocortisone, and beta-glycerophosphate can be used. If it is stained with alizarin red or osteopontin positive, it can be said that it has differentiated into osteoblasts, so the cell line is an immortalized mesenchymal stem cell-like pericyte, not stained with alizarin red, or osteopontin negative In this case, it can be said that the cell line did not differentiate into an osteoblast, and thus the cell line is an immortalized pericyte.

2. Immortalized Vascular Endothelial Cell Line The immortalized vascular endothelial cell line of the present invention obtained according to the above preparation method 1 is a capillary-derived cell line that expresses a marker for vascular endothelial cells such as CD146, rWF, and CD31. Retains function and properties as vascular endothelial cells. In addition, the immortalized vascular endothelial cell line of the present invention grows in a monolayer, has an adsorptivity to acetylated low-density lipoprotein (Acetyl LDL), and binds to lectin. In the case of mouse-derived cells, the immortalized vascular endothelial cell line of the present invention binds to Banderaea simplicifolia agglutinin (BS-1 lectin) among lectins.

  The immortalized vascular endothelial cell line of the present invention can be established by selecting a colony that grows in a single layer in the form of a paving stone or selecting using a known vascular endothelial cell marker, if necessary. For example, at least one expression selected from the group consisting of VEGFR2 (Flk1), vWF, VEGFR1 (Flt1), PECAM1 (CD31), VE-cadherin, TIE1, and TIE2, RT-PCR and / or Alternatively, an isolated immortalized vascular endothelial cell line can be identified by detection by Western blotting.

  For example, among the immortalized vascular endothelial cell lines obtained according to the preparation method described in 1 above, cell lines that are monolayered and proliferated in a cobblestone shape are adsorbed by VEGFR2 (Flk1) positive, vWF positive, and acetylated low-density lipoprotein. Cells having sex and / or BS-1 lectin binding ability may be selected and used as the immortalized vascular endothelial cell line of the present invention.

  When the immortalized vascular endothelial cell line of the present invention is cultured at a normal culture temperature using a three-dimensional medium containing VEGF, endothelial tube formation (EC tubing) occurs.

3. Immortalized pericytes (c-PC) and immortalized mesenchymal stem cell-like pericytes (MSC-PC)
3.1 Immortalized pericyte cell line The immortalized pericyte line of the present invention obtained according to the preparation method of 1 above is a capillary-derived cell line that expresses a pericellular marker such as NG2, PDGFRb, NGFR, It retains function and properties as a pericyte but does not have the ability to differentiate into mesenchymal cells. In addition, the immortalized pericytes of the present invention grow in multiple layers in a confluent state.

  The immortalized pericytes of the present invention can be established by further selecting colonies that multiply and proliferate or selecting using known pericellular markers as necessary. For example, at least one expression selected from the group consisting of PDGFRb, aSMA, Myocardin, Angiopoietin-1, Calponin, and Caldesmon is expressed by RT-PCR and / or Alternatively, an isolated immortalized pericyte cell line can be identified by detection by Western blotting.

  For example, among the immortalized pericytes obtained in accordance with the above preparation method 1, cell lines that multiply and proliferate are PDGFRb-positive, aSMA-positive, and myocardin-positive, and can differentiate into mesenchymal cells. A cell line that does not have a cell line may be selected and used as the immortalized pericyte line of the present invention.

  The immortalized pericytes of the present invention do not show changes in morphology even after continued passage.

  When the immortalized pericytes of the present invention are co-cultured with new blood vessels at normal culture temperature, they adhere to the blood vessel endothelium around the new blood vessels and form capillary-like structures.

3.2 Immortalized Mesenchymal Stem Cell-Like Peripheral Cell Line The immortalized mesenchymal stem cell-like pericyte cell line of the present invention obtained according to the preparation method of 1 above expresses a pericellular marker such as NG2, PDGFRb, and NGFR. A cell line derived from capillaries, which retains the functions and properties of pericytes and has the ability to differentiate into mesenchymal cells. In addition, the immortalized mesenchymal stem cell-like pericytes of the present invention multiply and proliferate in a confluent state.

