WO2016158670A1 - Composition for vascular regeneration therapy, containing dedifferentiated fat cells as active ingredient - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
Definitions
- the present invention relates to a composition for revascularization therapy.
- the present invention relates to a composition for revascularization therapy comprising dedifferentiated fat cells (DFAT) as an active ingredient.
- DFAT dedifferentiated fat cells
- G-CSF mobilized peripheral blood mononuclear cells (iii) adipose tissue derived basal basal fraction (SVF) cells, (iv) peripheral blood mononuclear cells, (v) G-CSF mobilized CD34 positive cells, Similar angiogenesis therapy using many autologous cells such as vi) bone marrow-derived CD133 positive cells, (vii) cultured bone marrow mesenchymal stem cells (MSC) (viii) cultured adipose tissue-derived stem cells (ASC) has been performed.
- MSC bone marrow mesenchymal stem cells
- ASC cultured adipose tissue-derived stem cells
- the cells of (i) to (vi) are not cultured, but a large amount of tissue is required to obtain the required number of cells, which is highly invasive to patients. For this reason, the number of treatments is usually limited to only one, except that the invasiveness associated with collection is relatively low (iv).
- the cells (i) to (iv) are a diverse cell population, the transplantation effect and safety are not constant.
- the cells of (v) and (vi) can be improved in cell uniformity by performing a sorting operation using a stem cell marker antibody, but the complexity and reduction in preparation efficiency associated with antibody selection and preparation are reduced. There is a problem that the cost becomes high.
- the cells of (vii) are used after collecting dozens of ml of bone marrow fluid, adherently cultured and proliferated, and the cells of (viii) are subjected to enzyme treatment of adipose tissue and adherently cultured and proliferated in the same manner as (vii). Use from. Therefore, both (vii) and (viii) cells have the merit that they can be prepared in large quantities from a relatively small amount of tissue, but this method is a method of culturing and proliferating stem cells present in minute amounts in adult tissues. For this reason, contamination with other cells is unavoidable, and it is necessary to repeat the subculture several times in order to obtain the number of cells necessary for treatment. The number of treatments is usually only once.
- rat adipose tissue-derived progenitor cells and rat adipose tissue-derived mesenchymal stem cells are effective for angiogenesis (Patent Documents 1 and 2).
- the present inventors have succeeded for the first time in establishing dedifferentiated adipocytes (DFAT) as preadipocytes by inducing dedifferentiation of mature adipocytes derived from adipose tissue of non-human animals such as mice (patents) Reference 3), it has been shown that by inducing differentiation of DFAT, functions of osteoblasts, myoblasts, chondrocytes, nerve cells and the like can be obtained (Patent Document 4).
- DFAT dedifferentiated adipocytes
- Non-Patent Documents 1 to 3 mouse and rat DFAT has angiogenic ability.
- DFAT in mice and rats is difficult to increase to the number required for transplantation because of its low growth ability after subculture, and since transformation (immortalization) occurs by subculture, it is tumorigenic. The possibility increases and it cannot be safely transplanted. For these reasons, it has been considered that the same phenomenon occurs in human DFAT as in mouse and rat DFAT and is not suitable for treatment.
- the present invention is a cell for revascularization therapy that exhibits an excellent blood flow improvement effect compared to bone marrow MSC, ASC, etc., which are conventional treatment cells, and it is easy to obtain a sufficient amount necessary for transplantation.
- An object of the present invention is to provide a cell for revascularization therapy having a stable quality.
- a blood flow improving agent comprising human dedifferentiated adipocytes (human DFAT) as an active ingredient.
- An angiogenesis promoter containing human dedifferentiated adipocytes (human DFAT) as an active ingredient.
- a therapeutic agent for ischemic disease comprising human dedifferentiated fat cells (human DFAT) as an active ingredient.
- human dedifferentiated adipocytes prepared from human mature adipocytes have excellent angiogenic potential, and are effective cells for cell therapy for ischemic diseases such as peripheral artery disease (PAD). Clarified that it can be a source. Therefore, the present invention has the following effects. (1) Large-scale cell preparation facilities, growth factors, and antibody selection for increasing purity are not necessary. Moreover, since a large amount can be adjusted in a short culture period, the preparation cost can be kept low. (2) Regenerative medical donor cells with high purity can be efficiently collected from a small amount of adipose tissue that can be easily collected, and the obtained cells have high angiogenic ability. (3) Multiple treatments can be performed by collecting a single tissue, and treatment that exhibits a certain level of effectiveness without being affected by donor age or underlying disease is possible. (4) Unlike existing cell sources, it can show long-term efficacy against PAD.
- the karyotype analysis result of human DFAT is shown.
- A Photograph of chromosome structure by Giemsa staining.
- B The table
- the comprehensive analysis result of the cytokine secreted in a human DFAT culture supernatant is shown.
- Human DFAT (hDFAT), human ASC (hASC), human preadipocyte (hPreadipocyte), human fibroblast (hFibroblast), secreted cytokine (HGF, SDF-1, The quantification results of MCP-1, IL-6, VEGF, and Leptin) are shown. Figures in parentheses indicate donor age.
- the examination result of the angiogenesis ability of a human DFAT culture supernatant is shown.
- A Photograph showing the proliferation of vascular endothelial cells by human DFAT culture supernatant.
- B A graph showing the total length (total lumen length) and total area (total lumen area) of the vascular lumen formed in the well by the human DFAT culture supernatant and human bone marrow MMC culture supernatant.
- the examination result of the angiogenesis ability of the human DFAT culture supernatant by the difference in donor age is shown.
- Donors are 2-year-old male (h-DFAT2yM), 29-year-old male (h-DFAT29yM), 56-year-old female (h-dFAT56yF), 75-year-old female (h-DFAT75yF), 82-year-old female (h-DFAT82yF).
- the comparative examination result of the angiogenesis ability of the human DFAT culture supernatant (hDFAT) and human ASC culture supernatant (hASC) derived from the same donor is shown.
- A Photograph showing histological observation results.
- the effect of human DFAT transplantation in an immunodeficient mouse lower limb ischemia model is shown.
- (B) The graph which shows the ischemic rate of the ischemic limb with respect to a mouse
- the quantification result of secretory cytokine (HGF) in the culture supernatant of human DFAT (hDFAT) and human ASC (hASC) is shown.
- the quantification result of secretory cytokine (VEGF) in the culture supernatant of human DFAT (hDFAT) and human ASC (hASC) is shown.
- Human DFAT The method for adjusting human DFAT in the present invention may be performed with reference to, for example, Japanese Patent Application Laid-Open No. 2000-83656 made by the present inventors. That is, after adipose tissue such as human subcutaneous or internal organs is treated with collagenase, a single fraction consisting only of monocystic adipocytes is collected by filtration with a mesh having a diameter of 100 to 200 ⁇ m. Human dedifferentiated adipocytes (human DFAT) can be obtained by further subculturing fibroblast-like adipocytes produced by ceiling culture of these monocystic adipocytes.
- human DFAT can also be obtained by a method of adjusting cells that do not have lipid droplets originating from mature adipocytes by culturing mature adipocytes by methods other than the above-described ceiling culture. Confirmation that the obtained cells are human DFAT can be determined, for example, based on whether or not they have the following characteristics specific to human DFAT. Human DFAT has adhesiveness to plastic and exhibits multipotency into osteoblasts, adipocytes, chondrocytes and smooth muscle cells in vitro.
- human DFAT has lost expression of marker genes of mature adipocytes such as lipoprotein lipase and GLUT4, but expresses early differentiation marker genes of fat, bone and cartilage such as PPARg, RUNX2, and SOX9. Furthermore, human DFAT had a high cell proliferation ability, and the cell doubling time was about 65 hours for the second passage cell and about 48 hours for the tenth passage.
- Human DFAT can be obtained from 10 ml of aspirated fat or 1 g of adipose tissue by the above-mentioned preparation method, and 10 8 cells can be obtained in primary culture for about 2 weeks. This is about 25 times the preparation efficiency of cultured ASC. Therefore, it is possible to obtain cells of the order of 10 9 by subculturing 2-3 times. Since the number of cells used for a single cell therapy is about 10 8 , treatment can be performed about 10 times if the obtained cells are subdivided and cryopreserved by collecting adipose tissue once (about 10 ml). I know that there is.
- human DFAT can obtain homogeneous cells from primary culture.
- Primary cultured ASC contains smooth muscle cells (18.6%), vascular endothelial cells (2.7%) and monocytes (13.3%).
- the contamination rate of these cells in primary cultured human DFAT is It was very low as 0.1% or less.
- human DFAT is not only subcultured but also primary culture cells are CD34 negative, and maintain stable traits compared to ASC that is CD34 positive at the start of culture but whose expression is reduced by subculture (See FIG. 9). When isolating mature adipocytes, if collagenase treatment and filtration are not performed properly and adequately, cells other than mature adipocytes attach to mature adipocytes and proliferate in ceiling culture.
- CD34 positive cells may increase. It was also revealed that human DFAT can be prepared without being affected by donor age or underlying disease. These characteristics are thought to contribute to eliminating individual differences in performance as therapeutic cells and reducing treatment refractory cases (standardization of therapeutic cells). Furthermore, human DFAT showed a significantly higher blood flow improvement effect than peripheral blood mononuclear cells and fibroblasts in transplantation experiments to lower limb ischemia model animals described later in Examples.
- angiogenesis refers to a phenomenon in which new blood vessels are formed from existing blood vessels.
- physiological blood vessel development in the embryonic period, as well as cancer and diabetic properties It also occurs in pathological conditions such as retinopathy.
- angiogenesis and vascular regeneration are used synonymously unless otherwise specified.
- revascularization or therapeutic angiogenesis
- ischemic diseases such as peripheral arterial disease (PAD), angina pectoris, and myocardial infarction.
- PED peripheral arterial disease
- the performance of cells having revascularization ability includes the expression and secretion amount of angiogenesis-promoting factors such as HGF, VEGF-A, FGF-2, SDF-1, and leptin, and vascular endothelial cells and pericytes that are vascular constituent cells. It can be confirmed by the ability to differentiate into.
- DFAT in-house DFAT
- DFAT in-house DFAT
- a method of improving the blood flow by thawing this periodically and injecting it into an ischemic site (muscle) can be mentioned.
- a DFAT cell bank for allogeneic transplantation using adipose tissue discarded by surgery or the like it is possible to construct a DFAT cell bank for allogeneic transplantation using adipose tissue discarded by surgery or the like.
- a therapeutic model is assumed in which cryopreserved DFAT fully matched with HLA is thawed and injected into the patient's ischemic site.
- the present invention relates to a drug for regenerating blood vessels and repairing damaged tissue caused by ischemia, comprising human DFAT of the present invention as an active ingredient.
- the drug of the present invention may be human DFAT itself, or a pharmaceutically acceptable carrier such as a preservative or stabilizer may be added.
- “Pharmaceutically acceptable” means a pharmaceutically acceptable material that itself does not have the above-mentioned activity and can be administered together with the above-mentioned drug.
- parenteral administration includes parenteral administration.
- parenteral administration include administration in the form of injections, and examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like.
- parenteral administration includes administration in the form of injections, and examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like.
- local administration may be performed targeting a part of a human body (one tissue such as an organ), or the cells of the present invention may be applied to an entire organism by administration into a blood vessel. May be circulated. Moreover, you may administer simultaneously to the target of several places.
- the cells of the present invention can be locally administered to a region where treatment is desired. For example, it can be administered by local injection during surgery or by use of a catheter.
- pH adjusters, buffers, stabilizers, preservatives, etc. are added as necessary, and subcutaneous, intramuscular and intravenous injections are prepared by conventional methods.
- the dose varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, type of active ingredient contained in the cells, etc., and is not particularly limited.
- the cells of the invention may be administered as part of a pharmaceutical composition with at least one known chemotherapeutic agent. In one embodiment, the cells of the invention and the known chemotherapeutic agent may be administered substantially simultaneously. It is also possible to administer the cell of the present invention to a part of a human removed from the human and return the part of the human to the removed human or other human.
- Example 1 Character analysis of human DFAT (1) Karyotype analysis (1-1) Test method (i) Preparation of human DFAT This was carried out with reference to Japanese Patent Application Laid-Open No. 2000-83656 made by the present inventors. . That is, a human adipose subcutaneous human tissue was treated with collagenase, and then filtered with a mesh having a diameter of 100 to 200 ⁇ m to collect a single fraction consisting only of monocystic adipocytes. Human dedifferentiated adipocytes (human DFAT) were prepared by subculturing fibroblast-like adipocytes produced by ceiling culture of these monocystic adipocytes.
- Example 2 Examination of angiogenic ability of human DFAT Analysis of DFAT-secreted cytokine (1) Protein array analysis of expressed cytokine (1-1) Test method (i) Preparation method of human ASC Precipitation fraction of human DFAT obtained in the same manner as in Example 1 (stromal vascular fraction) SVF cells were collected from the fractional basal fraction (SVF) and cultured for about 2 weeks in a culture flask to prepare human ASC. The method for adjusting human ASC is the same in the following test examples.
- hPreadipocyte Human preadipocytes purchased from DS Pharma Medical Co., Ltd. were prepared by thawing, washing and adherent culture. As the medium, DMEM containing 10% FBS was used. The method for preparing human preadipocytes is the same in the following test examples.
- hBM-MSC Human bone marrow MSC
- DMEM containing 10% FBS was used.
- the method for adjusting human bone marrow MSC is the same in the following test examples.
- HGF and SDF-1 were found to be characteristically higher in expression than preadipocyte, human ASC, and human bone marrow MSC (FIG. 4).
- Angiogenic ability of human DFAT culture supernatant in vitro (1) Comparison of human DFAT and human bone marrow MSC (hBM-MSC) (1-1) Test method Angiogenesis kit (KZ-1000, KURABO, Osaka, Japan) The in vitro angiogenesis ability of the human DFAT culture supernatant was examined.
- This kit is a design in which human fibroblasts and human vascular endothelial cells are co-cultured in advance in a 24-well plate, and luminal formation of vascular endothelial cells is induced by culturing in a dedicated angiogenic medium. When a sample medium containing a test substance is added to this and cultured, a difference in lumen forming ability occurs.
- the angiogenesis ability was measured by inducing tube formation according to the protocol of this kit, and tube formation was visualized by immunostaining using a mouse anti-human CD31 antibody.
