Perisite cells into which the basic fibroblast growth factor (bFGF) gene has been introduced.

Abstract

The present invention addresses the problem of providing a cell therapy expected to be useful as an angiogenic therapy for peripheral vessel diseases such as critical limb ischemia. Specifically, a pericyte having high angiogenic potentional and a method for producing the same are provided. A pericyte having a bFGF gene introduced therein and a method for producing the same are provided.

Description

[Technical Field]

[0001] The present invention relates to pericytes into which the basic fibroblast growth factor (bFGF) gene has been introduced, a pharmaceutical composition containing the pericytes, a method for producing the pericytes, and angiogenesis therapy characterized by administering the pericytes. [Background technology]

[0002] Capillaries connect arterioles and venules, forming a network that extends deep into body tissues to supply oxygen and nutrients to every corner of the body's periphery. Capillaries are composed of a single layer of vascular endothelial cells that form a tubular structure, surrounded by pericytes. Pericytes, as cells that cover vascular endothelial cells, play a crucial role in regulating normal blood flow, including vascular maturation and stabilization, and maintaining the blood-brain barrier. Severe lower limb ischemia is a serious disease that impairs blood flow, but effective drug therapy has not been established, and it is treated with bypass surgery or endovascular therapy. In recent years, there has been a desire to develop new treatment methods that induce the formation of new peripheral capillaries through cell therapy (Patent Document 1).

[0003] Basic fibroblast growth factor (bFGF), also known as FGF-2, is a growth factor family protein with various functions. It is known to contribute to angiogenesis by directly acting on vascular endothelial cells to promote their proliferation and lumen formation. Human bFGF / FGF-2 protein is a single-chain polypeptide consisting of 154 amino acids with a molecular weight of 18kD, and is known to be released from macrophages, endothelial cells, and damaged muscle fibers (Henke C et al., Am. J. Pathol., (1993), 143: 1189-1199, Wang YX et al., J. Cell Sci., (2014), 127: 4543-4548). The most characteristic function of bFGF / FGF-2 in angiogenesis is its direct action on vascular endothelial cells to promote their proliferation and lumen formation. Furthermore, bFGF / FGF-2 is thought to indirectly promote angiogenesis by regulating the expression of vascular endothelial growth factor (VEGF) in vascular smooth muscle cells (Non-Patent Literature 1). It has been reported that introducing the bFGF / FGF-2 gene into ischemic muscle tissue using a Sendai virus vector enhances the expression of endogenous VEGF and hepatocyte growth factor (HGF), leading to improvement in lower limb ischemia (Non-Patent Literature 2). Thus, bFGF / FGF-2 is a potent angiogenic factor and is expected to have applications in ischemic diseases. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2008 / 104064 [Non-patent literature]

[0005] [Non-Patent Document 1] Marco Presta et al., Cytokine & Growth Factor Reviews,(2005), 16: 159-178 [Non-Patent Document 2] Ichiro Masaki et al., Circulation Research, (2002), 90: 966-973 [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a cell therapy that is expected to be useful as angiogenesis therapy for peripheral vascular diseases such as severe lower limb ischemia. Specifically, the object is to provide pericytes with high angiogenic capacity and a method for producing the same. [Means for solving the problem]

[0007] In order to solve the above problems, the inventors conducted diligent studies, including introducing several genes encoding angiogenic factors into pericytes. As a result, they found that introducing the bFGF gene into pericytes significantly increases the angiogenic capacity of the transplanted pericytes, and that these pericytes can be applied to angiogenesis therapies for conditions such as severe lower limb ischemia. The present invention was completed based on these findings.

[0008] In other words, the present invention has the following features: [1] Pericytes into which the basic fibroblast growth factor (bFGF) gene has been introduced. [2] Perisite is the original perisite, as described in [1]. [3] Pericytes as described in [1], in which pericytes are pericyte-like cells differentiated from pluripotent stem cells. [4] Pericytes as described in [3], wherein the pluripotent stem cells are human pluripotent stem cells. [5] The pericyte according to [3] or [4], wherein the pluripotent stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells). A pharmaceutical composition for angiogenesis therapy, comprising perisite as described in any one of [6][1] to [5]. [7] The pharmaceutical composition according to [6], wherein angiogenesis therapy is used for the treatment of severe lower limb ischemia. [8] The pharmaceutical composition according to [6] or [7], used in combination with vascular endothelial cells. A pharmaceutical composition for angiogenesis therapy comprising a combination of pericytes and vascular endothelial cells as described in any one of [9][1] to [5].

[10] The pharmaceutical composition according to [9], wherein angiogenesis therapy is used for the treatment of severe lower limb ischemia. A method for producing pericite as described in any one of

[11] [1] to [5]. Angiogenesis therapy characterized by administering a therapeutically effective dose of pericyte as described in any one of

[12] [1] to [5].

[13] Angiogenesis therapy as described in

[12] , further comprising the administration of vascular endothelial cells.

[14] Angiogenesis therapy as described in

[12] or

[13] , for the treatment of severe lower limb ischemia.

[15] Use of perisite according to any one of [1] to [5] in the manufacture of a pharmaceutical composition for angiogenesis therapy.

[16] Use of perisite according to any one of [1] to [5] in the manufacture of a pharmaceutical composition for the treatment of severe lower limb ischemia.

[17] Perisites described in any one of [1] to [5] for use in angiogenesis therapy.

[18] Perisite described in any one of [1] to [5] for use in the treatment of severe lower limb ischemia.

[19] Pericytes with increased expression of endogenous bFGF. [Effects of the Invention]

[0009] In this invention, pericytes with high angiogenic capacity can be obtained and manufactured by introducing the bFGF gene into pericytes. Furthermore, the bFGF gene-modified pericytes obtained by the method of this invention can be used in angiogenesis therapy for conditions such as severe lower limb ischemia. [Brief explanation of the drawing]

[0010] [Figure 1]Fig. 1 shows the results of evaluation of the bFGF expression level in the bFGF gene-transfected human primary pericytes (bFGF-Primary pericyte) obtained in Example 3, together with the bFGF expression level in human primary pericytes (Primary pericyte) in Example 4. The vertical axis represents the bFGF expression level (ng / mL). Error bars indicate ± standard error of the mean. [Figure 2] Fig. 2 shows the results of qualitative evaluation of the angiogenic ability of the bFGF gene-transfected human primary pericytes obtained in Example 3 in Example 5. Specifically, Human Umbilical Vein Endothelial Cells (HUVEC), HUVEC and human primary pericytes (Primary pericyte / HUVEC), or HUVEC and bFGF gene-transfected human primary pericytes (bFGF-Primary pericyte / HUVEC) were administered together with Matrigel to NOG mice, and 14 days later, the Matrigel was recovered from the mice and photographed. The figure shows the obtained results. [Figure 3] Fig. 3 shows the results of quantitative evaluation of the angiogenic ability of the bFGF gene-transfected human primary pericytes obtained in Example 3 in Example 6. Specifically, the figure shows the results of measuring the hemoglobin concentration contained in the supernatant after centrifugation, obtained by crushing each Matrigel recovered by the procedure of Example 5. The vertical axis represents the hemoglobin concentration (μg / mL) contained in the Matrigel-derived supernatant. The P value indicated by ** in Fig. 3 is 0.0001. Error bars indicate ± standard error of the mean. [Figure 4]Figure 4 shows the therapeutic effect of administering the bFGF gene-transduced human primary pericytes obtained in Example 3 to lower limb ischemia model mice in Example 7. In the left panel of Figure 4, the horizontal axis represents the number of weeks elapsed after administration of control medium (Medium) or bFGF gene-transduced primary pericytes (bFGF-Primary pericyte) to the ischemic limb, and the vertical axis represents the blood flow ratio (%, Ischemic / normal) calculated by dividing the blood perfusion signal value of the ischemic limb by the blood flow signal value of the normal limb. The vertical axis in the right panel of Figure 4 represents AUC (Area Under Curve) after administration of control medium or bFGF gene-transduced primary pericytes, calculated based on the left panel. The P-value indicated by ** in the right panel of Figure 4 is 0.0139. Error bars represent the standard error of the mean ±. MODE FOR CARRYING OUT THE INVENTION

[0011] The present invention is described in detail below.