  The immortalized mesenchymal stem cell-like pericytes of the present invention can be established by further selecting a cell line that multiplies and proliferates, and using a known pericellular marker, as necessary. For example, by detecting at least one expression selected from the group consisting of PDGFRb, aSMA, Myocardin, angiopoietin-1, calponin, and caldesmon by RT-PCR and / or Western blotting, Isolated immortalized mesenchymal stem cell-like pericytes can be identified.

  For example, among immortalized mesenchymal stem cell-like pericytes obtained in accordance with the preparation method of 1 above, cell lines that multiply and proliferate are PDGFRb positive, aSMA positive, and myocardin positive, and mesenchymal A cell line capable of differentiating into cells may be selected and used as the immortalized mesenchymal stem cell-like pericytes of the present invention.

  The immortalized mesenchymal stem cell-like pericytes of the present invention (1) expresses mesenchymal stem cell markers Sca1, CD29, CD44, CD105, CD106, and (2) positive pericytes NG2, aSMA positive (3) It has a surface marker profile of blood cell lineage markers CD11 and CD45 negative. Therefore, the cells can be identified using flow cytometry or the like.

  The immortalized mesenchymal stem cell-like pericytes of the present invention are observed to tend to have a variety of cell forms when they are continuously passaged, but they are probably mesenchymal stem cell-like even after passage culture over 40 passages. Has abilities.

4). Reconstruction of capillary-like structure In the present invention, “capillary-like structure” means a structure in which peripheral cells surround a tube (EC tube) made of endothelial cells, such as a capillary, and its diameter. Is about 5 to 20 μm.

4.1 Preparation Method Using Immortal Vascular Endothelial Cell Line and Immortalized Peripheral Cell Line Capillary-like structure is obtained by three-dimensional co-culture of the immortalized vascular endothelial cell line and immortalized pericyte line of the present invention. Are formed and the capillaries can be reconstructed. Specifically, a capillary-like structure can be obtained by co-culturing an immortalized vascular endothelial cell line and an immortalized pericyte cell line at a normal culture temperature using a VEGF-containing three-dimensional gel medium. As the medium, a basal medium supplemented with VEGF can be used, and in order to prepare a three-dimensional gel medium, a commercially available kit such as Matrigel (registered trademark) (BD) is used. Can do.

4.2 Preparation method using an immortalized mesenchymal stem cell-like pericyte cell line A capillary-like structure is formed by three-dimensional culture of the immortalized mesenchymal stem cell-like pericyte cell line of the present invention, and a capillary vessel is formed. Can be rebuilt. Specifically, a capillary-like structure can be obtained by culturing an immortalized mesenchymal stem cell-like pericyte cell line at normal culture temperature using a VEGF-containing three-dimensional gel medium. Thereafter, when the medium is replaced with a TGF-β-containing three-dimensional gel medium and the culture is continued at the normal culture temperature, a thicker mature capillary-like structure can be obtained. As the medium, a basal medium supplemented with VEGF and a basal medium supplemented with TGF-β can be used. To prepare a three-dimensional gel medium, for example, Matrigel (registered trademark) (BD) A commercially available kit can be used.

1. Preparation of New Capillary Tissue SV40 Temperature Sensitive Mutant tsA58T Antigen Gene Expressed Mice (8 Weeks Old) Insert a wire (outer diameter 0.3 mm Cook) into the femoral artery and resume blood flow to restore impaired blood vessels. A modeling model is created (Arterioscler Thromb Vasc Biol 2010, 30, 464-470). A collagen-coated polypropylene tube (CCT) (outer diameter 0.7 mm) was placed so as to accompany the damaged blood vessel, and the skin was re-stitched. Two weeks after the operation, new blood vessels were distributed on the damaged adventitia and on the placed CCT (FIG. 1A). The CCT was isolated and the coating on the CCT surface was peeled off. In this coating (thickness 200 to 500 μm), it is observed that new blood vessels of various maturation levels are densely developed in two dimensions (FIGS. 1B and 1C).