- the DFAT culture supernatant and human bone marrow MSC culture supernatant were prepared by the above method and mixed 1: 1 with the attached angiogenesis medium to obtain a sample medium.
- Test Results The culture supernatant of human DFAT markedly promoted the proliferation and lumen formation of vascular endothelial cells.
- the angiogenic potential of DFAT was equal to or greater than that of bone marrow MSC (FIG. 5).
- SCID immunodeficient mice
- the cell therapy method with respect to ischemic diseases can be provided by utilizing the human dedifferentiated fat cell (human DFAT) prepared from a human mature fat cell. .
- human DFAT human dedifferentiated fat cell
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Abstract
The present invention addresses the problem of providing a cell for use in a vascular regeneration therapy, which has a superior blood flow-improving effect compared with a bone marrow-derived MSC, an ASC or the like that is a conventional cell for use in a therapy, and which can be obtained easily in an amount sufficient enough for transplantation, and which has stable quality.
It is found that a human DFAT has a high neovascularization ability, can exhibit a high proliferation ability after being subcultured and therefore can be produced easily in an amount needed for transplantation, and does not undergo transformation. Therefore, it is found for the first time that a human DFAT is effective for a vascular regeneration therapy for a human body. These findings lead to the accomplishment of the present invention.
That is, the present invention provides a blood flow-improving agent containing human dedifferentiated fat cells (human DFAT) as an active ingredient.
Description
本発明は、血管再生療法用組成物に関する。特に脱分化脂肪細胞（ｄｅｄｉｆｆｅｒｅｎｔｉａｔｅｄ ｆａｔ ｃｅｌｌ：ＤＦＡＴ）を有効成分とする血管再生療法用組成物に関する。
The present invention relates to a composition for revascularization therapy. In particular, the present invention relates to a composition for revascularization therapy comprising dedifferentiated fat cells (DFAT) as an active ingredient.
従来、薬物療法や外科的療法が適応とならない難治性末梢動脈疾患（以下、単にＰＡＤということがある）に対して、（ｉ）自己骨髄単核球を虚血筋肉内投与することによる血管新生療法が行われ、その有効性が確認されてきた（ＴＡＣＴスタディ）。その後、（ｉｉ）Ｇ－ＣＳＦ動員末梢血単核球、（ｉｉｉ）脂肪組織由来ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ ｆｒａｃｔｉｏｎ（ＳＶＦ）細胞、（ｉｖ）末梢血単核球、（ｖ）Ｇ－ＣＳＦ動員ＣＤ３４陽性細胞、（ｖｉ）骨髄由来ＣＤ１３３陽性細胞、（ｖｉｉ）培養骨髄間葉系幹細胞（ＭＳＣ）（ｖｉｉｉ）培養脂肪組織由来幹細胞（ＡＳＣ）など多くの自己細胞を用いた同様の血管新生療法が行われている。
上記（ｉ）～（ｖｉｉｉ）のうち、（ｉ）～（ｖｉ）の細胞は培養を行わないが、必要な細胞数を得るために多量の組織採取が必要となり患者に与える侵襲性が高い。このため採取に伴う侵襲性が比較的低い（ｉｖ）を除いて治療回数は通常１回のみに限られる。
また、（ｉ）～（ｉｖ）の細胞は、雑多な細胞集団であるため、移植効果や安全性が一定ではない。
また、（ｖ）、（ｖｉ）の細胞は、幹細胞マーカー抗体を利用して選別操作を行うことにより細胞の均一性を高めることができるが、抗体選別に伴う煩雑性や調製効率の低下、調製コストが高くなるといった問題がある。
（ｖｉｉ）の細胞は骨髄液を数１０ｍｌ採取し、付着培養し増殖させてから使用し、（ｖｉｉｉ）の細胞は、脂肪組織を酵素処理し、（ｖｉｉ）と同様に付着培養し増殖させてから使用する。そのため、（ｖｉｉ）、（ｖｉｉｉ）の細胞は共に、比較的少量の組織から大量調製が可能であるというメリットがあるが、この方法は成体組織に微量に存在する幹細胞を培養・増殖させる方法であるため、他細胞の混入が避けられず、治療に必要な細胞数を得るために継代培養を何回か繰り返す必要がある。治療回数は通常１回のみである。また増殖能など細胞の性能に個体差があり、年齢・基礎疾患による影響を受けやすいというデメリットがある。
（ｉ）～（ｖｉｉｉ）の細胞の共通する問題点は、治療コストがかかる割に、性能に個体差があり、必要細胞数が得られない症例や治療不応例（ｎｏｎ－ｒｅｓｐｏｎｄｅｒ）が存在すること、患者に対する侵襲性を考慮すると通常１回の治療にとどまること、である。現時点で、これらの細胞を用いた難治性末梢動脈疾患（ＰＡＤ）に対する臨床試験は多数行われているが、プラセボ・コントロール試験で有意な効果を示した細胞はまだない。
また、このほかにも、ラットの脂肪組織由来前駆細胞やラットの脂肪組織由来間葉系幹細胞が血管新生に有効であることが開示されている（特許文献１，２）。
本発明者らは、マウス等のヒト以外の動物の脂肪組織由来の成熟脂肪細胞の脱分化を誘導することで前駆脂肪細胞として脱分化脂肪細胞（ＤＦＡＴ）を樹立することに初めて成功し（特許文献３）、ＤＦＡＴを分化誘導することにより、骨芽細胞、筋芽細胞、軟骨細胞、神経細胞等の機能を獲得できることを示してきた（特許文献４）。
また、本発明者らは、マウスおよびラットのＤＦＡＴが血管新生能を有することについても明らかにしてきた（非特許文献１～３）。しかし、マウスやラットのＤＦＡＴは継代培養後の増殖能が低いことから移植に必要な数まで増やすことが困難であり、継代培養により形質転換（不死化）が起こることから造腫瘍性の可能性が高まり安全に移植できない。これらの理由から、ヒトＤＦＡＴにおいてもマウスやラットのＤＦＡＴと同様の現象が起こり、治療には適さないと考えられてきた。 Conventionally, for refractory peripheral arterial disease (hereinafter sometimes simply referred to as PAD) to which pharmacotherapy or surgical therapy is not applicable, (i) Angiogenesis by administering autologous bone marrow mononuclear cells into ischemic muscle Therapy has been performed and its effectiveness has been confirmed (TACT study). Then, (ii) G-CSF mobilized peripheral blood mononuclear cells, (iii) adipose tissue derived basal basal fraction (SVF) cells, (iv) peripheral blood mononuclear cells, (v) G-CSF mobilized CD34 positive cells, Similar angiogenesis therapy using many autologous cells such as vi) bone marrow-derived CD133 positive cells, (vii) cultured bone marrow mesenchymal stem cells (MSC) (viii) cultured adipose tissue-derived stem cells (ASC) has been performed.
Of the above (i) to (viii), the cells of (i) to (vi) are not cultured, but a large amount of tissue is required to obtain the required number of cells, which is highly invasive to patients. For this reason, the number of treatments is usually limited to only one, except that the invasiveness associated with collection is relatively low (iv).
In addition, since the cells (i) to (iv) are a diverse cell population, the transplantation effect and safety are not constant.
The cells of (v) and (vi) can be improved in cell uniformity by performing a sorting operation using a stem cell marker antibody, but the complexity and reduction in preparation efficiency associated with antibody selection and preparation are reduced. There is a problem that the cost becomes high.
The cells of (vii) are used after collecting dozens of ml of bone marrow fluid, adherently cultured and proliferated, and the cells of (viii) are subjected to enzyme treatment of adipose tissue and adherently cultured and proliferated in the same manner as (vii). Use from. Therefore, both (vii) and (viii) cells have the merit that they can be prepared in large quantities from a relatively small amount of tissue, but this method is a method of culturing and proliferating stem cells present in minute amounts in adult tissues. For this reason, contamination with other cells is unavoidable, and it is necessary to repeat the subculture several times in order to obtain the number of cells necessary for treatment. The number of treatments is usually only once. In addition, there is a demerit that there are individual differences in cell performance such as proliferative ability and it is easily affected by age and underlying disease.
The common problem of cells (i) to (viii) is that there are cases in which the required number of cells cannot be obtained and there are non-responders despite the high cost of treatment, despite the cost of treatment. In view of the invasiveness to the patient, it is usually only one treatment. At present, many clinical trials for refractory peripheral arterial disease (PAD) using these cells have been carried out, but no cells have yet shown significant effects in placebo-controlled trials.
In addition, it is disclosed that rat adipose tissue-derived progenitor cells and rat adipose tissue-derived mesenchymal stem cells are effective for angiogenesis (Patent Documents 1 and 2).
The present inventors have succeeded for the first time in establishing dedifferentiated adipocytes (DFAT) as preadipocytes by inducing dedifferentiation of mature adipocytes derived from adipose tissue of non-human animals such as mice (patents) Reference 3), it has been shown that by inducing differentiation of DFAT, functions of osteoblasts, myoblasts, chondrocytes, nerve cells and the like can be obtained (Patent Document 4).
The present inventors have also clarified that mouse and rat DFAT has angiogenic ability (Non-PatentDocuments 1 to 3). However, DFAT in mice and rats is difficult to increase to the number required for transplantation because of its low growth ability after subculture, and since transformation (immortalization) occurs by subculture, it is tumorigenic. The possibility increases and it cannot be safely transplanted. For these reasons, it has been considered that the same phenomenon occurs in human DFAT as in mouse and rat DFAT and is not suitable for treatment.
上記（ｉ）～（ｖｉｉｉ）のうち、（ｉ）～（ｖｉ）の細胞は培養を行わないが、必要な細胞数を得るために多量の組織採取が必要となり患者に与える侵襲性が高い。このため採取に伴う侵襲性が比較的低い（ｉｖ）を除いて治療回数は通常１回のみに限られる。
また、（ｉ）～（ｉｖ）の細胞は、雑多な細胞集団であるため、移植効果や安全性が一定ではない。
また、（ｖ）、（ｖｉ）の細胞は、幹細胞マーカー抗体を利用して選別操作を行うことにより細胞の均一性を高めることができるが、抗体選別に伴う煩雑性や調製効率の低下、調製コストが高くなるといった問題がある。
（ｖｉｉ）の細胞は骨髄液を数１０ｍｌ採取し、付着培養し増殖させてから使用し、（ｖｉｉｉ）の細胞は、脂肪組織を酵素処理し、（ｖｉｉ）と同様に付着培養し増殖させてから使用する。そのため、（ｖｉｉ）、（ｖｉｉｉ）の細胞は共に、比較的少量の組織から大量調製が可能であるというメリットがあるが、この方法は成体組織に微量に存在する幹細胞を培養・増殖させる方法であるため、他細胞の混入が避けられず、治療に必要な細胞数を得るために継代培養を何回か繰り返す必要がある。治療回数は通常１回のみである。また増殖能など細胞の性能に個体差があり、年齢・基礎疾患による影響を受けやすいというデメリットがある。
（ｉ）～（ｖｉｉｉ）の細胞の共通する問題点は、治療コストがかかる割に、性能に個体差があり、必要細胞数が得られない症例や治療不応例（ｎｏｎ－ｒｅｓｐｏｎｄｅｒ）が存在すること、患者に対する侵襲性を考慮すると通常１回の治療にとどまること、である。現時点で、これらの細胞を用いた難治性末梢動脈疾患（ＰＡＤ）に対する臨床試験は多数行われているが、プラセボ・コントロール試験で有意な効果を示した細胞はまだない。
また、このほかにも、ラットの脂肪組織由来前駆細胞やラットの脂肪組織由来間葉系幹細胞が血管新生に有効であることが開示されている（特許文献１，２）。
本発明者らは、マウス等のヒト以外の動物の脂肪組織由来の成熟脂肪細胞の脱分化を誘導することで前駆脂肪細胞として脱分化脂肪細胞（ＤＦＡＴ）を樹立することに初めて成功し（特許文献３）、ＤＦＡＴを分化誘導することにより、骨芽細胞、筋芽細胞、軟骨細胞、神経細胞等の機能を獲得できることを示してきた（特許文献４）。
また、本発明者らは、マウスおよびラットのＤＦＡＴが血管新生能を有することについても明らかにしてきた（非特許文献１～３）。しかし、マウスやラットのＤＦＡＴは継代培養後の増殖能が低いことから移植に必要な数まで増やすことが困難であり、継代培養により形質転換（不死化）が起こることから造腫瘍性の可能性が高まり安全に移植できない。これらの理由から、ヒトＤＦＡＴにおいてもマウスやラットのＤＦＡＴと同様の現象が起こり、治療には適さないと考えられてきた。 Conventionally, for refractory peripheral arterial disease (hereinafter sometimes simply referred to as PAD) to which pharmacotherapy or surgical therapy is not applicable, (i) Angiogenesis by administering autologous bone marrow mononuclear cells into ischemic muscle Therapy has been performed and its effectiveness has been confirmed (TACT study). Then, (ii) G-CSF mobilized peripheral blood mononuclear cells, (iii) adipose tissue derived basal basal fraction (SVF) cells, (iv) peripheral blood mononuclear cells, (v) G-CSF mobilized CD34 positive cells, Similar angiogenesis therapy using many autologous cells such as vi) bone marrow-derived CD133 positive cells, (vii) cultured bone marrow mesenchymal stem cells (MSC) (viii) cultured adipose tissue-derived stem cells (ASC) has been performed.
Of the above (i) to (viii), the cells of (i) to (vi) are not cultured, but a large amount of tissue is required to obtain the required number of cells, which is highly invasive to patients. For this reason, the number of treatments is usually limited to only one, except that the invasiveness associated with collection is relatively low (iv).
In addition, since the cells (i) to (iv) are a diverse cell population, the transplantation effect and safety are not constant.
The cells of (v) and (vi) can be improved in cell uniformity by performing a sorting operation using a stem cell marker antibody, but the complexity and reduction in preparation efficiency associated with antibody selection and preparation are reduced. There is a problem that the cost becomes high.