[0012] <Pericytes into which the bFGF gene has been introduced> The present invention provides pericytes into which the bFGF gene has been introduced (also referred to as "pericytes of the present invention").

[0013] The pericyte of the present invention is a pericyte into which the bFGF gene has been introduced. The nucleotide sequence of the bFGF gene and the amino acid sequence of bFGF are already publicly known and their sequences have been published in public databases, etc. For example, the nucleotide sequence and amino acid sequence of human bFGF have been published as GenBank Accession Number: M27968.1 and AAA52448.1, respectively. Specifically, human bFGF is encoded by the gene with GenBank Accession Number: M27968.1 (SEQ ID NO: 1) and has the amino acid sequence (SEQ ID NO: 2) shown by GenBank Accession Number: AAA52448.1. In the present invention, the bFGF gene introduced into the pericyte includes genes that encode naturally occurring bFGF and genes that encode modified forms of bFGF having the function of bFGF. In one embodiment, the bFGF gene introduced into the pericyte according to the present invention is a gene that has at least 80% or more identity, preferably 85% or more, 90% or more, 95% or more, or 98% or more identity with the amino acid sequence published as GenBank Accession Number: AAA52448.1, and encodes a protein that functions as bFGF. In one embodiment, the bFGF gene introduced into the pericyte according to the present invention is a gene that encodes a protein that functions as bFGF, consisting of an amino acid sequence in which 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is deleted, substituted, inserted, and / or added. In one embodiment, the bFGF gene introduced into the pericyte according to the present invention is a gene that encodes a protein consisting of the amino acid sequence shown in Sequence ID No. 2. The presence of bFGF function can be confirmed by known methods (Beenken A & Mohammadi M, Nat. Rev. Drug Discov., (2009), 8: 235-253). In one embodiment, the bFGF gene introduced into the pericyte in the present invention may be a gene encoding bFGF with a secretory signal added or a variant thereof.As the secretion signal, any secretion signal known to those skilled in the art can be used, and in one embodiment, the Bmp2 / 4 secretion signal (U.S. Patent US7816140) may be used. In one embodiment, the bFGF gene introduced into the pericyte in the present invention is a gene that encodes a protein having at least 80% or more identity, preferably 85% or more, 90% or more, 95% or more, or 98% or more identity with the amino acid sequence shown in SEQ ID NO: 4, and that functions as bFGF. In one embodiment, the bFGF gene introduced into the pericyte in the present invention is a gene that encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 4. In this specification, "bFGF" may also be referred to as "FGF-2".

[0014] With respect to nucleic acid sequences or amino acid sequences, "identity" as used herein means the value of Identity obtained using the default parameters provided by the NEEDLE program (Needleman SB et al., J. Mol. Biol., (1970), 48: 443-453) search. The parameters are as follows: Gap penalty = 10 Extend penalty = 0.5 Matrix = EBLOSUM62

[0015] Perisite Pericytes are cells that surround the walls of microvessels or capillaries in the brain, periphery, or retina, and are also called pericytes. As described above, their function is to coat vascular endothelial cells and play an important role in normal blood flow regulation, such as vascular maturation and stabilization, and maintenance of the blood-brain barrier (Daneman R et al., Nature, (2010), 468: 562-568, Armulik A et al., Dev. Cell, (2011), 21: 193-215). When pericyte function is impaired, the pericytes' original functions (e.g., vascular stabilization, maintenance of blood flow, maintenance of the blood-brain barrier, maintenance of the blood-nerve barrier, etc.) are impaired, leading to serious blood flow-related diseases such as diabetic retinopathy. Furthermore, the existence of cells called mesoangioblasts, which have the ability to differentiate into muscle and bone, has been revealed within skeletal muscle-derived pericytes (Gerli MFM et al., J. Vis. Exp., (2014), 83: 50523, Gerli MFM et al., Stem Cell Rep., (2019), 12: 461-473, Shimatani K et al., Am. J. Physiol. Heart Circ. Physiol., (2021), online: https: / / doi.org / 10.1152 / ajpheart.00470.2020).

[0016] -First Generation Perisite- In one embodiment, the pericyte of the present invention is a primary pericyte into which the bFGF gene has been introduced.

[0017] In this specification, "primary pericytes" refers to pericytes directly collected from an organism, or primary cultured cells and passaged cells obtained by culturing and growing such pericytes in vitro. Methods for isolating and culturing primary pericytes from organisms are described, for example, in Quattrocelli M et al., Methods Mol. Biol., (2012), 798: 65-76. The primary pericytes in this invention are not particularly limited, but in one embodiment, they are human primary pericytes. As human primary pericytes, it is desirable to use primary pericytes that are identical or substantially identical in human leukocyte antigen (HLA) genotype to that of the recipient organism, for example, the patient themselves, or from the viewpoint of preventing rejection. Here, "substantially identical" means that the HLA genotype matches to the extent that the immune response to the transplanted pericyte can be suppressed by an immunosuppressant, for example, a pericyte having an HLA type in which the three gene loci of HLA-A, HLA-B, and HLA-DR, or four gene loci including HLA-C, match.

[0018] -Pericyte-like cells- In one embodiment, the pericytes of the present invention are pericyte-like cells differentiated from pluripotent stem cells into which the bFGF gene has been introduced.

[0019] In this specification, "pericyte-like cells" refers to cells differentiated from pluripotent stem cells that possess properties similar to primary pericytes. The fact that pericyte-like cells possess properties similar to pericytes can be confirmed by known methods (Armulik A et al., Dev. Cell, (2011), 21: 193-215). Differentiation induction from pluripotent stem cells into pericyte-like cells can be carried out using methods known to those skilled in the art (WO2013 / 108039, WO2009 / 156151, US9771561, US9868939, US2015 / 0368609, US2017 / 0342384, US2019 / 0316094). For example, differentiation induction from pluripotent stem cells into pericyte-like cells can be carried out using the method described in the section "Method for Differentiating Pluripotent Stem Cells into Pericyte-like Cells" below.

[0020] ≪Pluripotent stem cells≫ In this specification, "pluripotent stem cells" means stem cells that possess pluripotency, enabling them to differentiate into many different types of cells with varying properties and morphologies present in living organisms, and also possess proliferative capacity. The preferred pluripotent stem cells in this invention are human pluripotent stem cells. In one embodiment, the pericytes of this invention are pericyte-like cells differentiated from human pluripotent stem cells into which the bFGF gene has been introduced.

[0021] The pluripotent stem cells used in the present invention are not particularly limited, but include, for example, embryonic stem cells (ES cells), embryonic stem cells produced using nuclear transfer technology (Nuclear transfer embryonic stem cells; ntES cells), spermatogonial stem cells (Germline stem cells; GS cells), embryonic germ cells (Embryonic germ cells; EG cells), induced pluripotent stem cells (iPS cells), and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Multi-lineage differentiating stress enduring cells; Muse cells). In the present invention, preferred pluripotent stem cells for inducing differentiation of pericyte-like cells are ES cells or iPS cells. In one embodiment, the pericyte of the present invention is a pericyte-like cell differentiated from an embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell) into which the bFGF gene has been introduced. In one embodiment, the pericyte of the present invention is a pericyte-like cell differentiated from a human ES cell or a human iPS cell into which the bFGF gene has been introduced.

[0022] -ES cells- ES cells are stem cells that possess pluripotency and the ability to proliferate through self-renewal, established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans and mice. ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Furthermore, cell maintenance through subculture can be carried out using a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and bFGF. Human ES cells can be established and maintained by known methods (e.g., Suemori H et al., Biochem. Biophys. Res. Commun., (2006), 345: 926-932, Kawasaki H et al., Proc. Natl. Acad. Sci. USA, (2002), 99: 1580-1585, etc.).