  FIG. 1A shows a state in which new blood vessels are formed on the surface of the CCT two weeks after the mouse femoral artery is wire-disturbed and CCT is placed in the vicinity thereof. FIG. 1B shows a coated tissue in which capillaries are developed in two dimensions, obtained by peeling the coating formed on the CCT surface. FIG. 1C shows a stained image obtained by staining the obtained capsular tissue with hematoxylin and eosin (HE), and it can be seen from the stained image that there are various levels of new blood vessels (bar = 100 μm).

2. Establishment of Capillary Cell Line Cell components were separated from the coating isolated in the above 1 by an enzyme solution for cell separation, and anti-CD146 antibody (rabbit monoclonal antibody, Abcam) and anti-NG2 antibody (rabbit polyclonal (rabbit polyclonal). ) Antibody, Abcam), and then CD146 positive cells and NG2 positive cells were separated by Magnet Cell Sorting System (Miltenyi Biotec). Each cell was immortalized after 10 passages using a medium for endothelial cells (Endothelial cell basal medium MV2, PromoCell) for CD146 positive cells and a basic medium (D-MEM, Gibco) for NG2 positive cells. Cells were separated again with CD146 antibody and NG2 antibody / MCSS, and then immortalized CD146 positive cell line and NG2 positive cell line were established. A single cell colony is formed from a CD146 positive cell in a dilution culture system, and VEGFR2 (Flk1), vWF positive and acetylated low density lipoprotein (Acetylated LDL-DiI, Biogenesis) is adsorbed in a cell colony that grows in a monolayer. A cell line capable of binding to sex / Lectin (Bandirea simplicifia agglutinin (BS-1 lectin) -FITC, Sigma) was established as an endothelial cell line (c-EC) (FIGS. 2A and B). When the cells are cultured in a three-dimensional gel medium, they have a tube forming ability (ECtubing) (FIG. 2C).

  FIG. 2A shows how the vascular endothelial cell line (c-EC) grows confluently and monolayers with polygonal cells. FIG. 2A shows that c-EC has an acetylated low-density lipoprotein-Dil (red) adsorptive property and a lectin-FITC (green) property characteristic of endothelial cells (bar = 100 μm). FIG. 2B confirms the gene expression of endothelial markers CD146, von Willebrand factor (von Willebrand factor, vWF), Flk1 (VEGFR2), and aSMA (α-smooth muscle actin) The results of RT-PCR performed are shown. c-EC expresses CD146, vWF, and Flk1, and does not express aSMA. FIG. 2C shows a microscopic image when c-EC is cultured at 37 ° C. in a VEGF-containing three-dimensional matrigel (bar = 100 μm). From FIG. 2C, it can be seen that when c-EC was cultured in a VEGF-containing three-dimensional medium, a tube-like structure was formed, that is, c-EC had the ability to form tubing.

  Cell colonies derived from single cells were formed from the NG2-positive cells in a dilution culture system. PDGFRb, aSMA, and Myocardin positivity was isolated as a pericyte cell line (c-PC) in a cell colony (FIG. 3A) that multilayered in a confluent state (FIG. 3B). When a rat aortic short-axis tissue section is co-cultured under a three-dimensional gel medium containing GFP-expressing c-PC cells, c-PC cells are wrapped around the neovascular endothelium generated from the aortic outer membrane, resulting in a capillary-like structure. Was formed (FIG. 3C).

  FIG. 3A shows that the pericyte cell line (c-PC) proliferates in a spindle-shaped cell in a smooth muscle cell-like “Hill & Valley” shape (undulation) (bar = 100 μm). FIG. 3B shows gene expression of NG2, a marker for pericytes, PDGFRb, aSMA, myocardin and SM22, which are markers common to pericytes and smooth muscle cells, and vWF and Flk1, which are markers for endothelial cells. -Shows the results of PCR. c-PC expresses NG2, PDGFRb, aSMA, myocardin, and SM22, but not vWF and Flk1. FIG. 3C shows that when c-PC is co-cultured with a new blood vessel (endothelium is stained with CD31 (red)) under a three-dimensional medium, pericytes (GFP (green)) are attached around the blood vessel endothelium, and capillary A state in which a blood vessel-like structure is formed is shown (bar = 50 μm).

