JP7217533B2 - Compositions for amplifying the effect of treatment with mesenchymal stem cells - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Publisher: Department of Plastic Surgery, Kindai University School of Medicine Title of publication: Program and Abstracts of the 26th Basic Academic Meeting of the Japanese Society of Plastic and Reconstructive Surgery Publication date: September 2017 Month Page 138

Application of Article 30, Paragraph 2 of the Patent Act (2) Name of Society: NINTH INTERNATIONAL CONFERENCE REGERATIVE SURGERY (9th International Conference on Regenerative Surgery) Venue: Eurostars Rome Aeterna Hotel (Rome, Italy) Date: 2017 (2017) I gave a presentation on December 15th

The present invention relates to compositions and uses thereof for amplifying the effects of treatment with mesenchymal stem cells.

In recent years, treatments using mesenchymal stem cells have been studied for many diseases. Mesenchymal stem cells are somatic stem cells derived from mesodermal tissue (mesenchyme) and have the ability to differentiate into cells belonging to mesenchymal cells. Each tissue to be collected is called adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, or the like. Mesenchymal stem cells are known to have pluripotency that can differentiate into various somatic cells such as bones, blood vessels, and myocardium, and secrete cytokines to exert immunosuppressive and anti-inflammatory effects. , and angiogenic effects. In addition, because of its high immune tolerance, not only autologous transplantation but also allotransplantation is possible. Due to these properties, mesenchymal stem cells are pluripotent stem cells with great potential in regenerative medicine.To date, many clinical studies have been conducted for various diseases, and they are used to treat a wide range of diseases. is expected. However, the reality is that currently, treatment with mesenchymal stem cells alone does not produce satisfactory effects.

For example, fat grafting is performed in the field of soft tissue reconstruction, such as breast reconstruction after breast cancer surgery, for the purpose of increasing volume and restoring contour. Adipose tissue is superior to exogenous materials because this technique uses the abundantly available adipose tissue as a self-filling material, so it can have a natural feel and is an inexpensive procedure with low infection rates. It has become a thing. However, during the liposuction harvesting process, fat cells are cut off from the original blood supply. As a result, the graft becomes transiently ischemic after injection, which causes partial necrosis and graft volume loss (Non-Patent Document 1). Despite the growing interest in fat grafting and its long-term clinical applications (2), no satisfactory solution to this ischemic volume loss has been developed. Secondary treatments are often necessary to achieve satisfactory results, and adipose-derived mesenchymal stem cells (ASCs) have been investigated as a cell therapy for enhancing fat grafting (20). ). Therapies that supplement ASC during adipocyte transplantation (ASC-supplemented fat grafts) are promising, but still need to be developed in order to obtain consistent clinical results [21-22]. ). ASCs have been observed to release pro-angiogenic cytokines (23) and endothelial differentiation in vitro (24). However, on the other hand, in vivo experiments with ASCs show that the effects on angiogenesis are mainly paracrine (25) or that ASCs enhance fat grafting, different, non-angiogenic It suggests that there is a mechanism (Non-Patent Document 26). However, the problem is that ASC alone is not sufficient for angiogenesis to engraft adipose tissue, and also causes problems due to fibrosis. If these problems can be improved, ASC supplementation can be expected to have a higher clinical effect in fat transplantation for purposes such as breast reconstruction.

An "ischemic disease" is a disease caused by tissue damage such as cell degeneration, atrophy, and fibrosis due to decreased blood flow in tissues. In particular, when "arteriosclerosis obliterans", which narrows or blocks arteries that supply blood to the legs, becomes severe, severe lower extremity ischemia occurs, causing symptoms such as pain and intractable ulcers. Amputation may be required. Treatment with mesenchymal stem cells has also been investigated for the treatment of ischemic diseases, but sufficient effects have not been obtained. Treatment with mesenchymal stem cells is expected to be effective through indirect angiogenesis through cytokine release, but is not expected to be effective through direct revascularization. Therefore, sufficient revascularization within tissue has not been observed in mesenchymal stem cell therapy. Furthermore, since fibrosis is observed in tissues after mesenchymal stem cell transplantation, it is possible that tissue damage due to fibrosis remains. expected to be effective.

The present inventors have found that vascular endothelial progenitor cells or anti-inflammatory/immune tolerance-inducing cells are enriched in serum-free culture from mononuclear cell fractions derived from bone marrow, cord blood, or peripheral blood, as shown in International Publication WO2014/0561154. The present inventors have invented a method for amplifying a group of cells that have undergone a transformation, with the aim of using it as a therapeutic agent for ischemic diseases, intractable ulcers, or diabetes-related diseases. However, the present invention has unexpectedly revealed that it has the effect of amplifying the effects of mesenchymal stem cells. Furthermore, surprisingly, this effect is not limited to one disease, and similar results were observed for multiple diseases for which mesenchymal stem cells are effective. It was clarified that the in vitro expanded cultured mononuclear cell fraction amplifies the characteristics of mesenchymal stem cells and improves the problem.

International publication WO2006/090882 International publication WO2014/0561154

Plast Reconstr Surg. 2013 Feb;131(2):185-191 Langenbecks Arch Klin Chir Ver Dtsch Z Chir. 1893;22:66-69 Otolaryngol Head Neck Surg. 1990 Apr;102(4):314-321 Ann Plast Surg. 2005 Jul;55(1):63-68 Exp Biol Med (Maywood). 2005 Nov;230(10):742-748 Dermatol Surg. 2006 Dec;32(12):1437-1443 Plast Reconstr Surg Glob Open. 2013 Oct 7;1(6):e40 Arch Plast Surg. 2015 Mar;42(2):150-158 Plast Reconstr Surg. 2012 May;129(5):1081-1092 Plast Reconstr Surg. 2015 Jun;135(6):1607-1617 Science. 1997 Feb 14;275(5302):964-967 Circulation. 2001 Feb 6;103(5):634-637 Nat Med. 2001 Apr;7(4):430-436 Circ Res. 2001 Jul 6;89(1):E1-7 J Am Coll Cardiol. 2003 Dec 17;42(12):2073-2080 FASEBJ. 2005 Jun;19(8):992-994 Plast Reconstr Surg. 2008 Jun;121(6):1929-1942 Diabetes. 2010 Aug;59(8);1974-1983 Stem Cells Transl Med. 2012 Feb;1(2):160-171 Mol Biol Cell. 2002 Dec;13(12):4279-4295 J Plast Reconstr Aesthet Surg. 2013 Nov;66(11):1494-1503 Aesthet SurgJ. 2017 Jul 1;37(suppl_3):S46-58 Stem Cells. 2009 Jan;27(1):266-274 Circulation. 2004 Feb 10;109(5):656-663 PLoS One. 2012;7(9):e45621 Lancet. 2013 Sep 28;382(9898):1113-1120 Diabetes. 2013 Sep;62(9):3207-3217 Circ Res. 2011 Jun 24;109(1):20-37 Stem Cell Res Ther. 2013 Feb 28;4(1):20 J Am Heart Assoc. 2014 Jun 25;3(3):e000743

Currently, mesenchymal stem cells are expected to be used for the treatment of a wide range of diseases due to their properties, but the expected effects have not yet been obtained by single treatment. Despite the need, no technology has existed that allows safe and effective amplification.