[0023] -iPS cells-iPS cells are a general term for pluripotent stem cell lines artificially induced by introducing specific genes into somatic cells that have lost their ability to differentiate. The method for producing iPS cells is well known in the field and can be produced by introducing reprogramming factors into any somatic cell. Examples of reprogramming factors include gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors may be used individually or in combination. Possible combinations of initialization factors include: WO2007 / 069666; WO2008 / 118820; WO2009 / 007852; WO2009 / 032194; WO2009 / 058413; WO2009 / 057831; WO2009 / 075119; WO2009 / 079007; WO2009 / 091659; WO2009 / 101084; WO2009 / 101407; WO2009 / 102983; WO2009 / 114949; WO2009 / 117439; WO2009 / 126250; WO2009 / 126251; WO2009 / 126655; WO2009 / 157593; WO2010 / 009015; WO2010 / 033906; WO2010 / 033920; WO2010 / 042800; WO2010 / 050626; WO 2010 / 056831; WO2010 / 068955; WO2010 / 098419;WO2010 / 102267;WO2010 / 111409;WO2010 / 111422;WO2010 / 115050;WO2010 / 124290;WO2010 / 147395;WO2010 / 147612; al., Nat. Biotechnol., (2008), 26: 795-797; Shi Y et al., Cell Stem Cell, (2008), 2: 525-528; Eminli S et al., Stem Cells, (2008), 26: 2467-2474; Huangfu D et al., Nat. Biotechnol., (2008), 26: 1269-1275; Shi Y et al., Cell Stem Cell, (2008), 3: 568-574; Zhao Y et al., Cell Stem Cell, (2008), 3: 475-479; Marson A Cell Stem Cell, (2008), 3: 132-135; Feng B et al., Nat. Cell Biol., (2009), 11: 197-203; Judson RL et al., Nat. Biotechnol., (2009), 27: 459-461; Lyssiotis CA et al., Proc. Natl. Acad. Sci. USA, (2009), 106: 8912-8917; Kim JB et al., Nature, (2009), 461: 649-643; Ichida JK et al., Cell Stem Cell, (2009), 5: 491-503; Heng JC et al., Cell Stem Cell, (2010), 6: 167-174; He J et al., Nature, (2010), 463: 1096-1100; Mali P et al., Stem Cells, (2010), 28: 713-720; Maekawa M et al., Nature, (2011), 474: 225-229.

[0024] The somatic cells used in the production of iPS cells may be, but are not limited to, somatic cells from newborns (children) or somatic cells from healthy individuals or patients. They may also be primary cultured cells, passaged cells, or cell lines derived from these. In one embodiment, the somatic cells used in the production of iPS cells may be, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, or (3) differentiated cells present in organs and tissues such as blood cells (peripheral blood cells, umbilical cord blood cells, etc.), muscle cells, skin cells, hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells, brain cells, lung cells, kidney cells, and adipocytes.

[0025] When using iPS cells as material for differentiating pericyte-like cells, it is desirable, but not always, to use somatic cell-derived iPS cells whose human leukocyte antigen (HLA) genotype is identical or substantially identical to that of the recipient individual, in order to avoid rejection. Here, "substantially identical" means that the HLA genotype matches to the extent that the immune response to the transplanted cells can be suppressed with immunosuppressants. For example, somatic cell-derived iPS cells having an HLA type in which the three gene loci of HLA-A, HLA-B, and HLA-DR, or the four gene loci of HLA-DR plus HLA-C, match.

[0026] As pluripotent stem cells, which are materials for inducing pericyte-like cells, for example, pluripotent stem cells prepared by the method described in Gornalusse GG et al., Nat. Biotechnol., (2017), 35: 765-772, which do not cause rejection in allogeneic transplantation, can also be used. As the pluripotent stem cells that do not cause rejection in allogeneic transplantation, ES cells or iPS cells that do not cause rejection in allogeneic transplantation are preferred, and human ES cells or human iPS cells that do not cause rejection in allogeneic transplantation are more preferred. In one embodiment, the pericyte of the present invention is a pericyte-like cell differentiated from a pluripotent stem cell into which a bFGF gene has been introduced and which does not cause rejection in allogeneic transplantation. In one embodiment, the pericyte of the present invention is a pericyte-like cell differentiated from a human ES cell or human iPS cell into which a bFGF gene has been introduced and which does not cause rejection in allogeneic transplantation.

[0027] <Pericytes with enhanced expression of endogenous bFGF> In one embodiment, the pericyte of the present invention is a pericyte with enhanced expression of endogenous bFGF. Here, "enhanced expression of endogenous bFGF" includes both direct enhancement of endogenous bFGF activity due to increased expression of endogenous bFGF protein in primary pericytes or pericyte-like cells differentiated from pluripotent stem cells, and indirect enhancement of endogenous bFGF activity due to release of relevant suppression systems. Enhancement of endogenous bFGF expression can be achieved by induction of endogenous bFGF expression by external factors. Examples of methods for inducing endogenous bFGF expression include a method of induction by acidosis (D'Arcangelo D et al., Circ. Res., (2000), 86: 312-318). There are no particular limitations on the expression level of bFGF protein in pericytes with enhanced endogenous bFGF expression. It should be noted that bFGF expression was below the detection limit in normal primary pericytes (see Example 4).

[0028] <Method for producing pericytes into which a bFGF gene has been introduced> The present invention also provides a method for producing perisites into which the bFGF gene has been introduced (also referred to as "the production method of the present invention").

[0029] In one embodiment, the manufacturing method of the present invention includes introducing the bFGF gene into pericytes. In one embodiment, the manufacturing method of the present invention includes introducing the bFGF gene into primary pericytes. In one embodiment, the manufacturing method of the present invention includes introducing the bFGF gene into pericyte-like cells differentiated from pluripotent stem cells. In one embodiment, the manufacturing method of the present invention includes introducing the bFGF gene into pericyte-like cells differentiated from ES cells or iPS cells. In one embodiment, the manufacturing method of the present invention includes introducing the bFGF gene into pericyte-like cells differentiated from human pluripotent stem cells. In one embodiment, the manufacturing method of the present invention includes introducing the bFGF gene into pericyte-like cells differentiated from human ES cells or human iPS cells.

[0030] In one embodiment, the manufacturing method of the present invention includes introducing a bFGF gene into pluripotent stem cells and inducing differentiation of the pluripotent stem cells into pericyte-like cells. In one embodiment, the manufacturing method of the present invention includes introducing a bFGF gene into ES cells or iPS cells and inducing differentiation of the ES cells or iPS cells into pericyte-like cells. In one embodiment, the manufacturing method of the present invention includes introducing a bFGF gene into human pluripotent stem cells and inducing differentiation of the human pluripotent stem cells into pericyte-like cells. In one embodiment, the manufacturing method of the present invention includes introducing a bFGF gene into human ES cells or human iPS cells and inducing differentiation of the human ES cells or human iPS cells into pericyte-like cells.

[0031] [Method for introducing the bFGF gene into cells] The bFGF gene can be constructed based on its nucleotide sequence information using methods known in the art. For example, the bFGF gene can be synthesized using gene synthesis methods known in the art.

[0032] In the present invention, the method for introducing the bFGF gene into pericytes or pluripotent stem cells can be a method commonly used for transfection of animal cells, such as the calcium phosphate method, lipofection method, electroporation method, microinjection method, or method using a viral vector. In one embodiment, a method using a viral vector can be used to introduce the bFGF gene into pericytes or pluripotent stem cells. Examples of viral vectors that can be used to introduce the bFGF gene into pericytes or pluripotent stem cells include lentiviruses, adenoviruses, adeno-associated viruses, or retroviruses. In one embodiment, a method using a lentiviral vector can be used to introduce the bFGF gene into pericytes or pluripotent stem cells. Specifically, as described in Example 3, the bFGF gene can be introduced into cells by infecting them with a lentivirus for bFGF gene introduction.