3. Reconstruction of capillaries with capillary-derived cells (c-EC and c-PC) When the cells established in 2 above are incubated in a Matrigel three-dimensional VEGF-containing culture system, c-EC forms a tube (left of FIG. 4). On the other hand, c-PC only forms cell clump colonies (FIG. 4 right). When c-EC and c-PC cells are produced with co-cultured DS-Red (red fluorescent) and GFP (green fluorescent) expressing cells by recombinant retrovirus, they are formed with c-EC (Ds-Red). In addition, c-PC (GFP) was surrounded around the tubing to form a capillary-like structure (center of FIG. 4).

  FIG. 4 shows a case where a vascular endothelial cell line (c-EC) is cultured alone (left), a pericyte cell line (c-PC) is cultured alone (right), and a vascular endothelial cell under three-dimensional gel culture. The microscopic observation image and the fluorescence microscopic observation image are shown when the strain (c-EC) and the pericytes (c-PC) are co-cultured (center). Vascular endothelial cell lines (Ds-Red (red) expression) undergo endothelial tube formation (EC tubing), but pericytes (GFP (green) expression) proliferated like cell clumps. When both cells were co-cultured, c-PC adhered and proliferated around the EC tube formed by c-EC, and a capillary-like structure was formed (bar = 100 μm).

4). Capillary-derived mesenchymal stem cell-like pericytes (MSC-PC) multipotency Among the 5 c-PC cell lines, 3 strains were found to have a particularly strong tendency to have various cell morphology, Cloning from a single cell maintained similar properties. The cells were confirmed to have the ability to efficiently differentiate into fat and osteoblasts when cultured in adipocyte differentiation medium and osteoblast differentiation medium (FIG. 5). In particular, in the case of adipocytes, 70 to 80% of cells are induced to differentiate within 7 days after differentiation induction, and differentiate into mature adipocytes that accumulate a large amount of lipid droplets in the cytoplasm in a culture for 2 to 3 weeks. (Fig. 5).

  FIG. 5 shows a microscopic image of mesenchymal stem cell-like pericytes (MSC-PC), an immunostained image with FABP4 after the cells were cultured in an adipocyte differentiation medium, an oil red stained image, and the cells as bone. An immunostained image and an alizarin red stained image with osteopontin after culturing in a blast differentiation medium are shown (bar = 50 μm). Among NG2-positive PC cell lines, cell lines having adipocyte differentiation ability (adipogenesis) and osteoblast differentiation ability (osteogenesis) were established. When the same cell line was cultured in a fat differentiation medium for 1 week, 70 to 80% of the cells were differentiated into FABP4-positive cells, and when cultured for 2 to 3 weeks, differentiated into mature adipocytes having a large amount of fat droplets (oil red staining) in the cytoplasm. . Similarly, when the established cell line was cultured in an osteoblast differentiation medium for 3 weeks, differentiation into osteopontin-positive osteoblasts was observed, and a part of the cell mass showed calcium deposition (alizarin red staining) (bar = 50 μm). .

  When the cell line is cultured in a VEGF-containing endothelial cell medium, it becomes a vWF-positive endothelial cell. Further, when cultured in a VEGF-containing three-dimensional gel culture system, not only EC tubing is formed, but also a capillary-like structure surrounding PC cells. Was formed (FIG. 6A).

  When observed with an electron microscope, a larger lumen than EC tubing surrounded by PC cells was formed around a part (FIG. 6B). At this point, when stimulated with TGF (transforming growth factor β), a thicker blood vessel structure was formed (FIG. 6C).