Supplementation of mesenchymal stem cell treatment with ex vivo expanded cultured mononuclear cell fraction stimulates angiogenesis and amplifies its effects safely and effectively by exerting anti-inflammatory and anti-immune effects. The following two were also found in the model.

In fat transplantation, the combination of mesenchymal stem cells and an ex vivo expanded cultured mononuclear cell fraction increased capillary density in the tissue by enhancing angiogenesis compared to treatment with mesenchymal stem cells alone. was found to have a stronger effect at In addition, when combined with the in vitro expanded cultured mononuclear cell fraction, not only the transplanted fat fibers were suppressed, but also the inflammatory response was suppressed. Due to these effects, the problems associated with mesenchymal stem cell treatment alone can be resolved, and higher clinical efficacy can be expected.

In addition, in lower extremity ischemia, the combination of mesenchymal stem cells and mononuclear cell fractions cultured in vitro increases angiogenesis compared to treatment with mesenchymal stem cells alone, thereby increasing blood flow in the lower extremities. It was found to have a strong lower extremity blood flow improvement effect by increasing the flow rate, reducing the occurrence of gangrene of the lower extremities, and increasing the density of capillaries in the tissue. Furthermore, blood vessels in the expanded tissue are observed to undergo not only angiogenesis due to the cytokine effect of mesenchymal stem cells, but also direct revascularization due to the mononuclear cell fraction cultured in vitro for expansion. Long-term survival of intra-tissue blood vessels amplified by direct angiogenesis is observed, and a long-term blood flow improvement effect can be expected. In addition, it is expected that the in vitro expanded cultured mononuclear cell fraction will amplify the ameliorative effect of mesenchymal stem cells on ischemia-associated ulcers due to its anti-inflammatory and anti-fibrotic effects.

Therefore, the present invention provides an in vitro enriched cultured vascular endothelial progenitor cell or anti-inflammatory/immune tolerance-inducing cell that is used alone for the treatment of ischemic diseases, intractable ulcers, and diabetes-related diseases. The mononuclear cell fraction, which is a cell group, is used for the purpose of amplifying the therapeutic effect of mesenchymal stem cells, which is a completely different application.

The present invention includes, without limitation, the following aspects.

[Aspect 1]
A composition comprising an in vitro expanded cultured mononuclear cell fraction for amplifying the effect of treatment with mesenchymal stem cells.

[Aspect 2]
A composition according to aspect 1, for use with mesenchymal stem cells, or a composition comprising mesenchymal cells.

[Aspect 3]
A composition according to aspect 1 or 2, wherein the mononuclear cell fraction is a bone marrow, peripheral blood or cord blood derived mononuclear cell fraction.

[Aspect 4]
The composition according to any one of aspects 1 to 3, wherein the bioenriched cultured mononuclear cell fraction is a cell population enriched in vascular endothelial progenitor cells or anti-inflammatory/immune tolerance-inducing cells.

[Aspect 5]
Aspects 1-4, wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, or cord blood-derived mesenchymal stem cells. composition.

[Aspect 6]
Any one of aspects 1-5, wherein the treatment with mesenchymal stem cells is a treatment whose effects are promoted by angiogenesis, anti-inflammatory action, anti-fibrosis, etc., by the ex vivo expanded cultured mononuclear cell fraction. 13. The composition of claim 1.

[Aspect 7]
Treatment with mesenchymal stem cells consists of tissue transplantation, tissue regeneration, treatment of ischemic disease, treatment of neurodegenerative disease, treatment of heart disease, treatment of malignant tumors, treatment of inflammatory disease, and treatment of immune disease. A composition according to any one of aspects 1-6, selected from the group.

[Aspect 8]
Treatment with mesenchymal stem cells can be used for fat transplantation, bone marrow transplantation, treatment of skeletal muscle wounds, treatment of skin wounds, tissue regeneration after prostate cancer surgery, treatment of osteoarthritis, treatment of lumbar disc deformity, and peripheral nerve injury. treatment, treatment of leg ischemia, treatment of ischemic ulcer, treatment of ischemic heart disease, treatment of cerebral infarction, treatment of amyotrophic lateral sclerosis (ALS), treatment of Parkinson's disease, treatment of Alzheimer's disease, progression treatment of supranuclear palsy, treatment of Huntington's disease, treatment of multiple system atrophy, treatment of spinocerebellar degeneration, treatment of spinal cord injury, treatment of heart failure, treatment of endocarditis, treatment of valvular heart disease, heart Treatment of meningitis, treatment of congenital heart disease, treatment of myocarditis, treatment of myocardial infarction, treatment of Crohn's disease, treatment of liver cirrhosis, treatment of hepatitis, treatment of ulcerative colitis, treatment of inflammatory bowel disease, collagen 8. The composition according to any one of aspects 1-7, selected from the group consisting of treatment of disease, treatment of graft versus host disease (GVHD) and treatment of multiple sclerosis.

[Aspect 9]
the treatment with mesenchymal stem cells is transplantation,
(i) Increased graft survival:
(ii) increase graft survival;
9. The composition of aspect 8, wherein at least one effect of (iii) inhibiting fibrosis of the graft, (iv) improving blood flow in the graft, or (v) reducing inflammation due to grafting is obtained. .

[Aspect 10]
treatment with mesenchymal stem cells is a treatment for ischemia,
(vi) improves blood flow:
9. The composition of aspect 8, wherein at least one effect of (vii) improving function; or (viii) improving tissue damage is obtained.

[Aspect 11]
A method for amplifying the effect of treatment with mesenchymal stem cells, comprising combining a composition comprising an ex vivo expanded cultured mononuclear cell fraction with a mesenchymal stem cell or a composition comprising mesenchymal cells. using.

Compositions comprising ex vivo expanded cultured mononuclear cell fractions of the present invention are effective for amplifying the effects of a wide variety of treatments with mesenchymal stem cells.