[0033] [Method for inducing differentiation of pluripotent stem cells into pericyte-like cells] In the present invention, pericyte-like cells can be obtained by differentiating pluripotent stem cells into pericyte-like cells, as described above. The method for differentiating pluripotent stem cells into pericyte-like cells is not particularly limited, but in one embodiment, they can be obtained by a method comprising the following steps (a) and (b): (a) A process for differentiating pluripotent stem cells into early mesodermal cells, and (b) A step of differentiating the early mesoderm cells obtained in step (a) into pericyte-like cells.

[0034] Processes (a) and (b) are described below. Step (a) is a step of differentiating pluripotent stem cells into primitive posterior mesoderm cells. In the present invention, the method for differentiating pluripotent stem cells into primitive posterior mesoderm cells is not particularly limited, but for example, the methods for inducing differentiation from pluripotent stem cells to primitive posterior mesoderm cells described in US9,868,939, US9,771,561, and Uenishi G et al., Stem Cell Reports, (2014), 3: 1073-1084 can be used. The differentiation of pluripotent stem cells into early mesodermal cells can be confirmed using surface antigen markers specific to early mesodermal cells (such as PDGFRα and APLNR), as described, for example, in Vodyanik MA et al., Cell Stem Cell, (2010), 7: 718-729, US9,771,561, and Uenishi G et al., Stem Cell Reports, (2014), 3: 1073-1084.

[0035] Step (b) is a step of inducing differentiation of the early mesoderm cells obtained in step (a) into pericyte-like cells. In the present invention, the method for differentiating early mesoderm cells into pericyte-like cells is not particularly limited, but for example, the method for inducing differentiation from early mesoderm cells to pericyte-like cells described in US9,868,939 can be used. Differentiation of early mesoderm cells into pericyte-like cells can be confirmed, for example, using surface antigen markers such as NG2 or CD146 (Herrmann M. et al., Eur. Cells Mater., (2016), 31: 236-249, Covas DT. et al., Exp. Hematol., (2008), 36: 642-654, Lv FJ. et al., Stem Cells, (2014), 32: 1408-1419).

[0036] In one embodiment, step (b) first forms spheroids of early mesoderm cells, and then induces differentiation of these spheroids into pericyte-like cells.

[0037] In the present invention, the method for spheroid formation of early mesoderm cells is not particularly limited, and known methods can be used. For example, a method using methylcellulose medium described in US9,771,561 can be used. The composition of the spheroid-forming medium can be determined by referring to known techniques (e.g., Vodyanik MA et al., Cell Stem Cell, (2010), 7: 718-729).

[0038] In the present invention, the method for differentiating spheroid-forming early mesoderm cells into pericyte-like cells is not particularly limited, and known methods can be used. For example, the method described in US9,868,939 can be used.

[0039] [Culture method for bFGF gene-transformed perisites] The method for culturing and propagating pericytes according to the present invention is not particularly limited, and methods for culturing and propagating pericytes known in the art can be used.

[0040] The culture medium for culturing the pericytes of the present invention is not particularly limited as long as it is a medium suitable for culturing pericytes, and as a basic medium, MEM medium, BME medium, D-MEM medium, α-MEM medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI medium, StemSpan medium, StemPro medium, and mixtures thereof can be used.

[0041] The culture medium for culturing the pericytes of the present invention may be appropriately supplemented with various nutrients necessary for the maintenance and proliferation of the cells. For example, the nutrients may include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, and dextrin; hydrocarbons such as fatty acids, oils and fats, lecithin, and alcohols; nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, and sodium nitrate; inorganic salts such as sodium chloride, potassium salts, phosphates, magnesium salts, calcium salts, iron salts, and manganese salts; monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate, and manganese sulfate; various vitamins; amino acids, etc. In one embodiment, amino acids such as glutamine can be used as a nutrient added to the culture medium for culturing the pericytes of the present invention.

[0042] The culture medium for culturing the pericytes of the present invention may be appropriately supplemented with growth factors such as FGF family growth factors necessary for cell proliferation. The growth factors to be added are not particularly limited, but in one embodiment, bFGF or a modified bFGF with enhanced thermal stability can be used as the growth factor added to the culture medium for culturing the pericytes of the present invention.

[0043] The pH of the culture medium for culturing the perisite of the present invention is in the range of 5.5 to 9.0, preferably 6.0 to 8.0, and more preferably 6.5 to 7.5.

[0044] Pericytes are adherent cells that have the characteristic of adhering to and proliferating on the extracellular matrix. Therefore, in one embodiment, a suitable scaffold can be used in the culture of pericytes according to the present invention. The scaffold is not particularly limited as long as it is a matrix, substrate, or carrier to which cells can adhere and divide and proliferate, and examples include fibronectin, vitronectin, collagen, proteoglycan, laminin, tenascin, enteractin, elastin, fibrillin, hyaluronic acid, gelatin, poly-L-lysine, poly-D-lysine, etc. In one embodiment, a collagen matrix can be used as the scaffold for the culture of pericytes according to the present invention.

[0045] In one embodiment, the perisite culture of the present invention can be carried out at 36°C to 38°C. In one embodiment, the perisite culture of the present invention can be carried out at 36.5°C to 37.5°C, under an atmosphere of 1% to 25% O2 and 1% to 15% CO2, while appropriately changing the culture medium.

[0046] <Pharmaceutical Compositions, etc. of the Invention> The present invention also provides a pharmaceutical composition comprising the pericyte of the present invention (also referred to as "the pharmaceutical composition of the present invention"). The pharmaceutical composition can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients and pharmaceutical carriers. In formulating the pharmaceutical composition, excipients, carriers, additives, etc., can be used according to these dosage forms, to the extent that is pharmaceutically acceptable. In one embodiment, the pharmaceutical composition of the present invention comprises primary pericyte into which the bFGF gene has been introduced. In one embodiment, the pharmaceutical composition of the present invention comprises pericyte-like cells differentiated from pluripotent stem cells into which the bFGF gene has been introduced. The pharmaceutical composition of the present invention comprises pericyte-like cells differentiated from ES cells or iPS cells into which the bFGF gene has been introduced. In one embodiment, the pharmaceutical composition of the present invention comprises pericyte-like cells differentiated from human pluripotent stem cells into which the bFGF gene has been introduced. In one embodiment, the pharmaceutical composition of the present invention comprises pericyte-like cells differentiated from human ES cells or human iPS cells into which the bFGF gene has been introduced.

[0047] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for angiogenesis therapy. This pharmaceutical composition includes a therapeutic agent for angiogenesis therapy containing the pericyte of the present invention. Angiogenesis therapy is a treatment method that promotes the generation of new blood vessels in an ischemic organ, tissue, or part of the human body in order to increase the amount of oxygen-rich blood reaching that organ, tissue, or part of the human body. Angiogenesis therapy includes the treatment of peripheral vascular diseases such as critical limb ischemia and diabetic retinopathy, and diseases such as pulmonary hypertension. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment of critical limb ischemia, peripheral vascular diseases such as diabetic retinopathy, and pulmonary hypertension. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment of critical limb ischemia.

[0048] This invention provides the use of pericite in the manufacture of pharmaceutical compositions for angiogenesis therapy. This invention provides pericite for use in angiogenesis therapy. Furthermore, this invention provides the use of pericite for angiogenesis therapy.

[0049] The present invention provides the use of pericite in the manufacture of pharmaceutical compositions for treating severe lower limb ischemia. The present invention provides pericite for use in the treatment of severe lower limb ischemia. Furthermore, the present invention provides the use of pericite for the treatment of severe lower limb ischemia.

[0050] The present invention also provides angiogenesis therapy (also referred to as "the method of treatment of the present invention") which includes administering a therapeutically effective amount of the perisite of the present invention to a subject. In the method of treatment of the present invention, "subject" means a human or other animal in need of the treatment. In one embodiment, "subject" means a human being in need of the method of treatment. When the perisite of the present invention is administered to a human, it can be administered to the subject in the form of a pharmaceutical composition comprising the perisite of the present invention and pharmaceutically acceptable excipients. The dosage and frequency of administration of the pharmaceutical composition of the present invention to a human can be appropriately adjusted according to the disease being treated, its severity, the age, weight and condition of the person receiving the treatment, etc.