The cells of (vii) are used after collecting dozens of ml of bone marrow fluid, adherently cultured and proliferated, and the cells of (viii) are subjected to enzyme treatment of adipose tissue and adherently cultured and proliferated in the same manner as (vii). Use from. Therefore, both (vii) and (viii) cells have the merit that they can be prepared in large quantities from a relatively small amount of tissue, but this method is a method of culturing and proliferating stem cells present in minute amounts in adult tissues. For this reason, contamination with other cells is unavoidable, and it is necessary to repeat the subculture several times in order to obtain the number of cells necessary for treatment. The number of treatments is usually only once. In addition, there is a demerit that there are individual differences in cell performance such as proliferative ability and it is easily affected by age and underlying disease.
The common problem of cells (i) to (viii) is that there are cases in which the required number of cells cannot be obtained and there are non-responders despite the high cost of treatment, despite the cost of treatment. In view of the invasiveness to the patient, it is usually only one treatment. At present, many clinical trials for refractory peripheral arterial disease (PAD) using these cells have been carried out, but no cells have yet shown significant effects in placebo-controlled trials.
In addition, it is disclosed that rat adipose tissue-derived progenitor cells and rat adipose tissue-derived mesenchymal stem cells are effective for angiogenesis (
The present inventors have succeeded for the first time in establishing dedifferentiated adipocytes (DFAT) as preadipocytes by inducing dedifferentiation of mature adipocytes derived from adipose tissue of non-human animals such as mice (patents) Reference 3), it has been shown that by inducing differentiation of DFAT, functions of osteoblasts, myoblasts, chondrocytes, nerve cells and the like can be obtained (Patent Document 4).
The present inventors have also clarified that mouse and rat DFAT has angiogenic ability (Non-Patent
本発明は、従来技術の治療用細胞である骨髄ＭＳＣやＡＳＣ等に比べ、優れた血流改善作用を示す血管再生療法用細胞であって、かつ移植に必要な十分量を取得することが容易であり、安定した品質の前記血管再生療法用細胞を提供することを目的とする。
The present invention is a cell for revascularization therapy that exhibits an excellent blood flow improvement effect compared to bone marrow MSC, ASC, etc., which are conventional treatment cells, and it is easy to obtain a sufficient amount necessary for transplantation. An object of the present invention is to provide a cell for revascularization therapy having a stable quality.
マウスやラットのＤＦＡＴは、血管新生能はあっても、上述の理由により、実際の治療には適さないことからヒトＤＦＡＴについても同様であろうと考えられてきた。しかし、本発明者らは意外にも、ヒトＤＦＡＴが高い血管新生能を有するのみならず、マウスやラットのＤＦＡＴとは異なり、継代培養後の増殖能が高く移植に必要な細胞数を容易に獲得できること、および形質転換も起こらないことを見出した。そして、これによりヒトＤＦＡＴがヒトの血管再生治療にも有効であることを初めて明らかにした。
すなわち、本発明は以下の構成を有する。
（１）ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする血流改善剤。
（２）ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする血管新生促進剤。
（３）ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする虚血性疾患治療剤。
（４）虚血性疾患が末梢動脈疾患（ＰＡＤ）または虚血性心筋症である、前記（３）に記載の虚血性疾患治療剤。
（５）ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）が、１～４代継代培養されたヒト脱分化脂肪細胞（ヒトＤＦＡＴ）である、前記（１）～（４）のいずれかに記載の剤。 Although DFAT in mice and rats has angiogenic potential, it has been considered that the same applies to human DFAT because it is not suitable for actual treatment for the reasons described above. However, the present inventors surprisingly not only have a high angiogenic ability of human DFAT, but also have a high proliferative ability after subculture, and easily the number of cells required for transplantation, unlike mouse and rat DFAT. And found that no transformation occurred. And this revealed for the first time that human DFAT is also effective for human blood vessel regeneration therapy.
That is, the present invention has the following configuration.
(1) A blood flow improving agent comprising human dedifferentiated adipocytes (human DFAT) as an active ingredient.
(2) An angiogenesis promoter containing human dedifferentiated adipocytes (human DFAT) as an active ingredient.
(3) A therapeutic agent for ischemic disease comprising human dedifferentiated fat cells (human DFAT) as an active ingredient.
(4) The therapeutic agent for ischemic disease according to (3), wherein the ischemic disease is peripheral arterial disease (PAD) or ischemic cardiomyopathy.
(5) The agent according to any one of (1) to (4) above, wherein the human dedifferentiated adipocyte (human DFAT) is a human dedifferentiated adipocyte (human DFAT) subcultured for 1 to 4 passages .
すなわち、本発明は以下の構成を有する。
（１）ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする血流改善剤。
（２）ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする血管新生促進剤。
（３）ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする虚血性疾患治療剤。
（４）虚血性疾患が末梢動脈疾患（ＰＡＤ）または虚血性心筋症である、前記（３）に記載の虚血性疾患治療剤。
（５）ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）が、１～４代継代培養されたヒト脱分化脂肪細胞（ヒトＤＦＡＴ）である、前記（１）～（４）のいずれかに記載の剤。 Although DFAT in mice and rats has angiogenic potential, it has been considered that the same applies to human DFAT because it is not suitable for actual treatment for the reasons described above. However, the present inventors surprisingly not only have a high angiogenic ability of human DFAT, but also have a high proliferative ability after subculture, and easily the number of cells required for transplantation, unlike mouse and rat DFAT. And found that no transformation occurred. And this revealed for the first time that human DFAT is also effective for human blood vessel regeneration therapy.
That is, the present invention has the following configuration.
(1) A blood flow improving agent comprising human dedifferentiated adipocytes (human DFAT) as an active ingredient.
(2) An angiogenesis promoter containing human dedifferentiated adipocytes (human DFAT) as an active ingredient.
(3) A therapeutic agent for ischemic disease comprising human dedifferentiated fat cells (human DFAT) as an active ingredient.
(4) The therapeutic agent for ischemic disease according to (3), wherein the ischemic disease is peripheral arterial disease (PAD) or ischemic cardiomyopathy.
(5) The agent according to any one of (1) to (4) above, wherein the human dedifferentiated adipocyte (human DFAT) is a human dedifferentiated adipocyte (human DFAT) subcultured for 1 to 4 passages .
本発明は、ヒト成熟脂肪細胞から調製されるヒト脱分化脂肪細胞（ヒトＤＦＡＴ）が、優れた血管新生能を有し、末梢動脈疾患（ＰＡＤ）などの虚血性疾患に対する細胞治療の有効な細胞ソースとなり得ることを明らかにした。したがって、本発明は、以下の効果を有する。
（１）大規模な細胞調製施設や、増殖因子、純度を高めるための抗体選別などが必要ない。また短い培養期間で大量調整が可能であるため、調製コストが低くおさえることができる。
（２）採取が容易な少量の脂肪組織から純度の高い再生医療用ドナー細胞を効率よく採取でき、得られた細胞は高い血管新生能を有する。
（３）一回の組織採取により複数回の治療が可能となり、ドナー年齢や基礎疾患に影響されずに一定の有効性を示す治療が可能となる。
（４）既存の細胞ソースと異なり、ＰＡＤに対して長期有効性を示すことが可能である。 In the present invention, human dedifferentiated adipocytes (human DFAT) prepared from human mature adipocytes have excellent angiogenic potential, and are effective cells for cell therapy for ischemic diseases such as peripheral artery disease (PAD). Clarified that it can be a source. Therefore, the present invention has the following effects.
(1) Large-scale cell preparation facilities, growth factors, and antibody selection for increasing purity are not necessary. Moreover, since a large amount can be adjusted in a short culture period, the preparation cost can be kept low.
(2) Regenerative medical donor cells with high purity can be efficiently collected from a small amount of adipose tissue that can be easily collected, and the obtained cells have high angiogenic ability.
(3) Multiple treatments can be performed by collecting a single tissue, and treatment that exhibits a certain level of effectiveness without being affected by donor age or underlying disease is possible.
(4) Unlike existing cell sources, it can show long-term efficacy against PAD.
（１）大規模な細胞調製施設や、増殖因子、純度を高めるための抗体選別などが必要ない。また短い培養期間で大量調整が可能であるため、調製コストが低くおさえることができる。
（２）採取が容易な少量の脂肪組織から純度の高い再生医療用ドナー細胞を効率よく採取でき、得られた細胞は高い血管新生能を有する。
（３）一回の組織採取により複数回の治療が可能となり、ドナー年齢や基礎疾患に影響されずに一定の有効性を示す治療が可能となる。
（４）既存の細胞ソースと異なり、ＰＡＤに対して長期有効性を示すことが可能である。 In the present invention, human dedifferentiated adipocytes (human DFAT) prepared from human mature adipocytes have excellent angiogenic potential, and are effective cells for cell therapy for ischemic diseases such as peripheral artery disease (PAD). Clarified that it can be a source. Therefore, the present invention has the following effects.
(1) Large-scale cell preparation facilities, growth factors, and antibody selection for increasing purity are not necessary. Moreover, since a large amount can be adjusted in a short culture period, the preparation cost can be kept low.
(2) Regenerative medical donor cells with high purity can be efficiently collected from a small amount of adipose tissue that can be easily collected, and the obtained cells have high angiogenic ability.
(3) Multiple treatments can be performed by collecting a single tissue, and treatment that exhibits a certain level of effectiveness without being affected by donor age or underlying disease is possible.
(4) Unlike existing cell sources, it can show long-term efficacy against PAD.
（ヒトＤＦＡＴ）
本発明におけるヒトＤＦＡＴの調整方法は、たとえば、本発明者らによってなされた特開２０００－８３６５６号公報を参考にしておこなうとよい。すなわち、ヒトの皮下または内臓などの脂肪組織をコラゲナーゼ処理したのち、口径１００～２００μｍのメッシュでフィルトレーションすることにより単胞性脂肪細胞のみからなる単一の画分を採取する。それらの単胞性脂肪細胞を天井培養することで産生された線維芽細胞様脂肪細胞を、さらに継代培養することによってヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を得ることができる。また、上記天井培養以外の方法によっても、成熟脂肪細胞を培養することにより、成熟脂肪細胞を起源とする脂肪滴を有さない細胞を調整する方法等によりヒトＤＦＡＴを得ることができる。
得られた細胞がヒトＤＦＡＴであることの確認は、例えば、以下のようなヒトＤＦＡＴ特有の性質を有するかどうかにより判断することができる。
ヒトＤＦＡＴはプラスチックへの付着性を有し、ｉｎ ｖｉｔｒｏで骨芽細胞、脂肪細胞、軟骨細胞、平滑筋細胞への多分化能を示す。また、細胞表面抗原として、ＣＤ１３，ＣＤ２９，ＣＤ４４，ＣＤ４９ｄ，ＣＤ７３，ＣＤ９０，ＣＤ１０５陽性、ＣＤ１１ｂ，ＣＤ１４，ＣＤ３４，ＣＤ４５，ＣＤ１９，ＨＬＡ－ＤＲ陰性であり、国際細胞治療学会（ＩＳＣＴ）が定めたＭＳＣの最小基準を満たす。また、ヒトＤＦＡＴはリポプロテインリパーゼ、ＧＬＵＴ４といった成熟脂肪細胞のマーカー遺伝子の発現が消失している一方、ＰＰＡＲｇ，ＲＵＮＸ２，ＳＯＸ９といった脂肪、骨、軟骨の初期分化マーカー遺伝子が発現している。さらに、ヒトＤＦＡＴは高い細胞増殖能を有し、細胞倍加時間は第２継代細胞で約６５時間、第１０継代で約４８時間であった。第５継代以後は多分化能が低下するため、第１継代～第４継代で使用することが望ましい。
ヒトＤＦＡＴは１０ｍｌの吸引脂肪または１ｇの脂肪組織より上記調整方法により、約２週間の初代培養で１０８の細胞が得られる。これは培養ＡＳＣの約２５倍の調製効率である。従って、２－３回継代培養することにより１０９オーダーの細胞を得ることが可能である。１回の細胞治療に用いる細胞数は約１０８個であるため、１回（約１０ｍｌ）の脂肪組織採取で、得られた細胞を小分けして凍結保存すれば、約１０回治療が可能であることがわかる。
また、ヒトＤＦＡＴは初代培養から均質な細胞が得られる。初代培養ＡＳＣは平滑筋細胞（１８．６％）、血管内皮細胞（２．７％）、単球（１３．３％）の混入があるが、初代培養ヒトＤＦＡＴのこれらの細胞の混入率は０．１％以下と非常に低いものであった。
また、ヒトＤＦＡＴは継代した細胞のみならず初代培養細胞もＣＤ３４陰性であり、培養開始時にＣＤ３４陽性であるが継代培養によって発現が低下するＡＳＣと比べて安定した形質を維持している（図９、参照）。成熟脂肪細胞を単離する際、コラゲナーゼ処理やフィルトレーションを適切、十分に行わないと、成熟脂肪細胞以外の細胞が成熟脂肪細胞に付着し、天井培養にて増殖するため本発明とは異なり、ＣＤ３４陽性細胞などが増加することがある。
また、ヒトＤＦＡＴはドナー年齢や基礎疾患に影響されず、調製可能であることも明らかになった。これらの特性は、治療用細胞としての性能の個体差をなくし、治療不応例を減らすこと（治療用細胞の標準化）に寄与すると考えられる。
さらには、実施例にて後述する下肢虚血モデル動物への移植実験において、ヒトＤＦＡＴは、末梢血単核球や線維芽細胞より有意に高い血流改善効果を示した。 (Human DFAT)
The method for adjusting human DFAT in the present invention may be performed with reference to, for example, Japanese Patent Application Laid-Open No. 2000-83656 made by the present inventors. That is, after adipose tissue such as human subcutaneous or internal organs is treated with collagenase, a single fraction consisting only of monocystic adipocytes is collected by filtration with a mesh having a diameter of 100 to 200 μm. Human dedifferentiated adipocytes (human DFAT) can be obtained by further subculturing fibroblast-like adipocytes produced by ceiling culture of these monocystic adipocytes. Moreover, human DFAT can also be obtained by a method of adjusting cells that do not have lipid droplets originating from mature adipocytes by culturing mature adipocytes by methods other than the above-described ceiling culture.
Confirmation that the obtained cells are human DFAT can be determined, for example, based on whether or not they have the following characteristics specific to human DFAT.