[Fig. 1] Fig. 1 is a schematic diagram of an embodiment of fat grafting in the examples of the present specification.
Healthy recipient mice were randomly divided into three groups. Each recipient mouse received two dorsal grafts and each graft consisted of 0.25 g of adipose tissue.
Fat grafts in the QQKSL+ASC group were grafted with 2×10 ⁴ QQ cultured KSL cells mixed with 4×10 ⁵ ASC suspended in 25 μl PBS, and fat grafts in the ASC group were grafted with 4×10 ⁵ ASC suspended in 25 μl PBS. transplanted. Fat grafts in the control group received 25 μl PBS without cells.
18 mice in each group were implanted. Specifically, two implants per mouse are injected superficially to the muscle as a lateral lumbar subcutaneous bolus with a sterile 1 ml syringe and blunt-end 16 gauge needle. The incisional wound is closed with 5/0 nylon sutures. During the 10-week follow-up, the transplanted fat and cells did not cause significant inflammation in the overlying skin.
Abbreviations: GFP, green fluorescent protein; KSL cells, c-Kit ⁺ Sca-1 ⁺ Lin ⁻ cells; QQ, ex vivo expanded culture; ASC, adipose-derived mesenchymal stem cells.
[Fig. 2] Fig. 2 shows the fat graft survival rate (%). Fat graft survival is calculated by dividing explant weight by original graft weight to yield weight persistence (N=10 grafts per group after 5 weeks; mean±SEM). ^* , p<0.05. Abbreviations: QQKSL, QQ cultured c-Kit ⁺ Sca-1 ⁺ Lin ⁻ cells; ASC, adipose-derived mesenchymal stem cells. Fat grafts implanted with QQKSL+ASC show significantly higher graft survival compared to the control group after 5 weeks.
[Fig. 3] Fig. 3 shows the integrity (%) of adipose tissue at 5 weeks and after fat grafting. Fat grafts implanted with QQKSL+ASC had significantly higher adipose tissue integrity after 5 weeks compared to control and ASC groups. ^* , p<0.05, Abbreviations: QQKSL, QQ-cultured c-Kit ⁺ Sca-1 ⁺ Lin ⁻ cells; ASC, adipose-derived mesenchymal stem cells.
[Fig. 4] Fig. 4 shows the incidence of fibrosis 5 weeks after fat transplantation. QQKSL+ASC-implanted fat grafts exhibited reduced fibrosis compared to ASC-only fat grafts during the first 5 weeks post-implantation. The percentage of fibrotic tissue in each graft was quantified by Azan staining (N=4 grafts per group after 5 weeks). Abbreviations: QQKSL, QQ cultured c-Kit ⁺ Sca-1 ⁺ Lin ⁻ cells; ASC, adipose-derived mesenchymal stem cells.
[Fig. 5] Fig. 5 shows the occurrence of local inflammation (inflammatory units/mm ² ) 5 weeks after fat transplantation. QQKSL+ASC and ASC-implanted fat grafts significantly suppressed inflammation compared to the control group. Furthermore, QQKSL+ASC-implanted fat grafts suppressed inflammation more significantly than ASC-only fat grafts. Local inflammatory units positive for the CD68 antigen were counted and presented 5 weeks after transplantation (N=4 grafts per group after 5 weeks; mean±SEM). ^* , p<0.05; ^** , p<0.01; ^*** , p<0.001. Abbreviations: QQKSL, QQ cultured c-Kit ⁺ Sca-1 ⁺ Lin ⁻ cells; ASC, adipose-derived mesenchymal stem cells.
[Fig. 6] Fig. 6 shows blood vessel density (vessels/ ^mm2 ) 5 weeks after fat grafting. Fat grafts implanted with QQKSL+ASC have significantly higher vascular density than fat grafts implanted with ASC alone and the control group. ^*** , p<0.001. Abbreviations: QQKSL, QQ cultured c-Kit ⁺ Sca-1 ⁺ Lin ⁻ cells; ASC, adipose-derived mesenchymal stem cells.
[Fig. 7] Fig. 7 shows blood flow images in the ischemic leg 10 days after cell transplantation, and blood flow values compared with healthy contralateral limb.
[Fig. 8] Fig. 8 shows macroscopic images 14 days after cell transplantation.

Modes for carrying out the present invention include the following modes

1. Compositions for Amplifying the Effects of Mesenchymal Stem Cell Treatment The present invention provides compositions for amplifying the effects of mesenchymal stem cell treatment. The composition comprises an in vitro expanded cultured mononuclear cell fraction.

"Mononuclear cell fraction" is a general term for cells with round nuclei contained in peripheral blood, bone marrow, cord blood, etc. obtained from adults, and includes lymphocytes, monocytes, macrophages, vascular endothelial progenitor cells, and hematopoietic stem cells. etc. are included. Mononuclear cells also contain CD34 and/or CD133 positive cells. It is obtained by collecting bone marrow, umbilical cord blood or peripheral blood from an animal and subjecting it to, for example, density gradient centrifugation to extract the fraction.

In one aspect, the mononuclear cell fraction is a bone marrow, peripheral blood or cord blood derived mononuclear cell fraction.

In one embodiment, the bioenriched cultured mononuclear cell fraction is a cell population enriched in vascular endothelial progenitor cells or anti-inflammatory and tolerance-inducing cells. This cell population includes vascular endothelial progenitor cells, anti-inflammatory macrophages, T lymphocyte subsets, and regulatory T cells.

"Vascular endothelial progenitor cells (EPC) are, among mononuclear cell fractions, particularly CD34-positive and/or CD133-positive cells. Also, differentiated EPC colony-forming cells are included. Vascular endothelial progenitor cells are known to be cells that can differentiate into blood vessels and are thought to be able to repair or replenish damaged blood vessels.

"Anti-inflammatory/tolerogenic cells" include M2 macrophages converted to anti-inflammatory properties, T lymphocyte subsets, and regulatory T cells.

"Anti-inflammatory macrophages" are M2 macrophages.

"Quality and Quantity (QQ)" refers to ex vivo culture of stem cells collected from the body in a serum-free medium containing factors such as stem cell growth factors and interleukins for a certain period of time. , to modify and amplify. "Modify and expand" means to increase the number and/or function of stem cells. For example, US Pat. No. 6,200,401 discloses mononuclear cells from bone marrow, cord blood or peripheral blood in serum-free medium containing stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor. A cell group obtained by culturing is described. The bioenriched cultured mononuclear cell fraction is preferably characterized by being enriched in vascular endothelial progenitor cells as well as anti-inflammatory and immune tolerance-inducing cells. In one aspect, it also contains anti-inflammatory M2 macrophages, T lymphocyte subsets and regulatory T cells. The bioexpanded cultured mononuclear cell fraction preferably has angiogenic, anti-fibrotic and/or anti-inflammatory effects.

The method for in vitro expansion culture of the nucleocyte fraction is not particularly limited, and any known method can be used. For example, Non-Patent Documents 19 and 27 disclose specific methods for in vitro expansion and culturing of vascular endothelial progenitor cells. For example, media supplemented with VEGF, SCF, Flt-3 ligand, TPO, IL-6 and 1% antibiotics can be used. In the examples herein, 1 week in StemSpan medium supplemented with 50 ng/ml VEGF, 100 ng/ml SCF, 100 ng/ml Flt-3 ligand, 20 ng/ml TPO, 20 ng/ml IL-6 and 1% antibiotics. cultured.

Alternatively, the method described in International Publication WO2006/090882 can also be used. The method comprises incubating blood vascular endothelial progenitor cells in a serum-free medium containing one or more factors selected from the group consisting of stem cell growth factor, interleukin 6, FMS-like tyrosine kinase 3 and thrombopoietin.

A “mesenchymal stem cell” is a somatic stem cell derived from a mesodermal tissue (mesenchyme) and has the ability to differentiate into cells belonging to mesenchymal cells. Each tissue to be collected is called adipose-derived mesenchymal stem cell (ASC), bone marrow-derived mesenchymal stem cell, or the like. Mesenchymal stem cells are included in stromal cells. For example, bone marrow stromal cells are induced to differentiate into mesenchymal cells (osteocytes, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.).

In one aspect, the mesenchymal stem cells are adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, or cord blood-derived mesenchymal stem cells.

The composition amplifies the effects of treatment with mesenchymal stem cells through the action of ex vivo expanded cultured mononuclear cell fractions.