[0051] The method of administering the pharmaceutical composition of the present invention is not particularly limited, and depending on the site of application, possible methods include local transplantation by surgical means, intravenous administration, lower limb puncture administration, local injection administration, subcutaneous administration, intradermal administration, intramuscular administration, etc.

[0052] The pharmaceutical composition of the present invention may be in sheet form and applied directly to the affected area. The sheet may contain not only cells but also a suitable support material.

[0053] The pharmaceutical composition of the present invention may contain scaffolding materials or components that assist in the maintenance and proliferation of cells and administration to the affected area, as well as other pharmaceutically acceptable carriers. Components necessary for the maintenance and proliferation of cells include culture medium components such as carbon sources, nitrogen sources, vitamins, minerals, salts, and various cytokines, or extracellular matrix preparations such as Matrigel.

[0054] The pericyte of the present invention or the pharmaceutical composition of the present invention may also be used in combination with vascular endothelial cells. In this specification, “combined use” means administering multiple types of pharmaceutically active ingredients simultaneously or separately to the same subject. In combined use, the multiple types of pharmaceutically active ingredients may be contained in the same composition or separately in different compositions. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition used in combination with vascular endothelial cells. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition further comprising vascular endothelial cells. In one embodiment, the therapeutic method of the present invention further comprises administering vascular endothelial cells.

[0055] The present invention also includes a pharmaceutical composition comprising a combination of the perisite and vascular endothelial cells of the present invention. In this specification, "combined" means that multiple types of pharmacoactive ingredients are contained in the same pharmaceutical composition, or that multiple types of pharmacoactive ingredients are contained separately in different pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprising a combination of the perisite and vascular endothelial cells of the present invention is a pharmaceutical composition containing the perisite and vascular endothelial cells of the present invention. In one embodiment, the pharmaceutical composition comprising a combination of the perisite and vascular endothelial cells of the present invention is a combination of pharmaceutical compositions in which the perisite and vascular endothelial cells of the present invention are contained separately in different pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprising a combination of the perisite and vascular endothelial cells of the present invention is a combination of a pharmaceutical composition containing the perisite and a pharmaceutical composition containing vascular endothelial cells. In one embodiment, the pharmaceutical composition comprising a combination of the perisite and vascular endothelial cells of the present invention is a pharmaceutical composition for angiogenesis therapy. In one embodiment, the pharmaceutical composition comprising a combination of the perisite and vascular endothelial cells of the present invention is a pharmaceutical composition for treating severe lower limb ischemia.

[0056] ≪Vascular endothelial cells≫ Endothelial cells are a single layer of flattened cells that line the lumen of blood vessels and have diverse functions, including regulating vascular tension and permeability, angiogenesis, anti-inflammatory effects, and blood coagulation. Large blood vessels at the arterial and venous levels have a three-layered structure consisting of the intima, media, and adventitia, which are mainly composed of endothelial cells, smooth muscle cells, and fibroblasts, respectively. On the other hand, in small blood vessels at the capillary level, the luminal structure of endothelial cells is surrounded by pericytes. In mature capillaries, pericytes share the basement membrane with endothelial cells and exist embedded within it. In recent years, it has been known that pericytes and endothelial cells communicate with each other through cell signaling, regulating differentiation and proliferation, and playing an important role in capillary maturation, stabilization, maintenance, basement membrane formation, and extracellular matrix deposition.

[0057] The vascular endothelial cells that can be combined with the perisite of the present invention or the pharmaceutical composition of the present invention are not particularly limited, but in one embodiment they are primary vascular endothelial cells or vascular endothelial cells differentiated from pluripotent stem cells, etc. The vascular endothelial cells used in combination with the perisite of the present invention or the pharmaceutical composition of the present invention are not particularly limited, but in one embodiment they are primary vascular endothelial cells or vascular endothelial cells differentiated from pluripotent stem cells, etc. In the angiogenesis therapy of the present invention, when the perisite of the present invention and vascular endothelial cells are used in combination, the vascular endothelial cells may be administered simultaneously with the administration of the perisite of the present invention, or before or after the administration of the perisite of the present invention.

[0058] "Primary vascular endothelial cells" refers to vascular endothelial cells directly collected from an individual organism, or primary cultured cells and passaged cells obtained by culturing and proliferating such vascular endothelial cells in vitro. Primary vascular endothelial cells are not particularly limited, but in some embodiments, they are human primary vascular endothelial cells. As human primary vascular endothelial cells, it is desirable to use primary vascular endothelial cells that are identical or substantially identical in human leukocyte antigen (HLA) genotype to that of the recipient individual, for example, from the viewpoint of preventing rejection. Here, "substantially identical" means that the HLA genotype matches to the extent that the immune response to the transplanted vascular endothelial cells can be suppressed by an immunosuppressant, for example, vascular endothelial cells having an HLA type in which the three gene loci of HLA-A, HLA-B, and HLA-DR, or the four gene loci of HLA-DR plus HLA-C, match. In one embodiment, the vascular endothelial cells that can be used in combination with or combined with the pericyte or pharmaceutical composition of the present invention are human primary vascular endothelial cells.

[0059] The "vascular endothelial cells differentiated from pluripotent stem cells, etc." that can be combined with the pericytes of the present invention or the pharmaceutical composition of the present invention are not particularly limited, but for example, vascular endothelial cells produced by the methods described in Ikuno T et al., Pros One, (2019), 12: e0173271 and Cho SW et al., Circulation, (2007), 116: 2409-2419 can be used. In one embodiment, the vascular endothelial cells that can be used in combination with the pericytes of the present invention or the pharmaceutical composition of the present invention are vascular endothelial cells differentiated from human pluripotent stem cells, etc.

[0060] Specific examples are provided hereby for reference to further understand the present invention, but these are for illustrative purposes only and do not limit the invention. [Examples]

[0061] Example 1: Establishment of primary human pericytes derived from skeletal muscle. A portion of human quadriceps femoris muscle was immersed in PBS, and the muscle was finely cut along its fibers using a scalpel and forceps. The cut muscle fibers were placed in a 50 mL centrifuge tube (Corning, 352070), 15 mL of collagenase solution (see below) was added, and after standing for 3 minutes, the supernatant was removed. The above procedure was repeated two more times. Next, the muscle fibers that had undergone the above procedure were placed in another 50 mL centrifuge tube, 15 mL of collagenase solution was added, and the mixture was left to stand in a 37°C warm bath for 1 hour. The cell suspension other than the muscle fibers was collected and designated as cell suspension 1. Collagenase solution was again added in two separate 15 mL increments, and after each addition of collagenase solution, the cell suspension was collected using the same procedure as for cell suspension 1 to obtain cell suspension 2 and cell suspension 3. Cell suspensions 1, 2, and 3 were each passed through a 100 μm cell strainer (Corning, 352360), and the resulting cell suspensions were centrifuged at 300 g, 4°C, and for 5 minutes. After removing the supernatant, 20 mL of pericyte establishment medium (see below) was added to each centrifuge tube, and the cells were divided into two collagen-coated dishes (Corning, 356450) and cultured at 37°C under a 5% CO2, 5% O2 atmosphere. Cells derived from cell suspensions 1, 2, and 3 will be referred to below as cells of condition 1, 2, and 3, respectively. On days 3, 6, and 8 of culture, the culture supernatant was removed from each and 10 mL of pericyte establishment medium was added. For cells of condition 1, on day 13 of culture, the culture supernatant on the dish was removed, washed with 5 mL of PBS, and 1 mL of cell dissociation reagent (TrypLE Express, ThermoFisher Scientific, 12604013) was added, and the mixture was allowed to stand on a 37°C plate for 5 minutes. 4 mL of pericyte establishment medium was added to the dish to collect the cell suspension, and it was centrifuged at 300 g, room temperature, for 5 minutes. After removing the supernatant, 5 mL of Cellbanker 1 (Takara Bio, CB011) was added, and the mixture was dispensed into 1 mL portions into cryotubes and stored in liquid nitrogen.