  FIG. 6A shows that when a mesenchymal stem cell-like pericyte cell line (MSC-PC) was cultured in a VEGF-containing three-dimensional gel, endothelial cell differentiation was induced (inset in A), and other regions around the EC tube. It shows that pericytes accumulated to form a capillary-like structure. FIG. 6B is an image obtained by observing a capillary-like structure using an electron microscope, and has a larger luminal structure than an EC tube, and it is observed that some peripheral cells are attached to the outside of the tube. FIG. 6C shows a capillary-like structure after 2 days after further replacing the medium with VEGF (−) TGF (+) medium, and it can be seen that a thicker capillary-like structure is formed compared to A. (Bar = 100 μm).

5. Evaluation of cell adhesion ability to c-EC cells After culturing cEC in a confluent state, bone marrow-derived endothelial progenitor cells (EPC) (derived from GFP-expressing mice) were added to this medium dish, and 6 hours later, the cells adhered to the endothelial cell surface. GFP positive EPC cells were counted. In EPC in which the expression of an adhesion molecule (integrins) necessary for adhesion to the endothelium is decreased, it was observed that the endothelial adhesion was significantly decreased (FIG. 7).

  FIG. 7 shows a culture solution containing EPC (GFP green luminescence) isolated from mouse bone marrow after culturing c-EC in a confluent monolayer state in order to examine the adhesion ability of endothelial progenitor cells (EPC) to vascular endothelium. The result of having evaluated the number of EPC adhering to the endothelial surface after incubating for 6 hours is shown. In EPC (subject) in which expression of adhesion molecules (integrin β1, etc.) is reduced, the ability to adhere to the surface of endothelial cells is significantly reduced (bar = 50 μm, Mean + SEM, p <0.05, n = 6).

6). Evaluation of pluripotency in vivo of MSC-PC Differentiation into various tissues was confirmed by introducing DsRed fluorescently labeled MSC-PC into mouse tissues.
(1) Differentiation into skeletal muscle cells Cardiotoxin (10 ug / mouse intramuscular injection) was applied to the SCID mouse gastrocnemius muscle to damage the skeletal muscle, and then MSC-PC was introduced into the same site. Two weeks later, skeletal muscle tissue was taken out and subjected to immunostaining analysis. The results are shown in FIG. 8A (bar = 50 μm). It was confirmed that some of the skeletal muscle fibers regenerating from the disorder were derived from MSC-PC (green = lectin staining = vascular endothelium with blood flow).

(2) Differentiation into capillaries (endothelial cells and pericytes) MSC-PC was introduced into the subcutaneous tissue part in the ischemic region of the lower extremity ligated with the SCID mouse femoral artery. Two weeks later, the tissue at the same site was taken out and subjected to immunostaining analysis. The results are shown in FIG. 8B (bar = 50 μm). Transduced cells (red signal) differentiate into neovascular endothelial cells (lectin staining = polymerized with blood vessel endothelium with blood flow) and pericytes (attached outside of green endothelial cells). It was confirmed that

(3) Differentiation into adipocytes Two weeks after introduction of MSC-PC into SCID mouse subcutaneous adipose tissue, the tissue was taken out and subjected to immunostaining analysis. The results are shown in FIG. 8C (bar = 50 μm). It was confirmed that cells introduced in the adipose tissue (red signal) were differentiated into adipocytes (green = Bodypy staining = adipose staining).

7). Cell marker profile of MSC-PC In MSC-PC, the expression of typical pericytes and mesenchymal stem cell markers was confirmed by flow cytometry analysis. The results are shown in FIG.

  Pericyte markers (NG2, aSMA) are positive, and representative mesenchymal stem cell markers such as Sca1, CD29, CD44, CD105, CD106 are all positive. In addition, CD11 and CD45, which are blood cell markers, were negative.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  Compared to the conventional experimental system for mere “endothelial cells”, this cell line, which is a capillary constituent cell, and the experimental system using these cell lines are more suitable for maintaining capillaries normally. It is very useful for elucidating the mechanism for regenerating mature blood vessels. Capillary dysplasia has been shown to be involved in the pathology of important intractable diseases such as tumors, retinopathy, arteriosclerosis, heart failure, insulin resistance, etc., so the cell lines of the present invention and these cell lines There is a possibility that a prophylactic or therapeutic drug for these diseases can be developed by an experimental system using. In addition, there is a possibility that a tissue vascular regeneration construction method required for organ regeneration can be developed by the cell line of the present invention and an experimental system using these cell lines.