"Treatment with mesenchymal stem cells" is not particularly limited as long as it is a disease or condition that is effective for treatment with mesenchymal stem cells. For example, mesenchymal cells can be used for a wide range of treatments, including application to regenerative medicine such as reconstruction of bones, blood vessels, and myocardium.

In one aspect, mesenchymal stem cell treatment is a treatment whose effects are enhanced by angiogenesis, anti-inflammatory action, anti-fibrosis, etc. by ex vivo expanded cultured mononuclear cell fractions.

In one aspect, treatment with mesenchymal stem cells is used for tissue transplantation, tissue regeneration, treatment of ischemic diseases, treatment of neurodegenerative diseases, treatment of heart diseases, treatment of malignant tumors, treatment of inflammatory diseases and treatment of immune diseases. is selected from the group consisting of In one aspect, treatment with mesenchymal stem cells consists of tissue regeneration, ischemic disease treatment, neurodegenerative disease treatment, heart disease treatment, malignant tumor treatment, inflammatory disease treatment and immune disease treatment. selected from the group. In one aspect, the treatment with mesenchymal stem cells is selected from the group consisting of ischemic disease treatment, neurodegenerative disease treatment, cardiac disease treatment, malignant tumor treatment, inflammatory disease treatment and immune disease treatment. be done. In one aspect, treatment with mesenchymal stem cells is treatment of ischemic disease.

The type of tissue in "tissue transplantation, tissue regeneration" is not particularly limited as long as it is a living tissue that can be transplanted/regenerated. Examples include fat, bone marrow, heart, kidney, and the like. It is performed for the purpose of fat transplantation such as breast reconstruction after breast cancer surgery, bone marrow transplantation (treatment of leukemia, lymphoma, myeloma, etc.).

The tissue regeneration includes, for example, treatment for skeletal muscle wounds, skin wounds, tissue regeneration after surgery for prostate cancer, osteoarthritis, lumbar intervertebral disc deformation, peripheral nerve injury, and the like.

An “ischemic disease” is a disease caused by tissue damage such as cell degeneration, atrophy, and fibrosis due to decreased blood flow in tissues. . Ischemia is roughly classified into obstructive ischemia, compression ischemia, convulsive ischemia, and compensatory ischemia, depending on the cause. Persistent ischemia causes cell degeneration, atrophy, and fibrosis. "Ischemic disease" includes leg ischemia, ischemic ulcer, ischemic heart disease, cerebral infarction, and the like. "Cerebral infarction" (or cerebral malacia) is caused by occlusion or stenosis of the arteries that supply the brain, resulting in cerebral ischemia, resulting in necrosis or near-necrosis of brain tissue due to lack of oxygen or nutrients. It is classified into cerebral thrombosis and cerebral embolism. “Lower limb ischemia” is a disease in which the arteries that supply blood to the legs become narrowed or blocked. and, in the worst case, lower extremity amputation may be required.

A “neurodegenerative disease” is a disease in which specific nerve cell groups in the central nervous system gradually die, and is a kind of cranial nerve disease. Including amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, progressive supranuclear palsy, Huntington's disease, multiple system atrophy, spinocerebellar degeneration, spinal cord injury and the like. “Spinal cord injury” is a pathological condition in which the spinal cord is damaged mainly by applying a strong external force to the spinal column, resulting in damage to the spinal cord. Spinal cord injury is said to be difficult to treat fundamentally at this stage, but the application of regenerative medicine using stem cells is being considered.

"Heart disease" is a general term for heart diseases, and includes heart failure, endocarditis, valvular heart disease, pericarditis, congenital heart disease, and other diseases (myocarditis, myocardial infarction, etc.).

"Inflammatory disease" is a general term for diseases that cause symptoms due to abnormalities such as tissue damage due to some cause. Inflammatory diseases include Crohn's disease, cirrhosis, hepatitis, ulcerative colitis, inflammatory bowel disease, and the like.

An "immune disease" (also called an "autoimmune disease") is one in which the immune system, which is responsible for recognizing and eliminating foreign substances, overreacts and attacks its own normal cells and tissues. It is a general term for diseases caused by the breakdown of immune tolerance, which causes symptoms when it is closed. Autoimmune diseases can be divided into two types: systemic autoimmune diseases that affect the whole body and organ-specific diseases that affect only specific organs. For example, collagen diseases represented by rheumatoid arthritis and systemic lupus erythematosus (SLE) are systemic autoimmune diseases. Immunological diseases also include graft versus host disease (GVHD), multiple sclerosis, and the like.

In one aspect, treatment with mesenchymal stem cells is used for fat transplantation, bone marrow transplantation, treatment of skeletal muscle wounds, treatment of skin wounds, tissue regeneration after prostate cancer surgery, treatment of osteoarthritis, treatment of lumbar disc deformity, Treatment of peripheral nerve injury, treatment of lower extremity ischemia, treatment of ischemic ulcer, treatment of ischemic heart disease, treatment of cerebral infarction, treatment of amyotrophic lateral sclerosis (ALS), treatment of Parkinson's disease, Alzheimer's disease treatment of progressive supranuclear palsy, treatment of Huntington's disease, treatment of multiple system atrophy, treatment of spinocerebellar degeneration, treatment of spinal cord injury, treatment of heart failure, treatment of endocarditis, valvular heart disease treatment of pericarditis, treatment of congenital heart disease, treatment of myocarditis, treatment of myocardial infarction, treatment of Crohn's disease, treatment of liver cirrhosis, treatment of hepatitis, treatment of ulcerative colitis, inflammatory bowel disease treatment of collagen disease, treatment of graft versus host disease (GVHD) or treatment of multiple sclerosis.

In one aspect, treatment with mesenchymal stem cells is used for treating skeletal muscle wounds, treating skin wounds, tissue regeneration after prostate cancer surgery, treating osteoarthritis, treating lumbar disc deformity, treating peripheral nerve injury, Treatment of lower extremity ischemia, treatment of ischemic ulcer, treatment of ischemic heart disease, treatment of cerebral infarction, treatment of amyotrophic lateral sclerosis (ALS), treatment of Parkinson's disease, treatment of Alzheimer's disease, progressive nucleus Treatment of supraparesis, Treatment of Huntington's disease, Treatment of multiple system atrophy, Treatment of spinocerebellar degeneration, Treatment of spinal cord injury, Treatment of heart failure, Treatment of endocarditis, Treatment of heart valve disease, Pericarditis treatment, congenital heart disease treatment, myocarditis treatment, myocardial infarction treatment, Crohn's disease treatment, liver cirrhosis treatment, hepatitis treatment, ulcerative colitis treatment, inflammatory bowel disease treatment, collagen disease treatment treatment, treatment of graft versus host disease (GVHD) or treatment of multiple sclerosis.

In one aspect, treatment with mesenchymal stem cells is used to treat lower extremity ischemia, to treat ischemic ulcers, to treat ischemic heart disease, to treat stroke, to treat amyotrophic lateral sclerosis (ALS), to treat Parkinson's disease. disease treatment, Alzheimer's disease treatment, progressive supranuclear palsy treatment, Huntington's disease treatment, multiple system atrophy treatment, spinocerebellar degeneration treatment, spinal cord injury treatment, heart failure treatment, endocarditis heart valve disease treatment pericarditis treatment congenital heart disease treatment myocarditis treatment myocardial infarction treatment Crohn's disease treatment liver cirrhosis treatment hepatitis treatment ulcerative colitis treatment treatment of inflammatory bowel disease, treatment of collagen disease, treatment of graft versus host disease (GVHD) or treatment of multiple sclerosis.