Human DFAT has adhesiveness to plastic and exhibits multipotency into osteoblasts, adipocytes, chondrocytes and smooth muscle cells in vitro. Further, as cell surface antigens, CD13, CD29, CD44, CD49d, CD73, CD90, CD105 positive, CD11b, CD14, CD34, CD45, CD19, HLA-DR negative, MSC defined by the International Society for Cell Therapy (ISCT) Meet the minimum criteria. In addition, human DFAT has lost expression of marker genes of mature adipocytes such as lipoprotein lipase and GLUT4, but expresses early differentiation marker genes of fat, bone and cartilage such as PPARg, RUNX2, and SOX9. Furthermore, human DFAT had a high cell proliferation ability, and the cell doubling time was about 65 hours for the second passage cell and about 48 hours for the tenth passage. Since the pluripotency decreases after the 5th passage, it is desirable to use in the 1st to 4th passages.
Human DFAT can be obtained from 10 ml of aspirated fat or 1 g of adipose tissue by the above-mentioned preparation method, and 10 8 cells can be obtained in primary culture for about 2 weeks. This is about 25 times the preparation efficiency of cultured ASC. Therefore, it is possible to obtain cells of the order of 10 9 by subculturing 2-3 times. Since the number of cells used for a single cell therapy is about 10 8 , treatment can be performed about 10 times if the obtained cells are subdivided and cryopreserved by collecting adipose tissue once (about 10 ml). I know that there is.
In addition, human DFAT can obtain homogeneous cells from primary culture. Primary cultured ASC contains smooth muscle cells (18.6%), vascular endothelial cells (2.7%) and monocytes (13.3%). The contamination rate of these cells in primary cultured human DFAT is It was very low as 0.1% or less.
Moreover, human DFAT is not only subcultured but also primary culture cells are CD34 negative, and maintain stable traits compared to ASC that is CD34 positive at the start of culture but whose expression is reduced by subculture ( (See FIG. 9). When isolating mature adipocytes, if collagenase treatment and filtration are not performed properly and adequately, cells other than mature adipocytes attach to mature adipocytes and proliferate in ceiling culture. CD34 positive cells may increase.
It was also revealed that human DFAT can be prepared without being affected by donor age or underlying disease. These characteristics are thought to contribute to eliminating individual differences in performance as therapeutic cells and reducing treatment refractory cases (standardization of therapeutic cells).
Furthermore, human DFAT showed a significantly higher blood flow improvement effect than peripheral blood mononuclear cells and fibroblasts in transplantation experiments to lower limb ischemia model animals described later in Examples.
本発明におけるヒトＤＦＡＴの調整方法は、たとえば、本発明者らによってなされた特開２０００－８３６５６号公報を参考にしておこなうとよい。すなわち、ヒトの皮下または内臓などの脂肪組織をコラゲナーゼ処理したのち、口径１００～２００μｍのメッシュでフィルトレーションすることにより単胞性脂肪細胞のみからなる単一の画分を採取する。それらの単胞性脂肪細胞を天井培養することで産生された線維芽細胞様脂肪細胞を、さらに継代培養することによってヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を得ることができる。また、上記天井培養以外の方法によっても、成熟脂肪細胞を培養することにより、成熟脂肪細胞を起源とする脂肪滴を有さない細胞を調整する方法等によりヒトＤＦＡＴを得ることができる。
得られた細胞がヒトＤＦＡＴであることの確認は、例えば、以下のようなヒトＤＦＡＴ特有の性質を有するかどうかにより判断することができる。
ヒトＤＦＡＴはプラスチックへの付着性を有し、ｉｎ ｖｉｔｒｏで骨芽細胞、脂肪細胞、軟骨細胞、平滑筋細胞への多分化能を示す。また、細胞表面抗原として、ＣＤ１３，ＣＤ２９，ＣＤ４４，ＣＤ４９ｄ，ＣＤ７３，ＣＤ９０，ＣＤ１０５陽性、ＣＤ１１ｂ，ＣＤ１４，ＣＤ３４，ＣＤ４５，ＣＤ１９，ＨＬＡ－ＤＲ陰性であり、国際細胞治療学会（ＩＳＣＴ）が定めたＭＳＣの最小基準を満たす。また、ヒトＤＦＡＴはリポプロテインリパーゼ、ＧＬＵＴ４といった成熟脂肪細胞のマーカー遺伝子の発現が消失している一方、ＰＰＡＲｇ，ＲＵＮＸ２，ＳＯＸ９といった脂肪、骨、軟骨の初期分化マーカー遺伝子が発現している。さらに、ヒトＤＦＡＴは高い細胞増殖能を有し、細胞倍加時間は第２継代細胞で約６５時間、第１０継代で約４８時間であった。第５継代以後は多分化能が低下するため、第１継代～第４継代で使用することが望ましい。
ヒトＤＦＡＴは１０ｍｌの吸引脂肪または１ｇの脂肪組織より上記調整方法により、約２週間の初代培養で１０８の細胞が得られる。これは培養ＡＳＣの約２５倍の調製効率である。従って、２－３回継代培養することにより１０９オーダーの細胞を得ることが可能である。１回の細胞治療に用いる細胞数は約１０８個であるため、１回（約１０ｍｌ）の脂肪組織採取で、得られた細胞を小分けして凍結保存すれば、約１０回治療が可能であることがわかる。
また、ヒトＤＦＡＴは初代培養から均質な細胞が得られる。初代培養ＡＳＣは平滑筋細胞（１８．６％）、血管内皮細胞（２．７％）、単球（１３．３％）の混入があるが、初代培養ヒトＤＦＡＴのこれらの細胞の混入率は０．１％以下と非常に低いものであった。
また、ヒトＤＦＡＴは継代した細胞のみならず初代培養細胞もＣＤ３４陰性であり、培養開始時にＣＤ３４陽性であるが継代培養によって発現が低下するＡＳＣと比べて安定した形質を維持している（図９、参照）。成熟脂肪細胞を単離する際、コラゲナーゼ処理やフィルトレーションを適切、十分に行わないと、成熟脂肪細胞以外の細胞が成熟脂肪細胞に付着し、天井培養にて増殖するため本発明とは異なり、ＣＤ３４陽性細胞などが増加することがある。
また、ヒトＤＦＡＴはドナー年齢や基礎疾患に影響されず、調製可能であることも明らかになった。これらの特性は、治療用細胞としての性能の個体差をなくし、治療不応例を減らすこと（治療用細胞の標準化）に寄与すると考えられる。
さらには、実施例にて後述する下肢虚血モデル動物への移植実験において、ヒトＤＦＡＴは、末梢血単核球や線維芽細胞より有意に高い血流改善効果を示した。 (Human DFAT)
The method for adjusting human DFAT in the present invention may be performed with reference to, for example, Japanese Patent Application Laid-Open No. 2000-83656 made by the present inventors. That is, after adipose tissue such as human subcutaneous or internal organs is treated with collagenase, a single fraction consisting only of monocystic adipocytes is collected by filtration with a mesh having a diameter of 100 to 200 μm. Human dedifferentiated adipocytes (human DFAT) can be obtained by further subculturing fibroblast-like adipocytes produced by ceiling culture of these monocystic adipocytes. Moreover, human DFAT can also be obtained by a method of adjusting cells that do not have lipid droplets originating from mature adipocytes by culturing mature adipocytes by methods other than the above-described ceiling culture.
Confirmation that the obtained cells are human DFAT can be determined, for example, based on whether or not they have the following characteristics specific to human DFAT.
Human DFAT has adhesiveness to plastic and exhibits multipotency into osteoblasts, adipocytes, chondrocytes and smooth muscle cells in vitro. Further, as cell surface antigens, CD13, CD29, CD44, CD49d, CD73, CD90, CD105 positive, CD11b, CD14, CD34, CD45, CD19, HLA-DR negative, MSC defined by the International Society for Cell Therapy (ISCT) Meet the minimum criteria. In addition, human DFAT has lost expression of marker genes of mature adipocytes such as lipoprotein lipase and GLUT4, but expresses early differentiation marker genes of fat, bone and cartilage such as PPARg, RUNX2, and SOX9. Furthermore, human DFAT had a high cell proliferation ability, and the cell doubling time was about 65 hours for the second passage cell and about 48 hours for the tenth passage. Since the pluripotency decreases after the 5th passage, it is desirable to use in the 1st to 4th passages.
Human DFAT can be obtained from 10 ml of aspirated fat or 1 g of adipose tissue by the above-mentioned preparation method, and 10 8 cells can be obtained in primary culture for about 2 weeks. This is about 25 times the preparation efficiency of cultured ASC. Therefore, it is possible to obtain cells of the order of 10 9 by subculturing 2-3 times. Since the number of cells used for a single cell therapy is about 10 8 , treatment can be performed about 10 times if the obtained cells are subdivided and cryopreserved by collecting adipose tissue once (about 10 ml). I know that there is.
In addition, human DFAT can obtain homogeneous cells from primary culture. Primary cultured ASC contains smooth muscle cells (18.6%), vascular endothelial cells (2.7%) and monocytes (13.3%). The contamination rate of these cells in primary cultured human DFAT is It was very low as 0.1% or less.
Moreover, human DFAT is not only subcultured but also primary culture cells are CD34 negative, and maintain stable traits compared to ASC that is CD34 positive at the start of culture but whose expression is reduced by subculture ( (See FIG. 9). When isolating mature adipocytes, if collagenase treatment and filtration are not performed properly and adequately, cells other than mature adipocytes attach to mature adipocytes and proliferate in ceiling culture. CD34 positive cells may increase.
It was also revealed that human DFAT can be prepared without being affected by donor age or underlying disease. These characteristics are thought to contribute to eliminating individual differences in performance as therapeutic cells and reducing treatment refractory cases (standardization of therapeutic cells).
Furthermore, human DFAT showed a significantly higher blood flow improvement effect than peripheral blood mononuclear cells and fibroblasts in transplantation experiments to lower limb ischemia model animals described later in Examples.
（血管新生）
本発明において血管新生とは、既存の血管より新しい血管が形成される現象をいい、虚血性疾患における側副血行路の形成のほか、胎生期における生理的な血管発達、さらには癌や糖尿病性網膜症などの病的状態でも起こる。動脈硬化や血栓などにより血管が閉塞し、局所組織が血流障害に陥った状態において、生理活性物質や細胞などを用いて人為的に血管新生を誘導することを特に治療的血管新生または血管再生と呼ぶ。本明細書において、血管新生と血管再生は特に断らない限り同義で用いられる。
血管再生（または治療的血管新生）のより具体的な例としては、末梢動脈疾患（ＰＡＤ）や狭心症、心筋梗塞などの虚血性疾患患者における血流改善が挙げられる。血管再生能を有する細胞の性能は、ＨＧＦ、ＶＥＧＦ－Ａ、ＦＧＦ－２、ＳＤＦ－１、ｌｅｐｔｉｎなどの血管新生促成因子の発現量・分泌量や、血管構成細胞である血管内皮細胞やペリサイトへの分化能などにより確認することができる。 (Angiogenesis)
In the present invention, angiogenesis refers to a phenomenon in which new blood vessels are formed from existing blood vessels. In addition to the formation of collateral blood circulation in ischemic diseases, physiological blood vessel development in the embryonic period, as well as cancer and diabetic properties It also occurs in pathological conditions such as retinopathy. Artificially induced angiogenesis using physiologically active substances or cells, etc., in the state where the blood vessel is blocked by arteriosclerosis or thrombus and the local tissue is damaged by blood flow, especially therapeutic angiogenesis or revascularization Call it. In this specification, angiogenesis and vascular regeneration are used synonymously unless otherwise specified.
More specific examples of revascularization (or therapeutic angiogenesis) include blood flow improvement in patients with ischemic diseases such as peripheral arterial disease (PAD), angina pectoris, and myocardial infarction. The performance of cells having revascularization ability includes the expression and secretion amount of angiogenesis-promoting factors such as HGF, VEGF-A, FGF-2, SDF-1, and leptin, and vascular endothelial cells and pericytes that are vascular constituent cells. It can be confirmed by the ability to differentiate into.
本発明において血管新生とは、既存の血管より新しい血管が形成される現象をいい、虚血性疾患における側副血行路の形成のほか、胎生期における生理的な血管発達、さらには癌や糖尿病性網膜症などの病的状態でも起こる。動脈硬化や血栓などにより血管が閉塞し、局所組織が血流障害に陥った状態において、生理活性物質や細胞などを用いて人為的に血管新生を誘導することを特に治療的血管新生または血管再生と呼ぶ。本明細書において、血管新生と血管再生は特に断らない限り同義で用いられる。
血管再生（または治療的血管新生）のより具体的な例としては、末梢動脈疾患（ＰＡＤ）や狭心症、心筋梗塞などの虚血性疾患患者における血流改善が挙げられる。血管再生能を有する細胞の性能は、ＨＧＦ、ＶＥＧＦ－Ａ、ＦＧＦ－２、ＳＤＦ－１、ｌｅｐｔｉｎなどの血管新生促成因子の発現量・分泌量や、血管構成細胞である血管内皮細胞やペリサイトへの分化能などにより確認することができる。 (Angiogenesis)
In the present invention, angiogenesis refers to a phenomenon in which new blood vessels are formed from existing blood vessels. In addition to the formation of collateral blood circulation in ischemic diseases, physiological blood vessel development in the embryonic period, as well as cancer and diabetic properties It also occurs in pathological conditions such as retinopathy. Artificially induced angiogenesis using physiologically active substances or cells, etc., in the state where the blood vessel is blocked by arteriosclerosis or thrombus and the local tissue is damaged by blood flow, especially therapeutic angiogenesis or revascularization Call it. In this specification, angiogenesis and vascular regeneration are used synonymously unless otherwise specified.
More specific examples of revascularization (or therapeutic angiogenesis) include blood flow improvement in patients with ischemic diseases such as peripheral arterial disease (PAD), angina pectoris, and myocardial infarction. The performance of cells having revascularization ability includes the expression and secretion amount of angiogenesis-promoting factors such as HGF, VEGF-A, FGF-2, SDF-1, and leptin, and vascular endothelial cells and pericytes that are vascular constituent cells. It can be confirmed by the ability to differentiate into.