  This cell line (MSC-PC) and the experimental system using this cell led to the elucidation of questions related to tissue-derived MSC, and not only the above-mentioned angiogenesis but also the intractable disease pathology that is a problem in an aging society Applications such as elucidation (fat and bone tissue remodeling (metabolic syndrome, osteoporosis), fibrosis disorders, etc.) and development of regenerative medicine (preparation of regenerative cell tools) are expected to be useful as a useful tool in a wide range of medicine and medical fields.

Claims (13)

A method for preparing an immortalized pericyte cell line and an immortalized mesenchymal stem cell-like pericyte cell line derived from capillaries of a non-human mammal, comprising the following steps:
(A) Separation of cells from tissue densely containing capillaries prepared by placing a carrier containing an extracellular matrix component around a damaged blood vessel of a non-human mammal introduced with the SV40 temperature-sensitive mutant tsA58T antigen gene The process of
(B) selecting a cell expressing at least one selected from NG2, PDGFRb, and NGFR, subculturing the cell to obtain an immortal cell line, and (c) from among the immortal cell lines Select cell lines that do not have the ability to differentiate into mesenchymal cells as immortalized pericytes, and select cell lines that have the ability to differentiate into mesenchymal cells as immortalized mesenchymal stem cell-like pericytes. Process. further,
(D) The preparation method of Claim 1 including the process of selecting the cell line which expresses the marker of a pericyte from an immortalized cell line.   The preparation method according to claim 2, wherein the marker of pericytes is at least one selected from the group consisting of PDGFRb, aSMA, myocardin, angiopoietin-1, calponin, caldesmon, ang-like4, and NGF. The carrier is one or more extracellular matrix components selected from collagen, fibronectin, vitronectin, proteoglycan, laminin, tenascin, entactin, elastin, fibrin, hyaluronic acid, or a reconstituted basement membrane matrix such as Matrigel (registered trademark) The preparation method in any one of Claims 1-3 containing this. It said carrier comprises a collagen or collagen gel, preparation method according to any one of claims 1 to 3. A method for preparing an immortalized vascular endothelial cell line derived from a capillary tube of a non-human mammal, comprising the following steps:
(A) Separation of cells from tissue densely containing capillaries prepared by placing a carrier containing an extracellular matrix component around a damaged blood vessel of a non-human mammal introduced with the SV40 temperature-sensitive mutant tsA58T antigen gene And (b) selecting a cell that expresses at least one selected from CD146, rWF, and CD31, and subculturing the cell to obtain an immortalized cell line. further,
(C) The preparation method according to claim 6 , comprising a step of selecting a cell line expressing a vascular endothelial cell marker from an immortalized cell line. Markers of endothelial cells VEGFR2 (Flk1), vWF, VEGFR1 (Flt1), PECAM1 (CD31), VE- cadherin, TIE1, and at least one selected from the group consisting of TIE2, according to claim 7 Preparation method. The carrier is one or more extracellular matrix components selected from collagen, fibronectin, vitronectin, proteoglycan, laminin, tenascin, entactin, elastin, fibrin, hyaluronic acid, or a reconstituted basement membrane matrix such as Matrigel (registered trademark) The preparation method in any one of Claims 6-8 containing this. The preparation method according to any one of claims 6 to 8 , wherein the carrier contains collagen or collagen gel. An immortalized vascular endothelial cell line prepared by the method according to any one of claims 6 to 10 and an immortalized pericyte cell line prepared by the method according to any one of claims 1 to 5 A method of preparing a capillary-like structure by culturing. A capillary-like structure is prepared by three-dimensionally culturing an immortalized mesenchymal stem cell-like pericyte cell line prepared by the method according to any one of claims 1 to 5 using a medium containing VEGF. Method. The method according to claim 12 , further comprising the step of three-dimensional culture using a medium containing VEGF and then exchanging the medium for a medium containing TGF without containing VEGF.