Alternatively, the composition is for amplifying the effects of treatment with mesenchymal stem cells. The compositions are used with mesenchymal stem cells or compositions comprising mesenchymal cells.

"Used" means applied to a subject, such as a human, in an appropriate manner according to the treatment. In one embodiment, the composition is preferably injected into the body site to be treated with mesenchymal stem cells (e.g., intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intracerebroventricular administration, administration into tissue). apply).

The phrase “with treatment with mesenchymal stem cells” means that the treatment with mesenchymal stem cells may be performed simultaneously with the mesenchymal stem cells, or the mesenchymal stem cells may It may be applied in vivo immediately before or after treatment with mesenchymal stem cells. Any use to the extent that it affects the effect of treatment with mesenchymal stem cells is included in "used in conjunction with treatment with mesenchymal stem cells."

A “composition containing mesenchymal cells” may be obtained from a living organism or artificially prepared, as long as it is a composition containing mesenchymal cells. An effective amount of mesenchymal cells may be contained so that the mesenchymal cells are effective for treatment. In one aspect, the composition comprising mesenchymal cells is a stromal vascular fraction (SVF) (also referred to as "stromal vascular cell population"). The stromal vascular cell fraction is a population of cells obtained by collagenase treatment of adipose tissue. The stromal vascular cell fraction is generally a heterogeneous cell group, and includes adipose tissue-derived cell groups in addition to blood-derived cells. Among the adipose tissue-derived cells, adipose stromal cells are included. , which contains adipose-derived mesenchymal stem cells (ASCs).

Compositions include any subject in need of treatment with mesenchymal cells. Including, without limitation, mammals such as humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, rodents (mice, rats, etc.). Preferably, the subject is human.

The composition of the present invention can be used by medical professionals (doctors, veterinarians) according to the appropriate dosage and administration, depending on the site to be treated, the severity of the disease/condition, and the condition of the subject (age, weight, height, medical history, physical condition, etc.). can be applied (used) to a subject in For example, when administered to humans, in one aspect, the total cell number of the ex vivo expanded cultured mononuclear cell fraction contained in the composition ranges from 1×10 ⁵ to 5×10 ¹¹ . The composition may be administered once or multiple times.

2. Effects of Compositions The compositions of the present invention amplify the effects of treatment with mesenchymal stem cells. By amplifying the effect is meant that the effect of treatment with mesenchymal stem cells is increased when the composition is applied with the mesenchymal stem cells as compared to when the mesenchymal stem cells alone are applied to the subject. .

In one aspect, the treatment with mesenchymal stem cells is, for example, transplantation,
(i) Increased graft survival:
(ii) increase graft survival;
(iii) inhibit fibrosis of the graft; (iv) improve blood flow in the graft; or (v) reduce graft-induced inflammation.

Preferably two, three, four or five (all) of the above effects are obtained.

In one aspect, the mesenchymal stem cell treatment is fat grafting,
(i) Increased survival of transplanted adipose tissue:
(ii) increase the survival rate of grafted fat tissue;
At least one effect of (iii) inhibiting fibrosis of the grafted adipose tissue graft, (iv) improving blood flow in the grafted adipose tissue, or (v) reducing inflammation due to grafting is obtained.

Preferably two, three, four or five (all) of the above effects are obtained.

In one aspect, the effect of treatment with mesenchymal stem cells, e.g., (i)-( At least one effect of v) is increased by 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.5-fold or more, 1.8-fold or more, 2-fold or more.

In one aspect, the treatment with mesenchymal stem cells is a treatment for ischemia,
(vi) improves blood flow:
9. The composition of claim 8, wherein at least one effect of (vii) improving function; or (viii) improving tissue damage is obtained.

Preferably, two or three (all) of the above effects are obtained.

In one aspect, the treatment with mesenchymal stem cells is treatment of lower extremity ischemia,
(vi) Improves lower extremity blood flow:
9. The composition of claim 8, wherein at least one effect of (vii) improving function; or (viii) improving tissue damage such as ischemic ulcer is obtained.

Preferably, two or three (all) of the above effects are obtained.

3. Methods for Amplifying the Effects of Mesenchymal Stem Cell Treatment The present invention also provides methods for amplifying the effects of mesenchymal stem cell treatment. The methods of the invention involve using a composition comprising an in vitro expanded cultured mononuclear cell fraction with mesenchymal stem cells or a composition comprising mesenchymal cells.

The definitions of "in vitro expanded cultured", "composition comprising a mononuclear cell fraction", "mesenchymal stem cells or a composition comprising mesenchymal cells" are defined as "1. Compositions for Amplifying Effects”.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. A person skilled in the art can easily make modifications and changes to the present invention based on the description of this specification, and they are included in the technical scope of the present invention.

Example 1 Fat Grafting This example confirms the amplifying effect of a composition comprising ex vivo expanded cultured EPCs on the efficacy of adipose-derived mesenchymal stem cells in fat grafting.

MATERIALS AND METHODS The following materials and methods were used in the examples herein unless otherwise stated.

(1) Animals Wild-type (WT) C57BL/6J mice and green fluorescent protein-expressing (GFP ⁺ ) C57BL/6-Tg (CAG-EGFP) mice aged 8-10 weeks were purchased from CLEA Japan. All experiments were approved by the Juntendo University School of Medicine Animal Care and Use Committee and were performed in accordance with the institutional animal care guidelines.

(2) Collection of adipose tissue and preparation of adipose-derived mesenchymal stem cells (ASC) WT adipose donor mice (wild-type (WT) C57BL/6J mice) were sacrificed by cervical dislocation. Inguinal adipose tissue was collected after an abdominal incision was made to remove the inguinal lymph nodes.
Adipose tissue for ASC isolation was excised from GFP ⁺ mice (green fluorescent protein-expressing (GFP ⁺ ) C57BL/6-Tg(CAG-EGFP) mice) in the same manner and washed in 0.12% type I collagenase. Digested. DMEM supplemented with 10% FBS and 1% antibiotic (penicillin-streptomycin solution (x100)) (0.85% saline solution containing 10,000 units/mL penicillin G and 10,000 μg/mL streptomycin sulfate) Collagenase activity was inhibited by stromal medium consisting of After straining and centrifugation, cells were collected as stromal vascular fraction (SVF) and cultured in stromal medium. Third passage adherent cells were collected and ASCs were characterized by morphology and by measuring surface marker expression on cells on flow cytometry (FACSCalibur, BD Biosciences). Flow cytometry showed that a CD73 ⁺ CD90.2 ⁺ CD105 ⁺ CD11b ⁻ CD31 ⁻ CD34 ⁻ CD45 ⁻ population was obtained.

(3) Preparation of KSL and QQKSL Cells c-Kit ⁺ Sca-1 ⁺ Lin ⁻ cells (KSL cells) were prepared as EPCs according to our previous report (Non-Patent Document 27).