本発明のヒトＤＦＡＴを使用した血管再生に係る治療方法は、例えば、患者自身から調製したＤＦＡＴ（自家ＤＦＡＴ）を複数のバイアルに入れ液体窒素タンクに凍結保存しておく。これを定期的に解凍して、虚血部位（筋肉）に注射することにより血流改善を図る方法が挙げられる。
また、外科手術などで破棄される脂肪組織を利用した他家移植用ＤＦＡＴ細胞バンクの構築も可能である。この場合は、何らかの理由で自家ＤＦＡＴが調製できない患者に対し、ＨＬＡフルマッチした凍結保存ＤＦＡＴを解凍し、患者の虚血部位に注射するといった治療モデルが想定される。 In the treatment method related to revascularization using human DFAT of the present invention, for example, DFAT (in-house DFAT) prepared from the patient himself is placed in a plurality of vials and stored frozen in a liquid nitrogen tank. A method of improving the blood flow by thawing this periodically and injecting it into an ischemic site (muscle) can be mentioned.
In addition, it is possible to construct a DFAT cell bank for allogeneic transplantation using adipose tissue discarded by surgery or the like. In this case, for a patient who cannot prepare autologous DFAT for some reason, a therapeutic model is assumed in which cryopreserved DFAT fully matched with HLA is thawed and injected into the patient's ischemic site.
また、外科手術などで破棄される脂肪組織を利用した他家移植用ＤＦＡＴ細胞バンクの構築も可能である。この場合は、何らかの理由で自家ＤＦＡＴが調製できない患者に対し、ＨＬＡフルマッチした凍結保存ＤＦＡＴを解凍し、患者の虚血部位に注射するといった治療モデルが想定される。 In the treatment method related to revascularization using human DFAT of the present invention, for example, DFAT (in-house DFAT) prepared from the patient himself is placed in a plurality of vials and stored frozen in a liquid nitrogen tank. A method of improving the blood flow by thawing this periodically and injecting it into an ischemic site (muscle) can be mentioned.
In addition, it is possible to construct a DFAT cell bank for allogeneic transplantation using adipose tissue discarded by surgery or the like. In this case, for a patient who cannot prepare autologous DFAT for some reason, a therapeutic model is assumed in which cryopreserved DFAT fully matched with HLA is thawed and injected into the patient's ischemic site.
本発明は、本発明のヒトＤＦＡＴを有効成分とする、血管を再生し虚血による障害組織を修復させる薬剤に関する。
本発明の薬剤は、ヒトＤＦＡＴそのものでもよいし、保存剤や安定剤等の製剤上許容しうる担体を添加してもよい。製剤上許容しうるとは、それ自体は上記の活性を有さない材料であって、上記の薬剤とともに投与可能な製剤上許容される材料を意味する。 The present invention relates to a drug for regenerating blood vessels and repairing damaged tissue caused by ischemia, comprising human DFAT of the present invention as an active ingredient.
The drug of the present invention may be human DFAT itself, or a pharmaceutically acceptable carrier such as a preservative or stabilizer may be added. “Pharmaceutically acceptable” means a pharmaceutically acceptable material that itself does not have the above-mentioned activity and can be administered together with the above-mentioned drug.
本発明の薬剤は、ヒトＤＦＡＴそのものでもよいし、保存剤や安定剤等の製剤上許容しうる担体を添加してもよい。製剤上許容しうるとは、それ自体は上記の活性を有さない材料であって、上記の薬剤とともに投与可能な製剤上許容される材料を意味する。 The present invention relates to a drug for regenerating blood vessels and repairing damaged tissue caused by ischemia, comprising human DFAT of the present invention as an active ingredient.
The drug of the present invention may be human DFAT itself, or a pharmaceutically acceptable carrier such as a preservative or stabilizer may be added. “Pharmaceutically acceptable” means a pharmaceutically acceptable material that itself does not have the above-mentioned activity and can be administered together with the above-mentioned drug.
本発明において、「投与する」とは、非経口的に投与することが含まれる。非経口的な投与としては、注射剤という形での投与を挙げることができ、注射剤としては、皮下注射剤、筋肉注射剤、あるいは腹腔内注射剤等を挙げることができる。注射剤を投与する方法としては、ヒト体内の一部分（臓器等の一組織）を標的として局所的に投与を行っても良いし、血管内に投与することにより、生物体全体に本発明の細胞を循環させてもよい。また、複数箇所の標的に同時に投与を行ってもよい。また、本発明の細胞を、処置を施したい領域に局所的に投与することもできる。例えば、手術中の局所注入、カテーテルの使用により投与することも可能である。
注射剤を調製する場合、必要により、ｐＨ 調製剤、緩衝剤、安定化剤、保存剤等を添加し、常法により、皮下、筋肉内、静脈内注射剤とする。 In the present invention, “administering” includes parenteral administration. Examples of parenteral administration include administration in the form of injections, and examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like. As a method for administering an injection, local administration may be performed targeting a part of a human body (one tissue such as an organ), or the cells of the present invention may be applied to an entire organism by administration into a blood vessel. May be circulated. Moreover, you may administer simultaneously to the target of several places. In addition, the cells of the present invention can be locally administered to a region where treatment is desired. For example, it can be administered by local injection during surgery or by use of a catheter.
When preparing injections, pH adjusters, buffers, stabilizers, preservatives, etc. are added as necessary, and subcutaneous, intramuscular and intravenous injections are prepared by conventional methods.
注射剤を調製する場合、必要により、ｐＨ 調製剤、緩衝剤、安定化剤、保存剤等を添加し、常法により、皮下、筋肉内、静脈内注射剤とする。 In the present invention, “administering” includes parenteral administration. Examples of parenteral administration include administration in the form of injections, and examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like. As a method for administering an injection, local administration may be performed targeting a part of a human body (one tissue such as an organ), or the cells of the present invention may be applied to an entire organism by administration into a blood vessel. May be circulated. Moreover, you may administer simultaneously to the target of several places. In addition, the cells of the present invention can be locally administered to a region where treatment is desired. For example, it can be administered by local injection during surgery or by use of a catheter.
When preparing injections, pH adjusters, buffers, stabilizers, preservatives, etc. are added as necessary, and subcutaneous, intramuscular and intravenous injections are prepared by conventional methods.
投与量は、患者の年齢、性別、体重および症状、治療効果、投与方法、処理時間、あるいは該細胞に含有される活性成分の種類などにより異なり、特に制限されるものではない。本発明の細胞は、少なくとも１つの既知の化学療法剤と共に薬学的組成物の一部として投与されてもよい。一つの態様において、本発明の細胞および既知の化学療法剤は、実質的に同時に投与されてもよい。また、ヒトより摘出されたヒトの一部分に本発明の細胞を投与し、摘出を行ったヒトまたは他のヒトに、該ヒトの一部分を戻すことも可能である。
The dose varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, type of active ingredient contained in the cells, etc., and is not particularly limited. The cells of the invention may be administered as part of a pharmaceutical composition with at least one known chemotherapeutic agent. In one embodiment, the cells of the invention and the known chemotherapeutic agent may be administered substantially simultaneously. It is also possible to administer the cell of the present invention to a part of a human removed from the human and return the part of the human to the removed human or other human.
［実施例１］ヒトＤＦＡＴの形質解析
（１）核型解析
（１－１）試験方法
（ｉ）ヒトＤＦＡＴの調整
本発明者らによってなされた特開２０００－８３６５６号公報を参考にして行った。すなわち、ドナーであるヒトの皮下脂肪組織をコラゲナーゼ処理したのち、口径１００～２００μｍのメッシュでフィルトレーションすることにより単胞性脂肪細胞のみからなる単一の画分を採取した。それらの単胞性脂肪細胞を天井培養して産生される線維芽細胞様脂肪細胞を継代培養することによってヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を調製した。
ドナー対象者は、日本大学医学部附属病院 形成外科、整形外科、小児外科にて外科手術を受けた１０歳から８２歳の患者である。ヒトＤＦＡＴの調整方法は以下の試験例において同じである。
（ｉｉ）（ｉ）により得られた培養ヒトＤＦＡＴ（ｎ＝３）をコルセミド処理し染色体標本を作製し、染色体数の測定およびギムザ染色による染色体構造の観察を行った。
（１－２）試験結果
５０細胞分の染色体数を測定した結果、すべての細胞において、染色体数は、正常ヒト染色体数である４６であった（図１（Ｂ））。また、ギムザ染色では顕著な染色体構造の異常は観察されなかった（図１（Ａ））。他の２細胞株でも同様の結果が得られた。 [Example 1] Character analysis of human DFAT (1) Karyotype analysis (1-1) Test method (i) Preparation of human DFAT This was carried out with reference to Japanese Patent Application Laid-Open No. 2000-83656 made by the present inventors. . That is, a human adipose subcutaneous human tissue was treated with collagenase, and then filtered with a mesh having a diameter of 100 to 200 μm to collect a single fraction consisting only of monocystic adipocytes. Human dedifferentiated adipocytes (human DFAT) were prepared by subculturing fibroblast-like adipocytes produced by ceiling culture of these monocystic adipocytes.
Donor subjects are patients aged 10 to 82 who have undergone surgery in plastic surgery, orthopedic surgery, and pediatric surgery at Nihon University Hospital. The method for adjusting human DFAT is the same in the following test examples.
(Ii) The cultured human DFAT (n = 3) obtained in (i) was treated with colcemid to prepare a chromosome sample, and the number of chromosomes was measured and the chromosome structure was observed by Giemsa staining.
(1-2) Test Results As a result of measuring the number of chromosomes for 50 cells, the number of chromosomes in all cells was 46, which is the number of normal human chromosomes (FIG. 1 (B)). In addition, no remarkable abnormality in the chromosomal structure was observed with Giemsa staining (FIG. 1 (A)). Similar results were obtained with the other two cell lines.
（１）核型解析
（１－１）試験方法
（ｉ）ヒトＤＦＡＴの調整
本発明者らによってなされた特開２０００－８３６５６号公報を参考にして行った。すなわち、ドナーであるヒトの皮下脂肪組織をコラゲナーゼ処理したのち、口径１００～２００μｍのメッシュでフィルトレーションすることにより単胞性脂肪細胞のみからなる単一の画分を採取した。それらの単胞性脂肪細胞を天井培養して産生される線維芽細胞様脂肪細胞を継代培養することによってヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を調製した。
ドナー対象者は、日本大学医学部附属病院 形成外科、整形外科、小児外科にて外科手術を受けた１０歳から８２歳の患者である。ヒトＤＦＡＴの調整方法は以下の試験例において同じである。
（ｉｉ）（ｉ）により得られた培養ヒトＤＦＡＴ（ｎ＝３）をコルセミド処理し染色体標本を作製し、染色体数の測定およびギムザ染色による染色体構造の観察を行った。
（１－２）試験結果
５０細胞分の染色体数を測定した結果、すべての細胞において、染色体数は、正常ヒト染色体数である４６であった（図１（Ｂ））。また、ギムザ染色では顕著な染色体構造の異常は観察されなかった（図１（Ａ））。他の２細胞株でも同様の結果が得られた。 [Example 1] Character analysis of human DFAT (1) Karyotype analysis (1-1) Test method (i) Preparation of human DFAT This was carried out with reference to Japanese Patent Application Laid-Open No. 2000-83656 made by the present inventors. . That is, a human adipose subcutaneous human tissue was treated with collagenase, and then filtered with a mesh having a diameter of 100 to 200 μm to collect a single fraction consisting only of monocystic adipocytes. Human dedifferentiated adipocytes (human DFAT) were prepared by subculturing fibroblast-like adipocytes produced by ceiling culture of these monocystic adipocytes.
Donor subjects are patients aged 10 to 82 who have undergone surgery in plastic surgery, orthopedic surgery, and pediatric surgery at Nihon University Hospital. The method for adjusting human DFAT is the same in the following test examples.
(Ii) The cultured human DFAT (n = 3) obtained in (i) was treated with colcemid to prepare a chromosome sample, and the number of chromosomes was measured and the chromosome structure was observed by Giemsa staining.
(1-2) Test Results As a result of measuring the number of chromosomes for 50 cells, the number of chromosomes in all cells was 46, which is the number of normal human chromosomes (FIG. 1 (B)). In addition, no remarkable abnormality in the chromosomal structure was observed with Giemsa staining (FIG. 1 (A)). Similar results were obtained with the other two cell lines.
（２）ＣＧＨマイクロアレイ
（２－１）試験方法
ヒト成熟脂肪細胞およびヒトＤＦＡＴ（ヒト成熟脂肪細胞から実施例１の方法により調製）（ｎ＝３）からゲノムＤＮＡを抽出し、ヒト成熟脂肪細胞とヒトＤＦＡＴのゲノムのコピー数の変化を、ＣＧＨマイクロアレイ（Ａｇｉｌｅｎｔ社）を用いて解析した。
（２－２）試験結果
上記培養前後におけるゲノムＤＮＡのコピー数の増幅はほとんど認められず、ＤＦＡＴのゲノムコピー数のプロファイルは成熟脂肪細胞とほぼ一致していた（図示せず）。成熟脂肪細胞の脱分化によりＤＦＡＴが産生される過程において、ゲノムコピー数はほとんど変化しないことが明らかになった。 (2) CGH microarray (2-1) Test method Genomic DNA was extracted from human mature adipocytes and human DFAT (prepared from the human mature adipocytes by the method of Example 1) (n = 3). Changes in the human DFAT genome copy number were analyzed using a CGH microarray (Agilent).
(2-2) Test Results Almost no amplification of the genomic DNA copy number was observed before and after the culture, and the profile of DFAT genomic copy number was almost the same as that of mature adipocytes (not shown). In the process of producing DFAT by dedifferentiation of mature adipocytes, it was revealed that the genome copy number hardly changed.
（２－１）試験方法
ヒト成熟脂肪細胞およびヒトＤＦＡＴ（ヒト成熟脂肪細胞から実施例１の方法により調製）（ｎ＝３）からゲノムＤＮＡを抽出し、ヒト成熟脂肪細胞とヒトＤＦＡＴのゲノムのコピー数の変化を、ＣＧＨマイクロアレイ（Ａｇｉｌｅｎｔ社）を用いて解析した。
（２－２）試験結果
上記培養前後におけるゲノムＤＮＡのコピー数の増幅はほとんど認められず、ＤＦＡＴのゲノムコピー数のプロファイルは成熟脂肪細胞とほぼ一致していた（図示せず）。成熟脂肪細胞の脱分化によりＤＦＡＴが産生される過程において、ゲノムコピー数はほとんど変化しないことが明らかになった。 (2) CGH microarray (2-1) Test method Genomic DNA was extracted from human mature adipocytes and human DFAT (prepared from the human mature adipocytes by the method of Example 1) (n = 3). Changes in the human DFAT genome copy number were analyzed using a CGH microarray (Agilent).