[0062] On day 10 of culture, the culture supernatant was removed from the cell dishes for conditions 2 and 3. Each dish was washed with 5 mL of PBS, and 1 mL of TrypLE Express was added to each. The mixture was then allowed to stand on a 37°C plate for 5 minutes. 4 mL of pericyte establishment medium was added to each dish to collect the cell suspension. The cell suspensions for conditions 2 and 3 were combined and mixed in the same centrifuge tube. The mixture was then centrifuged at 300 g, room temperature, for 5 minutes. After removing the supernatant, 5 mL of Cellbanker 1 was added, and the mixture was dispensed into 1 mL cryotubes and stored in liquid nitrogen. Later, 9 mL of pericyte growth medium (see below) was added to a 50 mL centrifuge tube. 1 mL of Cellbanker 1 solution containing the thawed cells from conditions 2 and 3 was added, and the mixture was centrifuged at 300 g, 4°C, for 5 minutes. After removing the supernatant, 20 mL of pericyte growth medium was added to the centrifuge tube to collect the cell suspension. The recovered cell suspension was divided into two collagen-coated dishes and cultured at 37°C under a 5% CO2, 5% O2 atmosphere. On the third day of culture, the culture supernatant was removed from each dish and 10 mL of pericyte growth medium was added. The following day, the culture supernatant was removed, washed with 5 mL of PBS, and 2 mL of cell dissociation reagent (Accutase®, Innovative Cell Technologies, AT104) was added, and the mixture was allowed to stand on a 37°C plate for 5 minutes. 8 mL of pericyte growth medium was added to each dish, and the cell suspension was collected in a centrifuge tube and centrifuged at 300 g, room temperature, for 5 minutes. The supernatant was removed, 100 μL of PBS was added to the centrifuge tube, and 20 μL of blocking reagent (FcR Blocking Reagent, Miltenyi Biotec, 130-059-901) was added, and the mixture was allowed to stand on ice for 15 minutes. Next, 20 μL each of anti-human ALP (Alkaline Phosphatase) antibody (R&D Systems, FAB1448P) and anti-CD56 APC antibody (Miltenyi Biotec, 130-090-843) were added, and the mixture was allowed to stand on ice for 20 minutes. After washing twice with PBS, the cells were resuspended in PBS, and ALP(+) and CD56(-) cells (i.e., primary human pericytes) were separated using a cell sorter (SH800S, Sony). The separated cells were then suspended in pericyte growth medium and seeded on collagen-coated dishes for culture.

[0063] [Collagenase solution] The solution was prepared by dissolving 100 mg of Collagenase, Type II (ThermoFisher Scientific, 17101015) in 200 mL of TrypLE select (ThermoFisher Scientific, 12563029).

[0064] [Perisite establishment medium] The composition is as follows: ·92% MegaCell Dulbecco's Modified Eagle's Medium(Sigma-Aldrich, M3942) 5% FBS (Sigma-Aldrich) ·1% GlutaMax(ThermoFisher Scinetific, 35050061) ·1% MEM Non-essential Amino Acid Solution(Sigma-Aldrich, M7145) ·1% Penicillin-Streptmycin (Sigma-Aldrich, P0781) ·100μM 2-Mercaptethanol (ThermoFisher Scientific, 21985023) ·5ng / mL FGF-basic(154a.a.), Human, Recombinant (Peprotech, 100-18B)

[0065] [Perisite growth medium] The composition is as follows: ·77% MegaCell Dulbecco's Modified Eagle's Medium 20% FBS 1% GlutaMax ·1% MEM Non-essential Amino Acid Solution • 1% Penicillin-Streptmycin • 100 μM 2-Mercaptethanol ·5ng / mL Animal-free Recombinant Human FGFbasic-TS(Proteintech, HZ-1285)

[0066] Example 2: Preparation of a lentiviral vector for introducing the bFGF gene. Lentivirus packaging cell line (Lenti-X 293T Cell Line, Takara Bio, 632180) was grown in 5 × 10⁶ cells using 10 mL of 293T growth medium per 10 cm dish (see below). 6 Cells were seeded individually and cultured in four dishes at 37°C under a 5% CO2 atmosphere. The following day, 28 μL of Lentiviral High Titer Packaging Mix (Takara Bio, 6194) and 15 μL of bFGF insertion plasmid (see below) were added to 6 mL of D-MEM (Fujifilm Wako Pure Chemical Industries, 045-30285), mixed, and allowed to stand at room temperature for 5 minutes. Then, 180 μL of transfection reagent (TransIT-293 Transfection Reagent, Takara Bio, MIR2704) was added and mixed, and allowed to stand at room temperature for 30 minutes (hereinafter referred to as "Transfection solution"). 1.5 mL of the Transfection solution was added to each of the four dishes, one 10 cm dish in which Lenti-X 293T Cell Line had been seeded the previous day. The following day, the supernatant from each dish was removed, and 10 mL of 293T growth medium was added. After two days, the culture supernatant was collected and centrifuged at room temperature at 300g for 5 minutes to remove detached cells. The supernatant was collected, and 1 / 3 the volume of lentivirus concentrate (Lenti-X Concentrator, Takara Bio, 631231) was added to it, and the mixture was centrifuged at 500g at 4°C for 45 minutes. The supernatant was removed, and 200 μL of D-MEM was added to obtain a lentivirus suspension for bFGF gene transfer.

[0067] [293T Growth Medium] The composition is as follows: · D-MEM 10% FBS • 1% GlutaMAX

[0068] [bFGF insertion plasmid] A plasmid (pLe6-Bmp-bFGF: SEQ ID NO: 5) was constructed by inserting the gene sequence (SEQ ID NO: 3) encoding the amino acid sequence (SEQ ID NO: 4) of Bmp-bFGF (Patent Document US 7816140 B2) using the pLenti6 / V5 Directional TOPO Cloning Kit (ThermoFisher Scientific, K495510).

[0069] Example 3: Production of primary human pericytes with bFGF gene transfer In Example 1, the human primary pericytes established were grown in pericyte growth medium at a rate of 1 × 10⁻⁶ 5Cells were seeded at a cell / well rate into two wells on a collagen-coated 6-well plate (Iwaki, 4810-010). Two days later, 1.2 μL of 10 mg / mL polyblen solution (Nacalai Tesque, 12996-81) and 62 μL of the bFGF gene transfer lentivirus suspension prepared in Example 2 were added to 3 mL of pericyte growth medium, mixed, and allowed to stand at room temperature for 5 minutes (Suspension A). The supernatant of the cultured human primary pericytes was removed, and 1.5 mL of Suspension A was aspirated from each well and added to each of the two wells. The plate was centrifuged at 1200 g for 60 minutes at room temperature, and then cultured at 37°C under a 5% CO2, 5% O2 atmosphere. The following day, the supernatant was removed, washed nine times with PBS, 1 mL of Accutase was added, and the plate was allowed to stand at 37°C for 5 minutes. Four milliliters of pericyte growth medium were added to a dish to collect the cell suspension. The cell suspension from two wells was collected in the same centrifuge tube and centrifuged at 300 g, room temperature, for 5 minutes. The supernatant was removed, and ten milliliters of pericyte growth medium were added. The collected cells were seeded into a collagen-coated dish and cultured. Culture was carried out at 37°C under a 5% CO2, 5% O2 atmosphere. The following day, 10 mg / mL of blastosidin S solution (Fujifilm Wako Pure Chemical Industries, 026-18711) was added to the collagen dish to a final concentration of 2.5 μg / mL, and culture was continued. The cells that proliferated through this procedure were designated as bFGF gene-transformed human primary pericytes.