Briefly, bone marrow contents were obtained from GFP ⁺ mice (green fluorescent protein-expressing (GFP ⁺ ) C57BL/6-Tg(CAG-EGFP) mice) and lineage-negative (Lin ⁻ ) cells were transfected with lineage-positive biotin conjugates. Lineage-positive cells were prepared by depleting lineage-positive cells on a MACS system (Miltenyi Biotec) using a cocktail of gated antibodies and anti-biotin microbeads (Miltenyi Biotec).

Lineage negative (Lin ⁻ ) cells were incubated with fluorescently labeled anti-Ly-6A/E (Sca-1) and anti-CD117 (c-Kit) antibodies. The following antibodies were used for fluorescence-activated cell sorting.

Double positive cells were then sorted as KSL cells on a FACSAria (BD Biosciences).

For QQ cultures, KSL cells were treated with 50 ng/ml VEGF, 100 ng/ml SCF, 100 ng/ml Flt-3 ligand, 20 ng/ml TPO, 20 ng/ml IL-6 and 1% antibiotics as previously reported. (Penicillin-streptomycin solution (×100)) and cultured in StemSpan medium for 1 week (Non-Patent Documents 19, 27). KSL and QQKSL cells were evaluated for angiogenic potential by colony formation assay.

(4) Colony Formation Assay KSL and QQKSL cells were assayed for angiogenic potential by EPC colony formation assay (EPC-CFA). 15% FBS on a 35 mm dish, 50 ng/ml VEGF, 50 ng/ml bFGF, 100 ng/ml SCF, 50 ng/ml IGF, 50 ng/ml EGF, 20 ng/ml IL-3 (all from PeproTech, Rocky Hill, New Jersey, USA). ), 500 to MethoCult™ SF M3236 solid culture medium (Stem Cell Technologies, Vancouver, British Columbia, Canada) supplemented with 2 IU/ml heparin, and 1% antibiotics (penicillin-streptomycin solution (x100)). Cells were seeded. After 7 days of culture at 37° C. in an atmosphere adjusted to saturated humidity and 5% CO ₂ , primitive EPC colony forming units and definitive EPC colony forming units (pEPC-CFU and dEPC- CFU) can be identified (Non-Patent Document 29). Colonies were observed and counted on an inverted microscope.

(5) Fat graft and sample collection, as described in "(2) Collection of adipose tissue and preparation of adipose-derived mesenchymal stem cells (ASC)", "(3) Preparation of KSL and QQKSL cells", fat graft, ASC and QQKSL were prepared and injected into wild-type (WT) C57BL/6J mice (Fig. 1).
Control: fat graft + PBS
QQKSL+ASC::fat graft+QQKSL+ASC
ASC: fat graft + ASC

Five weeks later, recipient mice (N=10) were sacrificed by cardiac perfusion fixation under anesthesia. Implanted samples were separated from endogenous tissue and collected. Fat graft survival was estimated by dividing the explant weight by the injected tissue weight.

(6) Histology PFA-fixed paraffin-embedded sections (4 μm) were stained with hematoxylin and eosin (HE) to examine adipose tissue integrity. Specifically, adipose tissue integrity was assessed on 2HE-stained full cross-sections per graft. High-resolution images were taken with a Keyence BZ-9000 Microscope (Keyence Japan Co, Ltd, Osaka, Japan). Intact adipocytes are identified as relatively large cells marked by a single unfragmented central adipose inclusion that is empty as a result of the staining reagent. They exhibit a characteristic signet ring appearance with a flattened nucleus along the edge of the cell. A region consisting of well-organized cellular tissue was drawn on a Keyence BZ-II Analyzer (Keyence Japan Co, Ltd, Osaka, Japan). Adipose tissue integrity was calculated by calculating the total surface area of the drawn area and dividing it by the total surface area of the tissue (percentage).

In addition, fibrosis was assessed by staining with Heidenhain's azan modification of Mallory's tricolor connective tissue stain. Specifically, the azan stain consists of orange G, azocarmine GX and aniline blue, which allows recognition of blue-stained collagen fibers. The Keyence BZ-II Analyzer measured the area composed of collagen fibers in two complete tissue cross-sections per implant and divided this by the total tissue surface area per cross-section to give the percent fibrosis. Calculated.

(7) Immunohistochemistry Sections were subjected to antigen retrieval at 95° C. in citrate buffer (pH 6.0), blocked for endogenous peroxidase with 0.3% H ₂ O ₂ and treated with 5% normal goat serum. blocked. Specimens were incubated with primary antibodies against CD68 (Abcam ab125212, 1:300) and CD31 (Abcam ab28364, 1:50). Biotinylated polyclonal IgG was used as secondary antibody, reacted with HRP-labeled streptavidin and visualized with 3,3'-diaminobenzidine (DAB). Double immunohistochemistry was performed with anti-GFP (Frontier Institute Af2020, 1:200) and anti-CD31 antibodies (Abcam ab56299, 1:50). DAB served as a chromogenic substrate for GFP and CD31 was detected with the Vector Blue AP substrate kit (Vector Laboratories SK-5300). Grafts in the QQKSL+ASC group were excluded from dual GFP-CD31 immunohistochemistry because both cell populations expressed GFP and were indistinguishable in our model.

High-resolution images were taken with a Keyence BZ-9000 Microscope (Keyence Japan Co, Ltd, Osaka, Japan) and processed with a BZ-II Analyzer. Sections were compared to their negative controls lacking primary antibody.

Each live or dead adipocyte surrounded by 2 or more CD68-positive macrophages was counted as a unit of local extension. Massive macrophages organized into crown-like structures, histologically characteristic of aggregates of moribund adipocyte-rich macrophages, were also counted as single units. Sixty random images were analyzed at 40× magnification per implant, corresponding to a total observed tissue surface of 6 mm ² per implant. Results were expressed as mean units of local inflammation per mm ² of tissue. Blood vessel density was assessed by manual quantification of blood vessels on the surface of the tissue. Blood vessels were recognized as lumenal structures surrounded by endothelial cells labeled by anti-CD31 antibodies. At 40x magnification, 250-500 sections were analyzed in each observed graft. This covers the complete cross-section of the implant. Results were presented as mean number of vessels per ^mm2 of tissue.

(8) Quantitative polymerase chain reaction (qPCR)
RNA was purified from uncultured KSL cells, QQ cultured KSL cells and ASCs and cDNA was synthesized to analyze TGF-β, SMAD3 and Col1A1 expression by qPCR in comparative CT experiments. RNA was extracted from the grafts followed by cDNA synthesis and qPCR. Comparative CT experiments were performed to determine gene expression of adiponectin, FABP4, PDGF, BAX and BCL-2.

Specifically, non-cultured KSL cells, QQ-cultured KSL cells and ASCs at passage 3 were cultured using spin technology in an RNeasy Mini Kit (Qiagen®, Valencia, California, USA) according to the manufacturer's instructions. RNA was purified from Total RNA was measured using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA). Quantitative polymerase chain reaction (qPCR) was performed by a singleplex two-step process. First, cDNA was synthesized from purified RNA using the Sensiscript Reverse Transcriptase Kit (Qiagen®) in a Bio-Rad T100 Thermal Cycler; (USA) and gene expression was detected by using the PowerUp SYBR Green Master Mix (Applied Biosystems, Carlsbad, California, USA). SYBR Green I dye was used, which binds to accumulated double-stranded DNA and produces detectable fluorescence. After an initial 2 min denaturation (95° C.), samples were subjected to 40 cycles of denaturation (1 sec, 95° C.) and annealing-extension (30 sec, 60° C.). A comparative CT experiment (△△CT method) was performed, and transforming growth factor (TGF-β), contraction of Sma and Mad (Mothers against decapentaplegic) in uncultured KSL cells, QQ cultured KSL cells and ASCs ) examined the expression levels of homolog 3) and collagen type 1 α1 (Col1A1),

Primer sequences are listed in the table below.