(2-2) Test Results Almost no amplification of the genomic DNA copy number was observed before and after the culture, and the profile of DFAT genomic copy number was almost the same as that of mature adipocytes (not shown). In the process of producing DFAT by dedifferentiation of mature adipocytes, it was revealed that the genome copy number hardly changed.
（３）ＤＮＡメチル化解析（ＭａｓｓＡＲＲＡＹ法）
（３－１）試験方法
ヒト成熟脂肪細胞およびヒトＤＦＡＴ（ｎ＝３）からゲノムＤＮＡを抽出し、既知の癌関連遺伝子（計９６遺伝子）のプロモーター領域ＣｐＧアイランドにおけるＤＮＡメチル化修飾を、ＭａｓｓＡＲＲＡＹ法（Ｓｅｑｕｅｎｏｍ社）を用いて網羅的に解析した（図３）。
（３－２）試験結果
成熟脂肪細胞とＤＦＡＴとの間には、癌関連遺伝子のプロモーター領域ＣｐＧのメチル化プロファイルは非常に類似しており、脱分化に伴う特徴的なメチル化変動は検討した遺伝子群（９２遺伝子ＣｐＧ）の中では全く確認されなかった。 (3) DNA methylation analysis (MassARRAY method)
(3-1) Test Method Genomic DNA is extracted from human mature adipocytes and human DFAT (n = 3), and DNA methylation modification in the promoter region CpG island of known cancer-related genes (96 genes in total) is performed by MassARRAY method. (Sequenom) was used for comprehensive analysis (FIG. 3).
(3-2) Test results The methylation profile of the promoter region CpG of cancer-related genes is very similar between mature adipocytes and DFAT, and characteristic methylation fluctuations associated with dedifferentiation were examined. It was not confirmed at all in the gene group (92 genes CpG).
（３－１）試験方法
ヒト成熟脂肪細胞およびヒトＤＦＡＴ（ｎ＝３）からゲノムＤＮＡを抽出し、既知の癌関連遺伝子（計９６遺伝子）のプロモーター領域ＣｐＧアイランドにおけるＤＮＡメチル化修飾を、ＭａｓｓＡＲＲＡＹ法（Ｓｅｑｕｅｎｏｍ社）を用いて網羅的に解析した（図３）。
（３－２）試験結果
成熟脂肪細胞とＤＦＡＴとの間には、癌関連遺伝子のプロモーター領域ＣｐＧのメチル化プロファイルは非常に類似しており、脱分化に伴う特徴的なメチル化変動は検討した遺伝子群（９２遺伝子ＣｐＧ）の中では全く確認されなかった。 (3) DNA methylation analysis (MassARRAY method)
(3-1) Test Method Genomic DNA is extracted from human mature adipocytes and human DFAT (n = 3), and DNA methylation modification in the promoter region CpG island of known cancer-related genes (96 genes in total) is performed by MassARRAY method. (Sequenom) was used for comprehensive analysis (FIG. 3).
(3-2) Test results The methylation profile of the promoter region CpG of cancer-related genes is very similar between mature adipocytes and DFAT, and characteristic methylation fluctuations associated with dedifferentiation were examined. It was not confirmed at all in the gene group (92 genes CpG).
（４）テロメラーゼ活性測定
（４－１）試験方法
市販されているテロメラーゼ測定キット（ＴｅｌｏＴＡＧＡＡ Ｔｅｌｏｍｅｒａｓｅ ＰＣＲ ＥＬＩＳＡｐｌｕｓ、ロシュ・ダイアグノスティックス株式会社）を用いて、異なった年齢から調整した培養ヒトＤＦＡＴ（ｎ＝３、細胞数：２×１０５）より細胞抽出液を採取し、テロメラーゼ活性を測定した。
（４－２）試験結果
キット内の内部スタンダードとコントロールテンプレートを用いてテロメラーゼ反応産物量を定量した結果、測定したすべての培養ヒトＤＦＡＴにおいてテロメラーゼ活性は検出されなかった。この結果により、癌細胞で高頻度に見られる既知の異常所見（染色体数の異常、ゲノムコピー数の異常、癌関連遺伝子のメチル化異常、テロメラーゼ活性の亢進）がヒトＤＦＡＴでは認められず、細胞の安全性を示す重要な所見が得られた。 (4) Measurement of telomerase activity (4-1) Test method Cultured human DFAT (n) adjusted from different ages using a commercially available telomerase measurement kit (TeloTAGAA Telomerase PCR ELISAplus, Roche Diagnostics Inc.) = 3, cell number: 2 × 10 5 ), cell extracts were collected and telomerase activity was measured.
(4-2) Test Results As a result of quantifying the amount of telomerase reaction product using the internal standard and control template in the kit, telomerase activity was not detected in all measured cultured human DFAT. As a result, known abnormal findings (abnormal number of chromosomes, abnormal number of genome copies, abnormal methylation of cancer-related genes, increased telomerase activity) frequently observed in cancer cells were not observed in human DFAT. The important findings showing the safety of
（４－１）試験方法
市販されているテロメラーゼ測定キット（ＴｅｌｏＴＡＧＡＡ Ｔｅｌｏｍｅｒａｓｅ ＰＣＲ ＥＬＩＳＡｐｌｕｓ、ロシュ・ダイアグノスティックス株式会社）を用いて、異なった年齢から調整した培養ヒトＤＦＡＴ（ｎ＝３、細胞数：２×１０５）より細胞抽出液を採取し、テロメラーゼ活性を測定した。
（４－２）試験結果
キット内の内部スタンダードとコントロールテンプレートを用いてテロメラーゼ反応産物量を定量した結果、測定したすべての培養ヒトＤＦＡＴにおいてテロメラーゼ活性は検出されなかった。この結果により、癌細胞で高頻度に見られる既知の異常所見（染色体数の異常、ゲノムコピー数の異常、癌関連遺伝子のメチル化異常、テロメラーゼ活性の亢進）がヒトＤＦＡＴでは認められず、細胞の安全性を示す重要な所見が得られた。 (4) Measurement of telomerase activity (4-1) Test method Cultured human DFAT (n) adjusted from different ages using a commercially available telomerase measurement kit (TeloTAGAA Telomerase PCR ELISAplus, Roche Diagnostics Inc.) = 3, cell number: 2 × 10 5 ), cell extracts were collected and telomerase activity was measured.
(4-2) Test Results As a result of quantifying the amount of telomerase reaction product using the internal standard and control template in the kit, telomerase activity was not detected in all measured cultured human DFAT. As a result, known abnormal findings (abnormal number of chromosomes, abnormal number of genome copies, abnormal methylation of cancer-related genes, increased telomerase activity) frequently observed in cancer cells were not observed in human DFAT. The important findings showing the safety of
［実施例２］ヒトＤＦＡＴの血管新生能の検討
１．ＤＦＡＴ分泌サイトカインの解析
（１）発現サイトカインのプロテインアレイ解析
（１－１）試験方法
（ｉ）ヒトＡＳＣの調整方法
実施例１と同様にして得られたヒトＤＦＡＴの沈降分画（間質血管分画 ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ ｆｒａｃｔｉｏｎ：ＳＶＦ）からＳＶＦ細胞を採取し、培養フラスコ内で約２週間付着培養を行い、ヒトＡＳＣを調製した。ヒトＡＳＣの調整方法は以下の試験例において同じである。
（ｉｉｉ）第２～第４継代のそれぞれの細胞がコンフルエントに到達した時点で、培地を５％ウシ胎仔血清（ＦＢＳ）含有ＤＭＥＭ培地５ｍｌに交換し、さらに７２時間培養した。
その後培養ヒトＤＦＡＴ，ヒトＡＳＣより培養上清を採取し、０．４５ｍｍフィルターで濾過した後、プロテインアレイ法にて発現、分泌するサイトカインを検出した。
（１－２）試験結果
ヒトＤＦＡＴからＴＩＭＰ－１、ＴＩＭＰ－２、ＩＬ－６、ＩＬ－８、ＭＣＰ－１などの種々のサイトカイン群の分泌が確認された（図３）。 [Example 2] Examination of angiogenic ability of human DFAT Analysis of DFAT-secreted cytokine (1) Protein array analysis of expressed cytokine (1-1) Test method (i) Preparation method of human ASC Precipitation fraction of human DFAT obtained in the same manner as in Example 1 (stromal vascular fraction) SVF cells were collected from the fractional basal fraction (SVF) and cultured for about 2 weeks in a culture flask to prepare human ASC. The method for adjusting human ASC is the same in the following test examples.
(Iii) When the cells of the second to fourth passages reached confluence, the medium was replaced with 5 ml of 5% fetal bovine serum (FBS) -containing DMEM medium, and further cultured for 72 hours.
Thereafter, the culture supernatant was collected from cultured human DFAT and human ASC, filtered through a 0.45 mm filter, and then the cytokine expressed and secreted by the protein array method was detected.
(1-2) Test Results Secretion of various cytokine groups such as TIMP-1, TIMP-2, IL-6, IL-8 and MCP-1 was confirmed from human DFAT (FIG. 3).
１．ＤＦＡＴ分泌サイトカインの解析
（１）発現サイトカインのプロテインアレイ解析
（１－１）試験方法
（ｉ）ヒトＡＳＣの調整方法
実施例１と同様にして得られたヒトＤＦＡＴの沈降分画（間質血管分画 ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ ｆｒａｃｔｉｏｎ：ＳＶＦ）からＳＶＦ細胞を採取し、培養フラスコ内で約２週間付着培養を行い、ヒトＡＳＣを調製した。ヒトＡＳＣの調整方法は以下の試験例において同じである。
（ｉｉｉ）第２～第４継代のそれぞれの細胞がコンフルエントに到達した時点で、培地を５％ウシ胎仔血清（ＦＢＳ）含有ＤＭＥＭ培地５ｍｌに交換し、さらに７２時間培養した。
その後培養ヒトＤＦＡＴ，ヒトＡＳＣより培養上清を採取し、０．４５ｍｍフィルターで濾過した後、プロテインアレイ法にて発現、分泌するサイトカインを検出した。
（１－２）試験結果
ヒトＤＦＡＴからＴＩＭＰ－１、ＴＩＭＰ－２、ＩＬ－６、ＩＬ－８、ＭＣＰ－１などの種々のサイトカイン群の分泌が確認された（図３）。 [Example 2] Examination of angiogenic ability of human DFAT Analysis of DFAT-secreted cytokine (1) Protein array analysis of expressed cytokine (1-1) Test method (i) Preparation method of human ASC Precipitation fraction of human DFAT obtained in the same manner as in Example 1 (stromal vascular fraction) SVF cells were collected from the fractional basal fraction (SVF) and cultured for about 2 weeks in a culture flask to prepare human ASC. The method for adjusting human ASC is the same in the following test examples.
(Iii) When the cells of the second to fourth passages reached confluence, the medium was replaced with 5 ml of 5% fetal bovine serum (FBS) -containing DMEM medium, and further cultured for 72 hours.
Thereafter, the culture supernatant was collected from cultured human DFAT and human ASC, filtered through a 0.45 mm filter, and then the cytokine expressed and secreted by the protein array method was detected.
(1-2) Test Results Secretion of various cytokine groups such as TIMP-1, TIMP-2, IL-6, IL-8 and MCP-1 was confirmed from human DFAT (FIG. 3).
（２）発現サイトカインのＥＬＩＳＡ法による定量
（２－１）試験方法
培養ヒトＤＦＡＴ（各ドナーにつきｎ＝３），ヒトＡＳＣ（ヒトＤＦＡＴと同一ドナー由来、各ドナーにつきｎ＝３），ヒト前駆脂肪細胞（ｈＰｒｅａｄｉｐｏｃｙｔｅ），ヒト骨髄ＭＳＣ（ｈＢＭ－ＭＳＣ），ヒト線維芽細胞（ｈＦｉｂｒｏｂｌａｓｔ）より培養上清を採取し、プロテインアレイで分泌が確認されたサイトカインを中心に培養上清中の濃度を、ＥＬＩＳＡキットを用いて測定した。検討対象サイトカインとしてＨＧＦ，ＶＥＧＦ，ｂＦＧＦ，ＳＤＦ－１，ＩＬ－６，ＩＬ－８，ＭＣＰ－１，Ｌｅｐｔｉｎ，ＩＧＦ－１，ＴＧＦβ１を測定した。各細胞の調整方法を以下に示す。測定は、各ドナーにつき３つの検体を調整して（ｎ＝３）測定し、得られた３つのデータの平均値と標準偏差を求めた。
（ｉ）ヒト前駆脂肪細胞（ｈＰｒｅａｄｉｐｏｃｙｔｅ）
ＤＳファーマメディカル株式会社より購入したヒト前駆脂肪細胞を解凍、洗浄し、付着培養して調製した。培地は、１０％ＦＢＳ含有ＤＭＥＭを用いた。ヒト前駆脂肪細胞の調整方法は以下の試験例において同じである。
（ｉｉ）ヒト骨髄ＭＳＣ（ｈＢＭ－ＭＳＣ）
市販されている初代培養細胞（Ｌｏｎｚａ社）を解凍、洗浄し、付着培養して調製した。培地は、１０％ＦＢＳ含有ＤＭＥＭを用いた。ヒト骨髄ＭＳＣの調整方法は以下の試験例において同じである。
（２－２）試験結果
ヒトＤＦＡＴからはＨＧＦ，ＶＥＧＦ，ＳＤＦ－１，ＩＬ－６，ＩＬ－８，ＭＣＰ－１，ｌｅｐｔｉｎが高発現（１－１０ｎｇ／ｍｌ）していることが確認された。特に、ＨＧＦおよびＳＤＦ－１はｐｒｅａｄｉｐｏｃｙｔｅ、ヒトＡＳＣ、ヒト骨髄ＭＳＣに比べて特徴的に発現が高いことが明らかになった（図４）。 (2) Quantification of expressed cytokines by ELISA method (2-1) Test method Cultured human DFAT (n = 3 for each donor), human ASC (derived from the same donor as human DFAT, n = 3 for each donor), human preadip Culture supernatants are collected from cells (hPreadiposite), human bone marrow MSC (hBM-MSC), and human fibroblasts (hFibroblast). Measurement was performed using a kit. HGF, VEGF, bFGF, SDF-1, IL-6, IL-8, MCP-1, Leptin, IGF-1, and TGFβ1 were measured as cytokines to be examined. The adjustment method of each cell is shown below. In the measurement, three specimens were adjusted for each donor (n = 3), and the average value and standard deviation of the obtained three data were obtained.