[0070] Example 4: Determination of bFGF expression levels in primary human pericytes with bFGF gene transfer. The bFGF gene-transformed human primary pericytes prepared in Example 3 and the human primary pericytes (control) prepared in Example 1 were subjected to 3 × 10⁻¹⁶ growth in pericyte growth medium. 5Cells were seeded at a cell / dish rate in collagen-coated dishes and cultured at 37°C under a 5% CO2, 5% O2 atmosphere. On day 3 of culture, the culture supernatant was collected and passed through a syringe filter (IWAKI, 2053-025). The concentration of bFGF in the supernatant after passing through the syringe filter was measured using an ELISA kit (Human FGF basic Quantikine ELISA KIT, R&D Systems, DFB50). The measurement of bFGF concentration was performed according to the protocol provided with the kit.

[0071] bFGF expression was not detected in control human primary pericytes, whereas very high bFGF expression was observed in human primary pericytes with the bFGF gene introduced (Figure 1).

[0072] Example 5: Qualitative evaluation of the angiogenic capacity of primary human pericytes with bFGF gene transfer. Human umbilical vein endothelial cells (HUVEC, PromoCell, C-12200) were cultured in endothelial cell growth medium (PromoCell, C-22111) at 37°C under a 5% CO2 atmosphere. In addition, primary human pericytes (control) established in Example 1 and primary human pericytes with bFGF gene modification prepared in Example 3 were cultured on collagen-coated dishes using pericyte growth medium. After confirming that each of the three cell types had grown to a confluent state, the supernatant was removed, washed with PBS, and 2 mL of Accutase was added, followed by standing on a 37°C plate for 5 minutes. Next, 8 mL of endothelial cell growth medium was added to each dish for HUVEC, and 8 mL of pericyte growth medium was added to each dish for primary human pericytes and primary human pericytes with bFGF gene modification. The cell suspensions were collected from each dish, centrifuged at 300g, room temperature for 5 minutes, and the supernatant was removed. HUVEC was then suspended in endothelial cell growth medium, and the two types of human primary pericytes were suspended in pericyte growth medium. Nine 1.5 mL tubes (Eppendorf, 0030120.086) were used to store 5.5 × 10⁶ HUVEC cells. 5Each of the following was added: three of them contained human primary pericytes, and the other three contained bFGF gene-modified human primary pericytes, each containing 5.5 × 10¹⁶ of cells. 5 The cells were added and mixed. Each tube was centrifuged at 300g, 4°C, and for 5 minutes. The supernatant was removed, and after washing once with PBS, it was centrifuged again under the same conditions, and the supernatant was removed. Next, 400 μL of extracellular matrix (Matrigel® Growth factor reduced, Corning, 356231, hereinafter referred to as "Matrigel") was added to each tube, mixed on ice, and the Matrigel containing the cells was aspirated using a syringe with a 25-gauge needle. A three-component anesthetic solution was prepared by adding 3 mL of Domitol (Nippon Zenyaku Kogyo), 8 mL of Dormicum injection (Maruishi Pharmaceutical), and 10 mL of Betolfar (Meiji Seika Pharma) to 79 mL of physiological saline (Otsuka Pharmaceutical Co., Ltd.). Nine NOG mice (NOD.Cg-Prkdc scid Il2rg tm1Sug 300 μL of the three-component mixed anesthetic solution was administered intraperitoneally to each of the ShiJic mice (In-Vivo Science). Three anesthetized mice were then subcutaneously administered the entire amount of Matrigel containing either the prepared HUVEC only (HUVEC), HUVEC and control human primary pericyte (Primary pericyte / HUVEC), or HUVEC and bFGF-gene-modified human primary pericyte (bFGF-Primary pericyte / HUVEC). After 14 days, the administered Matrigel was collected from each mouse and photographed (Figure 2).

[0073] Matrigel containing bFGF-transformed human primary pericytes (bFGF-Primary pericyte / HUVEC in Figure 2) showed enhanced angiogenesis compared to the control Matrigel containing human primary pericytes (Primary pericyte / HUVEC in Figure 2). Furthermore, no angiogenesis was observed with Matrigel containing only HUVEC (HUVEC in Figure 2).

[0074] Example 6: Quantitative evaluation of angiogenesis in bFGF gene-transformed human primary pericytes. Each Matrigel collected using the procedure of Example 5 was placed in a 2 mL tube (Eppendorf, 0030120.094) and cut several times with dissecting scissors. One stainless steel bead (Qiagen, 69989) was added, and 350 μL of 0.1% Brij® L23 solution (Sigma-Aldrich, B4184) was added. The Matrigel was crushed using a TissueLyser II (Qiagen, 85300), centrifuged at 10,000 g, 4°C, and for 5 minutes, and the supernatant was collected. The hemoglobin concentration in the collected supernatant was measured using a QuantiChrom Hemoglobin Assay Kit (BioAssay Systems, DIHB-250). The method followed the product protocol. For statistical analysis, t-tests were performed between two groups: HUVEC and control human primary pericytes (Primary pericyte / HUVEC), and between HUVEC and bFGF-gene-transformed human primary pericytes (bFGF-Primary pericyte / HUVEC).

[0075] The hemoglobin concentration in Matrigel containing bFGF gene-transformed human primary pericytes was significantly higher than that in Matrigel containing a control group of primary pericytes (Figure 3).

[0076] Example 7: Evaluation of blood flow improvement in bFGF gene-transformed human primary pericytes in a lower limb ischemia model. 300 μL of a three-component mixed anesthetic solution with the same composition as used in Example 5 was administered into the peritoneal cavity of NOG mice. After anesthesia, the body hair around the left lower limb was removed using depilatory cream. Then, 300 μL of an antisedan preparation solution, prepared by adding 150 μL of antisedan (Nippon Zenyaku Kogyo) to 24.8 mL of physiological saline, was administered subcutaneously to awaken the NOG mice. The following day, the NOG mice were again anesthetized using the three-component mixed anesthetic solution in the same manner as the previous day. They were placed supine under a stereomicroscope, and the skin of the left lower limb was incised to expose the femoral artery and vein, and the saphenous artery and vein. After ligating the blood vessels branching from the femoral artery and vein, the femoral artery and vein and the saphenous artery and vein were excised, and the skin was sutured. Subsequently, the NOG mice were placed prone, the skin near the gastrocnemius muscle was incised, and the marginal veins were cut. The skin was sutured again, and 300 μL of the antisedan preparation solution was administered subcutaneously to awaken the NOG mice. Two weeks post-surgery, NOG mice were anesthetized with the aforementioned three-component mixed anesthetic solution, kept warm on a 36°C incubator for 10 minutes, and their lower limb blood flow was analyzed using a blood flow imaging device (moorLDI2-IR, moor instruments). The blood flow signal value of the treated ischemic limb was divided by the blood flow signal value of the untreated normal limb to calculate the blood flow ratio (converted to a percentage). Simultaneously, the number of necrotic nails on the toes of the NOG mice whose blood flow was measured was recorded. From the multiple lower limb ischemia model mice that underwent the aforementioned procedure, individuals with a blood flow ratio of 20% to 40% and 4 or 5 necrotic nails were selected as evaluation mice, and the mice were divided into two groups so that the average blood flow ratio was the same between the two groups. The following day, the two groups of NOG mice were anesthetized with the three-component mixed anesthetic solution, the skin of the left lower limb was incised, and the muscle was exposed. One group contained 3 × 10¹⁶ cells suspended in 100 μL of Megacell Dulbecco's Modified Eagle's Medium. 6One bFGF gene-transformed human primary pericytes were administered in 10 locations: 9 locations from the muscles of the lower left femur to the gastrocnemius muscle, and 1 location on the sole of the left lower limb (cell administration group; 8 animals). In the other group, Megacell Dulbecco's Modified Eagle's Medium was administered in the same manner as a control (culture medium administration group; 7 animals). At weeks 2, 4, and 6 after cell or culture medium administration, blood flow in the lower limbs was analyzed using the moorLDI2-IR blood flow imaging system in the manner described above. The blood flow ratio (converted to a percentage) (Figure 4 left) was calculated by dividing the signal value of blood flow in the ischemic limb by the signal value of blood flow in the normal limb, and the AUC (Area Under Curve) up to week 6 after cell administration was calculated (Figure 4 right). Here, AUC is the area under the curve drawn when time is plotted on the X axis and the blood flow ratio on the Y axis. A t-test was performed as a statistical test.