Data were normalized to the ribosomal RNA of a housekeeping gene, glycerolaldehyde-3'-phosphate dehydrogenase (GAPDH), as an endogenous control with constant expression in different tissues and cells. Explants for RNA extraction and quantitative PCR were stored at −30° C. in RNase inhibitor TRIzol® (Life Technologies, Invitrogen, Carlsbad, California, USA). After the homogenization and centrifugation steps of the grafts, chloroform was added to purify the nucleic acids and remove proteins, followed by propanol and then ethanol to precipitate the nucleic acids. Total RNA was measured using a NanoDrop ND-1000 spectrophotometer. This was then used to perform cDNA synthesis with the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Vilnius, Lithuania) in a Bio-Rad T100 Thermal Cycler. Expression levels of adiponectin, fatty acid binding protein (FABP) 4, platelet-derived growth factor (PDGF), BCL-2-associated X protein (BAX) and B-cell lymphoma 2 (BCL-2) in the five groups studied were described above. Measured using StepOnePlus and PowerUP SYBR Green Master Mix. The expression of all genes in different samples was measured three times each with nearly identical results. The amount of normalized target in the experimental group was compared to the amount of normalized target in the control group. Results are expressed as fold change compared to the control group.

(9) Statistics

Experimental data are presented as mean±SEM. Data were analyzed for equal variances and then pairwise compared with a two-tailed t-test. Differences were considered statistically significant if p<0.05.

Effect of QQ culture on KSL cell proliferation Expansion is very important to obtain enough cells before EPC therapy. In this example, the quantitative effect of QQ culture on KSL cell proliferation was determined.

KSL cell numbers increased by a factor of 6.2±3.1×10 ² after 1 week. A colony forming assay was performed to study angiogenic potential. Compared to uncultured KSL cells, QQKSL cells presented more dEPC-CFU (9.9±1.3 vs 3.0±0.6/plate; p<0.01). This result indicates that QQKSL contains spindle-shaped EPCs with high potential for vascular endothelial lineage differentiation, cell adhesion and tubular structure formation and is a highly vasogenic population (17). , 27-29).

This example showed that QQ culture stimulates the proliferation and angiogenic potential of KSL cells.

Effects on Fat Graft Survival Tissue loss is a major problem in fat grafting. In this example, fat graft survival is calculated by calculating fat graft weight retention, specifically by dividing the explant weight (maintained for 5 weeks) by the injected tissue weight. and examined the effect of QQKSL+ASC or ASC injection on fat graft survival.

Five weeks after fat grafting, there was a significant improvement in the QQKSL+ASC group (57.8±4.0%; p<0.05) compared to the control (40.3±4.8%) (Fig. 2). ASC alone (51.1±4.8%) did not significantly increase fat graft weight, suggesting that QQ-cultured KSL cells were responsible for the higher debris weight.

This example shows that QQKSL+ASC cells improve fat graft survival.

Effect on Fat Graft Integrity In this example, the effect of QQKSL+ASC cells on fat graft integrity was investigated.

The clinical outcome of fat grafts is determined not only by weight retention, but also by tissue quality. In this example, the effect of QQKSL+ASC, or transplantation of ASC, on fat graft quality (integrity) was investigated. The integrity of the fat graft was evaluated by the method described in "(5) Histology".

The percentage of adipose tissue meeting histological integrity requirements 5 weeks after transplantation is shown in FIG. After 5 weeks, the percentage of adipose tissue meeting histological integrity requirements was 22.5±2.4% in the QQKSL+ASC group compared to controls (16.9±3.0%; p<0.05). ) increased significantly only in On the other hand, it remained unchanged in the ASC group (16.7±1.7%).

This example shows that QQKSL+ASC improves adipose tissue integrity of fat grafts.

Expression of Markers Characteristic of Viable Adipocytes As described above in "Effect on fat graft integrity", the integrity of adipose tissue differs depending on the presence or absence of QQKSL+ASC and ASC injected during fat grafting. In this example, gene expression of adiponectin and FABP4 was examined in adipocytes of live grafts of fat grafts. Both adiponectin and FABP4 are characteristic markers of viable adipocytes. Expression of each gene was performed by the method described in "(7) Quantitative polymerase chain reaction (qPCR)".

Five weeks after fat grafting, adipocyte-specific adiponectin showed higher expression in the QQKSL+ASC group (2.3-fold compared to control; p<0.05).

Impact on Tumorigenicity In general terms, transplanted cells may be tumorigenic. In this example, the effect of QQKSL+ASC or ASC injection on the tumorigenicity of transplanted cells in fat grafts was investigated. Specifically, the BAX/BCL-2 ratio, which determines the apoptosis induction of cells, was calculated. BCL-2 overexpression or decreased expression of BAX has been reported to lead to cell accumulation and to be found in malignant processes. The BAX/BCL-2 ratios in the two experimental groups were not significantly different from the control group. That is, the presence or absence of QQKSL+ASCASC injection did not change (increase) tumorigenicity.

Effect on Fibrosis Hypoxia induces fibrosis in adipose tissue. This example investigated the effect of QQKSL+ASC, or injection of ASC, on fibrosis in fat grafts, specifically whether it could prevent fibrotic transformation of the graft.

The results are shown in FIG. Five weeks after fat grafting, the percentage of fibrosis increased in the ASC group (28.2±3.2%; p<0.01) compared to controls (21.9±2.5%; p<0.01). .01) and the QQKSL+ASC group (26.2±2.5%; p<0.01).

This example shows that QQKSL+ASC has the effect of suppressing the fibrotic transformation of the transplanted tissue against the fibrosis of the ASC transplant in the fat graft.

Effects on Inflammation In this example, CD68 immunohistochemistry was performed to examine the inflammatory status of fat grafts upon injection of QQKSL+ASC or ASC.

The results are shown in FIG. Five weeks after fat grafting, minimal local inflammatory units were detected in the QQKSL+ASC group (22.4±1.8/mm ² ). ASC group (28.0±1.8/mm ² ) and control (42.8±4.3/mm ² ). These data indicate that the QQKSL+ASC combination has the strongest anti-inflammatory effect on grafts.

Effect on Angiogenesis In this example, the effect of injection of QQKSL+ASC or ASC on angiogenesis in fat grafts was investigated. CD31 immunohistochemistry was used to determine whether post-implantation revascularization of grafts was successful.

The results are shown in FIG. These data indicate that QQKSL cells containing ASC have a strong angiogenic (vasogenic) effect.

Example 2 Lower Limb Ischemia In this example, the enhancing effect of a composition containing an in vitro expanded cultured mononuclear cell fraction on the ameliorating effect of bone marrow-derived mesenchymal stem cells in lower limb ischemia is confirmed.