(I) Human preadipocytes (hPreadipocyte)
Human preadipocytes purchased from DS Pharma Medical Co., Ltd. were prepared by thawing, washing and adherent culture. As the medium, DMEM containing 10% FBS was used. The method for preparing human preadipocytes is the same in the following test examples.
(Ii) Human bone marrow MSC (hBM-MSC)
Commercially available primary cultured cells (Lonza) were prepared by thawing, washing and adherent culture. As the medium, DMEM containing 10% FBS was used. The method for adjusting human bone marrow MSC is the same in the following test examples.
(2-2) Test results From human DFAT, it was confirmed that HGF, VEGF, SDF-1, IL-6, IL-8, MCP-1, and leptin were highly expressed (1-10 ng / ml). . In particular, HGF and SDF-1 were found to be characteristically higher in expression than preadipocyte, human ASC, and human bone marrow MSC (FIG. 4).
（２－１）試験方法
培養ヒトＤＦＡＴ（各ドナーにつきｎ＝３），ヒトＡＳＣ（ヒトＤＦＡＴと同一ドナー由来、各ドナーにつきｎ＝３），ヒト前駆脂肪細胞（ｈＰｒｅａｄｉｐｏｃｙｔｅ），ヒト骨髄ＭＳＣ（ｈＢＭ－ＭＳＣ），ヒト線維芽細胞（ｈＦｉｂｒｏｂｌａｓｔ）より培養上清を採取し、プロテインアレイで分泌が確認されたサイトカインを中心に培養上清中の濃度を、ＥＬＩＳＡキットを用いて測定した。検討対象サイトカインとしてＨＧＦ，ＶＥＧＦ，ｂＦＧＦ，ＳＤＦ－１，ＩＬ－６，ＩＬ－８，ＭＣＰ－１，Ｌｅｐｔｉｎ，ＩＧＦ－１，ＴＧＦβ１を測定した。各細胞の調整方法を以下に示す。測定は、各ドナーにつき３つの検体を調整して（ｎ＝３）測定し、得られた３つのデータの平均値と標準偏差を求めた。
（ｉ）ヒト前駆脂肪細胞（ｈＰｒｅａｄｉｐｏｃｙｔｅ）
ＤＳファーマメディカル株式会社より購入したヒト前駆脂肪細胞を解凍、洗浄し、付着培養して調製した。培地は、１０％ＦＢＳ含有ＤＭＥＭを用いた。ヒト前駆脂肪細胞の調整方法は以下の試験例において同じである。
（ｉｉ）ヒト骨髄ＭＳＣ（ｈＢＭ－ＭＳＣ）
市販されている初代培養細胞（Ｌｏｎｚａ社）を解凍、洗浄し、付着培養して調製した。培地は、１０％ＦＢＳ含有ＤＭＥＭを用いた。ヒト骨髄ＭＳＣの調整方法は以下の試験例において同じである。
（２－２）試験結果
ヒトＤＦＡＴからはＨＧＦ，ＶＥＧＦ，ＳＤＦ－１，ＩＬ－６，ＩＬ－８，ＭＣＰ－１，ｌｅｐｔｉｎが高発現（１－１０ｎｇ／ｍｌ）していることが確認された。特に、ＨＧＦおよびＳＤＦ－１はｐｒｅａｄｉｐｏｃｙｔｅ、ヒトＡＳＣ、ヒト骨髄ＭＳＣに比べて特徴的に発現が高いことが明らかになった（図４）。 (2) Quantification of expressed cytokines by ELISA method (2-1) Test method Cultured human DFAT (n = 3 for each donor), human ASC (derived from the same donor as human DFAT, n = 3 for each donor), human preadip Culture supernatants are collected from cells (hPreadiposite), human bone marrow MSC (hBM-MSC), and human fibroblasts (hFibroblast). Measurement was performed using a kit. HGF, VEGF, bFGF, SDF-1, IL-6, IL-8, MCP-1, Leptin, IGF-1, and TGFβ1 were measured as cytokines to be examined. The adjustment method of each cell is shown below. In the measurement, three specimens were adjusted for each donor (n = 3), and the average value and standard deviation of the obtained three data were obtained.
(I) Human preadipocytes (hPreadipocyte)
Human preadipocytes purchased from DS Pharma Medical Co., Ltd. were prepared by thawing, washing and adherent culture. As the medium, DMEM containing 10% FBS was used. The method for preparing human preadipocytes is the same in the following test examples.
(Ii) Human bone marrow MSC (hBM-MSC)
Commercially available primary cultured cells (Lonza) were prepared by thawing, washing and adherent culture. As the medium, DMEM containing 10% FBS was used. The method for adjusting human bone marrow MSC is the same in the following test examples.
(2-2) Test results From human DFAT, it was confirmed that HGF, VEGF, SDF-1, IL-6, IL-8, MCP-1, and leptin were highly expressed (1-10 ng / ml). . In particular, HGF and SDF-1 were found to be characteristically higher in expression than preadipocyte, human ASC, and human bone marrow MSC (FIG. 4).
２．ヒトＤＦＡＴ培養上清のｉｎ ｖｉｔｒｏにおける血管新生能
（１）ヒトＤＦＡＴとヒト骨髄ＭＳＣ（ｈＢＭ－ＭＳＣ）の比較
（１－１）試験方法
血管新生キット（ＫＺ－１０００，ＫＵＲＡＢＯ，Ｏｓａｋａ，Ｊａｐａｎ）を用いて、ヒトＤＦＡＴ培養上清のｉｎ ｖｉｔｒｏにおける血管新生能を検討した。本キットは２４ウェルプレートにあらかじめヒト線維芽細胞とヒト血管内皮細胞が共培養され、専用の血管新生培地で培養することで血管内皮細胞の管腔形成が誘導されるデザインである。これに被検物質を含むサンプル培地を加えて培養することで、管腔形成能に差が生じる。血管新生能の測定は本キットのプロトコールに従って管腔形成を誘導し、マウス抗ヒトＣＤ３１抗体を用いた免疫染色により管腔形成を可視可した。ＤＦＡＴ培養上清やヒト骨髄ＭＳＣ培養上清は上記の方法で調整し、付属の血管新生培地に１：１で混合してサンプル培地とした。
（１－２）試験結果
ヒトＤＦＡＴの培養上清は血管内皮細胞の増殖、管腔形成を著明に促進した。ＤＦＡＴの血管新生能は、骨髄ＭＳＣと同等またはそれ以上であった（図５）。 2. Angiogenic ability of human DFAT culture supernatant in vitro (1) Comparison of human DFAT and human bone marrow MSC (hBM-MSC) (1-1) Test method Angiogenesis kit (KZ-1000, KURABO, Osaka, Japan) The in vitro angiogenesis ability of the human DFAT culture supernatant was examined. This kit is a design in which human fibroblasts and human vascular endothelial cells are co-cultured in advance in a 24-well plate, and luminal formation of vascular endothelial cells is induced by culturing in a dedicated angiogenic medium. When a sample medium containing a test substance is added to this and cultured, a difference in lumen forming ability occurs. The angiogenesis ability was measured by inducing tube formation according to the protocol of this kit, and tube formation was visualized by immunostaining using a mouse anti-human CD31 antibody. The DFAT culture supernatant and human bone marrow MSC culture supernatant were prepared by the above method and mixed 1: 1 with the attached angiogenesis medium to obtain a sample medium.
(1-2) Test Results The culture supernatant of human DFAT markedly promoted the proliferation and lumen formation of vascular endothelial cells. The angiogenic potential of DFAT was equal to or greater than that of bone marrow MSC (FIG. 5).
（１）ヒトＤＦＡＴとヒト骨髄ＭＳＣ（ｈＢＭ－ＭＳＣ）の比較
（１－１）試験方法
血管新生キット（ＫＺ－１０００，ＫＵＲＡＢＯ，Ｏｓａｋａ，Ｊａｐａｎ）を用いて、ヒトＤＦＡＴ培養上清のｉｎ ｖｉｔｒｏにおける血管新生能を検討した。本キットは２４ウェルプレートにあらかじめヒト線維芽細胞とヒト血管内皮細胞が共培養され、専用の血管新生培地で培養することで血管内皮細胞の管腔形成が誘導されるデザインである。これに被検物質を含むサンプル培地を加えて培養することで、管腔形成能に差が生じる。血管新生能の測定は本キットのプロトコールに従って管腔形成を誘導し、マウス抗ヒトＣＤ３１抗体を用いた免疫染色により管腔形成を可視可した。ＤＦＡＴ培養上清やヒト骨髄ＭＳＣ培養上清は上記の方法で調整し、付属の血管新生培地に１：１で混合してサンプル培地とした。
（１－２）試験結果
ヒトＤＦＡＴの培養上清は血管内皮細胞の増殖、管腔形成を著明に促進した。ＤＦＡＴの血管新生能は、骨髄ＭＳＣと同等またはそれ以上であった（図５）。 2. Angiogenic ability of human DFAT culture supernatant in vitro (1) Comparison of human DFAT and human bone marrow MSC (hBM-MSC) (1-1) Test method Angiogenesis kit (KZ-1000, KURABO, Osaka, Japan) The in vitro angiogenesis ability of the human DFAT culture supernatant was examined. This kit is a design in which human fibroblasts and human vascular endothelial cells are co-cultured in advance in a 24-well plate, and luminal formation of vascular endothelial cells is induced by culturing in a dedicated angiogenic medium. When a sample medium containing a test substance is added to this and cultured, a difference in lumen forming ability occurs. The angiogenesis ability was measured by inducing tube formation according to the protocol of this kit, and tube formation was visualized by immunostaining using a mouse anti-human CD31 antibody. The DFAT culture supernatant and human bone marrow MSC culture supernatant were prepared by the above method and mixed 1: 1 with the attached angiogenesis medium to obtain a sample medium.
(1-2) Test Results The culture supernatant of human DFAT markedly promoted the proliferation and lumen formation of vascular endothelial cells. The angiogenic potential of DFAT was equal to or greater than that of bone marrow MSC (FIG. 5).
（２）ドナー年齢の違いによる比較
（２－１）試験方法
ドナー年齢の違いによるＤＦＡＴ培養上清の血管新生能について検討した。ポジテイブコントロールとしてＶＥＧＦ－Ａ（１０ｎｇ／ｍｌ Ｒ＆Ｄ Ｓｙｓｔｅｍｓ）を使用した。
（ｉ）ドナー年齢および性別
２歳男性、２９歳女性、５６歳女性、７５歳女性、８２歳女性の５人である。
（２－２）試験結果
高齢ドナー（７５歳、８２歳）由来のヒトＤＦＡＴ培養上清が、若年者と比較しても同等の血管新生能を有することが明らかになった（図６）。すなわち、ヒトＤＦＡＴ培養上清の血管内皮細胞の管腔形成能に年齢による違いはなかった。 (2) Comparison by difference in donor age (2-1) Test method The angiogenic ability of the DFAT culture supernatant by difference in donor age was examined. VEGF-A (10 ng / ml R & D Systems) was used as a positive control.
(I) Donor age and sex There are five people: a 2-year-old man, a 29-year-old woman, a 56-year-old woman, a 75-year-old woman, and an 82-year-old woman.
(2-2) Test Results It was revealed that the human DFAT culture supernatant derived from an elderly donor (75 years old, 82 years old) has the same angiogenic ability as compared with younger ones (FIG. 6). That is, there was no difference with age in the lumen forming ability of vascular endothelial cells of the human DFAT culture supernatant.
（２－１）試験方法
ドナー年齢の違いによるＤＦＡＴ培養上清の血管新生能について検討した。ポジテイブコントロールとしてＶＥＧＦ－Ａ（１０ｎｇ／ｍｌ Ｒ＆Ｄ Ｓｙｓｔｅｍｓ）を使用した。
（ｉ）ドナー年齢および性別
２歳男性、２９歳女性、５６歳女性、７５歳女性、８２歳女性の５人である。
（２－２）試験結果
高齢ドナー（７５歳、８２歳）由来のヒトＤＦＡＴ培養上清が、若年者と比較しても同等の血管新生能を有することが明らかになった（図６）。すなわち、ヒトＤＦＡＴ培養上清の血管内皮細胞の管腔形成能に年齢による違いはなかった。 (2) Comparison by difference in donor age (2-1) Test method The angiogenic ability of the DFAT culture supernatant by difference in donor age was examined. VEGF-A (10 ng / ml R & D Systems) was used as a positive control.
(I) Donor age and sex There are five people: a 2-year-old man, a 29-year-old woman, a 56-year-old woman, a 75-year-old woman, and an 82-year-old woman.
(2-2) Test Results It was revealed that the human DFAT culture supernatant derived from an elderly donor (75 years old, 82 years old) has the same angiogenic ability as compared with younger ones (FIG. 6). That is, there was no difference with age in the lumen forming ability of vascular endothelial cells of the human DFAT culture supernatant.
（３）ヒトＤＦＡＴとＡＳＣ（同一ドナー由来）の比較
（３－１）試験方法
同一ドナーに由来するヒトＤＦＡＴとヒトＡＳＣの血管新生能の比較検討を行った。ヒトＤＦＡＴとヒトＡＳＣの調整方法は、実施例１と同じである。
（３－２）試験結果
ヒトＤＦＡＴ培養上清はヒトＡＳＣ培養上清と比較して管腔長、管腔面積、分枝数および接合部数などが有意に高値を示した（図７）。 (3) Comparison between human DFAT and ASC (from the same donor) (3-1) Test method A comparative study was conducted on the angiogenic ability of human DFAT and human ASC derived from the same donor. The method for adjusting human DFAT and human ASC is the same as in Example 1.
(3-2) Test Results Human DFAT culture supernatant showed significantly higher lumen length, lumen area, number of branches, number of junctions, etc. than human ASC culture supernatant (FIG. 7).