[0077] In a mouse model of lower limb ischemia, administration of bFGF-modified human primary pericyte significantly improved blood flow.

[0078] Example 8: Differentiation induction from ES cells to early mesodermal cells Add 230 µL of Matrigel Human ES Cell Optimized Matrix (Corning, 354277) to 25 mL of DMEM / Ham's F12 medium (Nacalai Tesque, 11581-15), add 1.5 mL each to 3 wells of a 6-well plate (Iwaki, 3810-006), and leave the mixture to stand at room temperature for 3 hours to prepare a Matrigel-coated plate. Seed human ES cells on said Matrigel-coated plate, and culture the cells in STEMdiff Mesoderm Induction Medium (STEMCELL Technologies, 05220) under an atmosphere of 37°C and 5% CO₂ to induce differentiation of ES cells into early mesoderm cells. Check whether the obtained cells are differentiated into early mesoderm cells by flow cytometry. Specifically, CD140α and APLNR are selected as cell surface markers of early mesoderm cells, and the progress of differentiation induction from ES cells into early mesoderm is confirmed by confirming an increase in the proportion of CD140α(+) and APLNR(+) cells via flow cytometry using antibodies against each cell surface marker.

[0079] Example 9: Spheroid formation from early mesoderm cells 1.7×10 early mesoderm cells obtained in Example 8 5 cells are added with the spheroid formation medium described in the literature (Vodyanik MA et al., Cell Stem Cell, (2010), 7: 718-729), and cultured on an EZSPHERE (registered trademark) dish (Iwaki, 11-0434) under an atmosphere of 37°C, 5% CO₂ and 5% O₂. Once spheroids are formed, pass the culture solution containing spheroids through a 100 µm cell strainer to collect spheroids with a size of 100 µm or larger.

[0080] Example 10: Differentiation induction from spheroids to pericyte-like cells Based on U.S. Patent US9868939, differentiation induction from spheroids to pericyte-like cells is performed. Specifically, all spheroids recovered in Example 9 are suspended in pericyte differentiation induction medium (see below) and seeded in a dish coated with Fibronectin and Human type 1 Collagen for culture. Once monolayer cell proliferation is observed at the bottom of the dish, the supernatant is removed, washed with PBS, and Accutase is added to detach the cells. The cell suspension is collected in the dish using pericyte growth medium (see Example 1) and centrifuged. The supernatant is removed, pericyte growth medium is added, and the cells are seeded in a collagen-coated dish and cultured further.

[0081] [Pericyte Differentiation Induction Medium] The composition is as follows: • 50% Stemline(registered trademark) II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich, S0192) ·50% Human Endothelial SFM (ThermoFisher Scientific, 11111044) 1% GlutaMax ·0.05% Ex-CYTE NZ Growth Enhancement Media Supplement (Merck, 81150N) • 100 μM Monothioglycerol (Fujifilm Wako Pure Chemical Industries, 195-15791) ·10ng / mL Animal-free Recombinant Human FGFbasic-TS ·50ng / mL Recombinant Human PDGF-BB Protein(R&D Systems, 220-BB)

[0082] Example 11: Transfer of the bFGF gene into human ES cell-derived pericyte-like cells For the human ES cell-derived pericyte-like cells prepared in Example 10, the bFGF gene was introduced into the human ES cell-derived pericyte-like cells using a lentiviral suspension for bFGF gene transfer prepared in the same manner as in Example 2, in the same way as for the human primary pericytes in Example 3.

[0083] Example 12: Determination of bFGF expression in pericyte-like cells derived from human ES cells with bFGF gene transfection. The bFGF expression level of the bFGF gene-transformed human ES cell-derived pericyte-like cells prepared in Example 11, and the human ES cell-derived pericyte-like cells obtained in Example 10 as a control, was quantified using the same method as in Example 4. The bFGF gene-transformed human ES cell-derived pericyte-like cells showed a higher bFGF expression level compared to the control.

[0084] Example 13: Evaluation of the angiogenic capacity of pericyte-like cells derived from human ES cells with the bFGF gene introduced. The angiogenic capacity of bFGF-modified human ES cell-derived pericyte-like cells can be evaluated in the same manner as in Examples 5 and 6, which evaluated the angiogenic capacity of bFGF-modified human primary pericytes. bFGF-modified human ES cell-derived pericyte-like cells show higher angiogenic capacity compared to control human ES cell-derived pericyte-like cells.

[0085] Example 14: Evaluation of blood flow improvement in bFGF gene-transformed human ES cell-derived pericyte-like cells in a lower limb ischemia model. The evaluation of in vivo blood flow improvement in bFGF-gene-transformed human ES cell-derived pericyte-like cells can be performed in the same manner as in Example 7, in which blood flow improvement of bFGF-gene-transformed human primary pericytes was evaluated using a lower limb ischemia model mouse. Administration of bFGF-gene-transformed human ES cell-derived pericyte-like cells exhibits a high blood flow improvement effect in the aforementioned model mouse. [Industrial applicability]

[0086] Perisites into which the bFGF gene of the present invention has been introduced have excellent angiogenic activity and can be used in angiogenesis therapy for conditions such as severe lower limb ischemia. [Sequence Listing Free Text]

[0087] The nucleotide sequence shown in Sequence ID No. 1 of the sequence listing is the gene sequence of human bFGF represented by GenBank Accession Number: M27968.1, and the amino acid sequence shown in Sequence ID No. 2 is the amino acid sequence of human bFGF represented by GenBank Accession Number: AAA52448.1. Also, the numerical headings in the following sequence listings... <223> This section provides an explanation of "Artificial Sequence". Specifically, the nucleotide sequence shown in Sequence ID No. 3 of the sequence listing encodes the amino acid sequence of the human bFGF analog shown in Sequence ID No. 4. Furthermore, the nucleotide sequence shown in Sequence ID No. 5 of the sequence listing is the nucleotide sequence of the human bFGF expression plasmid (pLe6-Bmp-bFGF) used in the present invention's embodiment.

Claims

1. Pericytes into which the basic fibroblast growth factor (bFGF) gene has been introduced, The pericyte is a gene that encodes a protein having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and the protein has the function of bFGF.

2. The perisite according to claim 1, wherein the perisite is the original perisite.

3. The pericyte according to claim 1, wherein the pericyte is a pericyte-like cell differentiated from a pluripotent stem cell.

4. The pericyte according to claim 3, wherein the pluripotent stem cell is a human pluripotent stem cell.

5. The pericyte according to claim 3 or 4, wherein the pluripotent stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).

6. A pharmaceutical composition for angiogenesis therapy comprising perisite according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, wherein the angiogenesis therapy is a treatment for severe lower limb ischemia.

8. The pharmaceutical composition according to claim 6 or 7, which is used in combination with vascular endothelial cells.

9. A pharmaceutical composition for angiogenesis therapy comprising a combination of pericytes and vascular endothelial cells as described in any one of claims 1 to 5.

10. The pharmaceutical composition according to claim 9, wherein the angiogenesis therapy is a treatment for severe lower limb ischemia.

11. A method for producing perisite according to any one of claims 1 to 5.

12. Use of perisite according to any one of claims 1 to 5 in the manufacture of a pharmaceutical composition for angiogenesis therapy.

13. Use of perisite according to any one of claims 1 to 5 in the manufacture of a pharmaceutical composition for the treatment of severe lower limb ischemia.

14. A pericyte according to any one of claims 1 to 5, for use in angiogenesis therapy.

15. A pericyte according to any one of claims 1 to 5, for use in the treatment of severe lower limb ischemia.

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