(1) Preparation of human MNC and MNC-QQ cells EPC-containing mononuclear cells (MNC) were prepared from peripheral blood obtained from a healthy subject by a conventional blood collection method using density gradient centrifugation.

For QQ cultures, MNCs were mixed with 50 ng/ml VEGF, 100 ng/ml SCF, 100 ng/ml Flt-3 ligand, 20 ng/ml TPO, 20 ng/ml IL-6 and 1% antibiotics (penicillin-streptomycin solution (x100) ) for 1 week in Stemline II medium (Non-Patent Document 30). MNC and MNC-QQ cells were assessed for angiogenic potential by colony formation assay

(2) Culture of MSCs Human mesenchymal stem cells (MSCs) were purchased from LONZA (Basel, Switzerland) and cultured using Mesenchymal Stem Cell Growth Medium BulletKit (trademark) medium according to the culture method provided by LONZA. , up to the 5th generation were used.

(3) Lower Limb Ischemia Model Mouse Eight-week-old BALB/cAJcl-nu/nu mice (nude mice) used to prepare lower limb ischemia model mice were purchased from Clea Japan, Inc. (Tokyo, Japan). All experiments were approved by the Juntendo University School of Medicine Animal Care and Use Committee and were performed in accordance with the institutional animal care guidelines.

The femoral artery and vein were ligated and cauterized from the left inguinal region of a nude mouse to prepare an ischemic lower limb. After that, the blood flow is measured by laser Doppler over time, and the degree of improvement in blood flow is used as the test endpoint, and the tissue of the ischemic site (lower leg) is sampled.

Table 4 shows specific test implementation procedures.

Effect of MNC-QQ cells on improvement of lower limb ischemia In a blood flow test by laser Doppler immediately after lower limb ischemia, the blood flow in the ischemic limb decreased to 15% when compared to 100% in the healthy contralateral limb. There was no difference between groups. When the three groups were compared on day 10 after transplantation, blood flow in the ischemic limb in the saline group was 26% compared to that in the healthy contralateral limb, showing no significant improvement, whereas the MSC group showed no significant improvement. A constant improvement of 47% was observed in , and a significant improvement of 83% was observed in the MSC+MNC-QQ group compared to the other groups (Fig. 7).

Further, in macroscopic findings 14 days after cell transplantation, necrosis of the toes was confirmed in the physiological saline group, and necrosis of the fingertips was confirmed in the MSC group although it was improved compared to the physiological saline group. On the other hand, no necrosis was observed in the MSC+MNC-QQ group, and macroscopically effective and clear synergistic effects were shown. (Fig. 8).

This example demonstrated that MNC-QQ cells have a synergistic effect on blood flow improvement by MSCs and ameliorate ischemia.
[Sequence list]

Claims (12)

1. A composition for amplifying the effect of treatment with mesenchymal stem cells comprising an in vitro expanded cultured mononuclear cell fraction, comprising:
The in vitro expanded cultured mononuclear cell fraction is cultured in a medium containing one or more factors selected from the group consisting of stem cell growth factor, interleukin 6, FMS-like tyrosine kinase 3 and thrombopoietin. is a nucleocytic fraction, and
Treatment with said mesenchymal stem cells is fat transplantation, treatment of skeletal muscle wounds, treatment of skin wounds, tissue regeneration after prostate cancer surgery, treatment of osteoarthritis, treatment of lower extremity ischemia, treatment of ischemic ulcers, Treatment of ischemic heart disease, treatment of cerebral infarction, treatment of heart failure, treatment of endocarditis, treatment of heart valve disease, treatment of pericarditis, treatment of congenital heart disease, treatment of myocarditis, treatment of myocardial infarction treatment of Crohn's disease, treatment of ulcerative colitis, treatment of inflammatory bowel disease, treatment of connective tissue disease, treatment of graft versus host disease (GVHD) and treatment of multiple sclerosis. selected from the group
said composition. 2. The composition of claim 1, for use with a composition comprising mesenchymal stem cells. 3. The composition of claim 1 or 2, wherein the mononuclear cell fraction is a bone marrow, peripheral blood or cord blood derived mononuclear cell fraction. 4. The method according to any one of claims 1 to 3, wherein the bioenriched cultured mononuclear cell fraction is vascular endothelial progenitor cells, M2 macrophages, a T lymphocyte subset, or a cell group enriched with regulatory T cells. The described composition. 5. The mesenchymal stem cell of any one of claims 1-4, wherein the mesenchymal stem cell is an adipose-derived mesenchymal stem cell, a bone marrow-derived mesenchymal stem cell, a peripheral blood-derived mesenchymal stem cell, or a cord blood-derived mesenchymal stem cell. The described composition. The in vitro expanded cultured mononuclear cell fraction is a mononuclear cell fraction cultured in a medium containing one or more factors selected from the group consisting of FMS-like tyrosine kinase 3 and thrombopoietin. The composition according to any one of Items 1-5. Claim 1-5, wherein the ex vivo expanded cultured mononuclear cell fraction is a mononuclear cell fraction cultured in a medium containing stem cell growth factor, interleukin 6, FMS-like tyrosine kinase 3 and thrombopoietin. The composition according to any one of Claims 1 to 3. 8. The composition of any one of claims 1-7, wherein the in vitro expanded cultured mononuclear cell fraction further comprises VEGF. The composition according to any one of claims 1-8, wherein said medium is a serum-free medium. treatment with mesenchymal stem cells is fat grafting or treatment of graft-versus-host disease,
(i) Increased graft survival:
(ii) increase graft survival;
10. The composition of claim 9, which has at least one effect of (iii) inhibiting fibrosis of the graft, (iv) improving blood flow in the graft; or (v) reducing inflammation due to grafting. thing. wherein the treatment with mesenchymal stem cells is treatment of lower extremity ischemia, treatment of ischemic ulcers, or treatment of ischemic heart disease,
(vi) improves blood flow:
10. The composition of claim 9, wherein at least one effect of (vii) improving function; or (viii) improving tissue damage is obtained. A composition comprising an in vitro expanded cultured mononuclear cell fraction in combination with mesenchymal stem cells for use in a method for amplifying the effects of treatment with mesenchymal stem cells, comprising:
The in vitro expanded cultured mononuclear cell fraction is cultured in a medium containing one or more factors selected from the group consisting of stem cell growth factor, interleukin 6, FMS-like tyrosine kinase 3 and thrombopoietin. is a nucleocytic fraction, and
Treatment with said mesenchymal stem cells is fat transplantation, treatment of skeletal muscle wounds, treatment of skin wounds, tissue regeneration after prostate cancer surgery, treatment of osteoarthritis, treatment of lower extremity ischemia, treatment of ischemic ulcers, Treatment of ischemic heart disease, treatment of cerebral infarction, treatment of heart failure, treatment of endocarditis, treatment of heart valve disease, treatment of pericarditis, treatment of congenital heart disease, treatment of myocarditis, treatment of myocardial infarction treatment of Crohn's disease, treatment of ulcerative colitis, treatment of inflammatory bowel disease, treatment of connective tissue disease, treatment of graft versus host disease (GVHD) and treatment of multiple sclerosis. selected from the group
a combination of the foregoing;

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