（３－１）試験方法
同一ドナーに由来するヒトＤＦＡＴとヒトＡＳＣの血管新生能の比較検討を行った。ヒトＤＦＡＴとヒトＡＳＣの調整方法は、実施例１と同じである。
（３－２）試験結果
ヒトＤＦＡＴ培養上清はヒトＡＳＣ培養上清と比較して管腔長、管腔面積、分枝数および接合部数などが有意に高値を示した（図７）。 (3) Comparison between human DFAT and ASC (from the same donor) (3-1) Test method A comparative study was conducted on the angiogenic ability of human DFAT and human ASC derived from the same donor. The method for adjusting human DFAT and human ASC is the same as in Example 1.
(3-2) Test Results Human DFAT culture supernatant showed significantly higher lumen length, lumen area, number of branches, number of junctions, etc. than human ASC culture supernatant (FIG. 7).
［実施例３］免疫不全マウス下肢虚血モデルにおけるヒトＤＦＡＴ移植実験
１．ヒトＤＦＡＴのモデルマウスの筋肉内への移植
（１）試験方法
免疫不全（ＳＣＩＤ）マウス下肢虚血モデル（日本クレア株式会社、ｎ＝２０）に対し、虚血作成６時間後にヒトＤＦＡＴ（１ｘ１０５）を虚血筋肉内に移植し、レーザードップラー血流計による血流測定および組織学的検討を行った。そして生理食塩水を投与するコントロール群、ヒト末梢血単核球を移植する群との比較検討を行った。
（ｉ）ヒト末梢血単核球の調整方法
健常人の血液から血球分離溶液Ｌｙｍｐｈｏｐｒｅｐ（コスモバイオ株式会社製）を用いた比重遠心法により調整した。
（２）試験結果
ＤＦＡＴ移植群ではコントロール群に比べ、移植３週後より有意に血流改善が認められた。また、ＤＦＡＴ移植した虚血組織では血管密度が有意に増加していた。ＤＦＡＴ移植による血流改善効果は、ヒト末梢血単核球移植に比べても優れていることが明らかになった（図８）。 [Example 3] Human DFAT transplantation experiment in an immunodeficient mouse lower limb ischemia model Transplantation of human DFAT into muscles of model mice (1) Test method Human DFAT (1 × 10 5 ) 6 hours after ischemia generation against immunodeficient (SCID) mouse lower limb ischemia model (CLEA Japan, n = 20) ) Was transplanted into ischemic muscle, and blood flow measurement and histological examination were performed with a laser Doppler blood flow meter. Then, a comparative study was performed between a control group administered with physiological saline and a group transplanted with human peripheral blood mononuclear cells.
(I) Method for adjusting human peripheral blood mononuclear cells The blood was adjusted from a healthy person by a specific gravity centrifugation method using a blood cell separation solution Lymphoprep (manufactured by Cosmo Bio Co., Ltd.).
(2) Test results Significant improvement in blood flow was observed in the DFAT transplant group compared to thecontrol group 3 weeks after transplantation. Further, the blood vessel density was significantly increased in the ischemic tissue transplanted with DFAT. It was revealed that the blood flow improvement effect by DFAT transplantation was superior to that of human peripheral blood mononuclear cell transplantation (FIG. 8).
１．ヒトＤＦＡＴのモデルマウスの筋肉内への移植
（１）試験方法
免疫不全（ＳＣＩＤ）マウス下肢虚血モデル（日本クレア株式会社、ｎ＝２０）に対し、虚血作成６時間後にヒトＤＦＡＴ（１ｘ１０５）を虚血筋肉内に移植し、レーザードップラー血流計による血流測定および組織学的検討を行った。そして生理食塩水を投与するコントロール群、ヒト末梢血単核球を移植する群との比較検討を行った。
（ｉ）ヒト末梢血単核球の調整方法
健常人の血液から血球分離溶液Ｌｙｍｐｈｏｐｒｅｐ（コスモバイオ株式会社製）を用いた比重遠心法により調整した。
（２）試験結果
ＤＦＡＴ移植群ではコントロール群に比べ、移植３週後より有意に血流改善が認められた。また、ＤＦＡＴ移植した虚血組織では血管密度が有意に増加していた。ＤＦＡＴ移植による血流改善効果は、ヒト末梢血単核球移植に比べても優れていることが明らかになった（図８）。 [Example 3] Human DFAT transplantation experiment in an immunodeficient mouse lower limb ischemia model Transplantation of human DFAT into muscles of model mice (1) Test method Human DFAT (1 × 10 5 ) 6 hours after ischemia generation against immunodeficient (SCID) mouse lower limb ischemia model (CLEA Japan, n = 20) ) Was transplanted into ischemic muscle, and blood flow measurement and histological examination were performed with a laser Doppler blood flow meter. Then, a comparative study was performed between a control group administered with physiological saline and a group transplanted with human peripheral blood mononuclear cells.
(I) Method for adjusting human peripheral blood mononuclear cells The blood was adjusted from a healthy person by a specific gravity centrifugation method using a blood cell separation solution Lymphoprep (manufactured by Cosmo Bio Co., Ltd.).
(2) Test results Significant improvement in blood flow was observed in the DFAT transplant group compared to the
２．蛍光ラベルしたヒトＤＦＡＴのモデルマウス組織への移植
（１）試験方法
ＳＣＩＤマウス下肢虚血モデル（ｎ＝１６）に対し、ＱＴｒａｃｋｅｒ（商品名、Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ社製）にて蛍光ラベルしたヒトＤＦＡＴ（１ｘ１０５）を左側虚血組織および右側健常組織内に移植し、移植２日、７日、１５日、１ヶ月、２ヶ月、３ヶ月、４ヶ月、６ヶ月後に２匹ずつ両則筋組織および肺、肝臓、腎臓、脾臓、腎臓を採取し、肉眼的、組織学的に移植細胞の局在、分布、腫瘍形成の有無について検討を行った。組織学的なヒトＤＦＡＴの同定はＱＴｒａｃｋｅｒによる蛍光および抗ＨＬＡ抗体（市販）による免疫染色で行った。
（２）試験結果
虚血組織内では移植１ヶ月、正常筋組織では移植１５日後までは移植部位を中心にＤＦＡＴが検出された。一方、虚血組織内では移植２ヶ月、正常筋組織では移植１ヶ月後からは移植したＤＦＡＴはほとんど検出されなくなり、その後も検討した全ての組織において、細胞の増殖性変化や腫瘍形成などの有害事象は認められなかった。 2. Transplantation of fluorescently labeled human DFAT into model mouse tissue (1) Test method For SCID mouse lower limb ischemia model (n = 16), fluorescently labeled human DFAT (1 × 10) by QTracker (trade name, manufactured by Life technologies) 5 ) transplanted into the left ischemic tissue and right healthy tissue, 2 days after transplantation, 2 days, 7 days, 15 days, 1 month, 2 months, 3 months, 4 months, 6months 2 mice each The liver, kidney, spleen, and kidney were collected, and the localization and distribution of transplanted cells and the presence or absence of tumor formation were examined macroscopically and histologically. Histological identification of human DFAT was performed by fluorescence with QTracker and immunostaining with anti-HLA antibody (commercially available).
(2) Test results DFAT was detected mainly in the transplantation site until 1 month after transplantation in ischemic tissue and 15 days after transplantation in normal muscle tissue. On the other hand, transplanted DFAT is hardly detected after 2 months of transplantation in ischemic tissue and 1 month after transplantation in normal muscle tissue, and harmful effects such as cell proliferative changes and tumor formation are observed in all tissues examined thereafter. There were no events.
（１）試験方法
ＳＣＩＤマウス下肢虚血モデル（ｎ＝１６）に対し、ＱＴｒａｃｋｅｒ（商品名、Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ社製）にて蛍光ラベルしたヒトＤＦＡＴ（１ｘ１０５）を左側虚血組織および右側健常組織内に移植し、移植２日、７日、１５日、１ヶ月、２ヶ月、３ヶ月、４ヶ月、６ヶ月後に２匹ずつ両則筋組織および肺、肝臓、腎臓、脾臓、腎臓を採取し、肉眼的、組織学的に移植細胞の局在、分布、腫瘍形成の有無について検討を行った。組織学的なヒトＤＦＡＴの同定はＱＴｒａｃｋｅｒによる蛍光および抗ＨＬＡ抗体（市販）による免疫染色で行った。
（２）試験結果
虚血組織内では移植１ヶ月、正常筋組織では移植１５日後までは移植部位を中心にＤＦＡＴが検出された。一方、虚血組織内では移植２ヶ月、正常筋組織では移植１ヶ月後からは移植したＤＦＡＴはほとんど検出されなくなり、その後も検討した全ての組織において、細胞の増殖性変化や腫瘍形成などの有害事象は認められなかった。 2. Transplantation of fluorescently labeled human DFAT into model mouse tissue (1) Test method For SCID mouse lower limb ischemia model (n = 16), fluorescently labeled human DFAT (1 × 10) by QTracker (trade name, manufactured by Life technologies) 5 ) transplanted into the left ischemic tissue and right healthy tissue, 2 days after transplantation, 2 days, 7 days, 15 days, 1 month, 2 months, 3 months, 4 months, 6
(2) Test results DFAT was detected mainly in the transplantation site until 1 month after transplantation in ischemic tissue and 15 days after transplantation in normal muscle tissue. On the other hand, transplanted DFAT is hardly detected after 2 months of transplantation in ischemic tissue and 1 month after transplantation in normal muscle tissue, and harmful effects such as cell proliferative changes and tumor formation are observed in all tissues examined thereafter. There were no events.
［実施例４］ヒトＤＦＡＴ分泌サイトカインの解析
（１）ドナー年齢の違いによる比較
（１－１）試験方法
培養ヒトＤＦＡＴ（各ドナーの継代数２，４，６，８につきｎ＝３）およびヒトＡＳＣ（ヒトＤＦＡＴと同一ドナー由来、各ドナーの継代数２，４，６，８につきｎ＝３）より培養上清を採取し、実施例２、（２）と同様の方法により、分泌されたサイトカイン（ＨＧＦ，ＶＥＧＦ）の濃度を測定した。
（ｉ）ドナー年齢および性別
生後一ヶ月男性、生後６ヶ月男性、３４歳女性、６１歳男性、７４歳女性の５人である。
（１－２）試験結果
ＨＧＦについては、いずれのドナー由来のヒトＤＦＡＴも、同一ドナー由来のヒトＡＳＣと比べて高発現している傾向が確認された。特に継代早期（継代数２、継代数４）や高齢ドナー（７４歳）では、ＨＧＦ濃度の差が顕著であることが確認された（図１０）。
また、ＶＥＧＦについては、ヒトＤＦＡＴは、同一ドナー由来のヒトＡＳＣと明らかな差はなく、ドナー年齢や継代数に影響されず、ほぼ同等に発現していることが確認された（図１１）。 [Example 4] Analysis of cytokine secreted by human DFAT (1) Comparison by difference in donor age (1-1) Test method Cultured human DFAT (n = 3 for passage numbers 2, 4, 6, 8 of each donor) and human Culture supernatant was collected from ASC (derived from the same donor as human DFAT, n = 3 per passage number 2, 4, 6, 8 of each donor) and secreted by the same method as in Example 2 (2). The concentration of cytokine (HGF, VEGF) was measured.
(I) Donor age and gender There are five men: one month old male, six month old male, 34 year old female, 61 year old male and 74 year old female.
(1-2) Test Results Regarding HGF, it was confirmed that the human DFAT derived from any donor tends to be highly expressed compared to human ASC derived from the same donor. In particular, in the early passage (passage number 2, passage number 4) and elderly donors (74 years old), it was confirmed that the difference in HGF concentration was remarkable (FIG. 10).
As for VEGF, human DFAT was not clearly different from human ASC derived from the same donor, and it was confirmed that it was expressed almost equally without being influenced by donor age or passage number (FIG. 11).
（１）ドナー年齢の違いによる比較
（１－１）試験方法
培養ヒトＤＦＡＴ（各ドナーの継代数２，４，６，８につきｎ＝３）およびヒトＡＳＣ（ヒトＤＦＡＴと同一ドナー由来、各ドナーの継代数２，４，６，８につきｎ＝３）より培養上清を採取し、実施例２、（２）と同様の方法により、分泌されたサイトカイン（ＨＧＦ，ＶＥＧＦ）の濃度を測定した。
（ｉ）ドナー年齢および性別
生後一ヶ月男性、生後６ヶ月男性、３４歳女性、６１歳男性、７４歳女性の５人である。
（１－２）試験結果
ＨＧＦについては、いずれのドナー由来のヒトＤＦＡＴも、同一ドナー由来のヒトＡＳＣと比べて高発現している傾向が確認された。特に継代早期（継代数２、継代数４）や高齢ドナー（７４歳）では、ＨＧＦ濃度の差が顕著であることが確認された（図１０）。
また、ＶＥＧＦについては、ヒトＤＦＡＴは、同一ドナー由来のヒトＡＳＣと明らかな差はなく、ドナー年齢や継代数に影響されず、ほぼ同等に発現していることが確認された（図１１）。 [Example 4] Analysis of cytokine secreted by human DFAT (1) Comparison by difference in donor age (1-1) Test method Cultured human DFAT (n = 3 for
(I) Donor age and gender There are five men: one month old male, six month old male, 34 year old female, 61 year old male and 74 year old female.
(1-2) Test Results Regarding HGF, it was confirmed that the human DFAT derived from any donor tends to be highly expressed compared to human ASC derived from the same donor. In particular, in the early passage (
As for VEGF, human DFAT was not clearly different from human ASC derived from the same donor, and it was confirmed that it was expressed almost equally without being influenced by donor age or passage number (FIG. 11).
本発明によれば、ヒト成熟脂肪細胞から調製されるヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を利用することで、末梢動脈疾患（ＰＡＤ）などの虚血性疾患に対する細胞治療方法を提供することができる。
ADVANTAGE OF THE INVENTION According to this invention, the cell therapy method with respect to ischemic diseases, such as a peripheral artery disease (PAD), can be provided by utilizing the human dedifferentiated fat cell (human DFAT) prepared from a human mature fat cell. .
Claims (5)
- ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする血流改善剤。 A blood flow improving agent comprising human dedifferentiated fat cells (human DFAT) as an active ingredient.
- ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする血管新生促進剤。 An angiogenesis promoter comprising human dedifferentiated adipocytes (human DFAT) as an active ingredient.
- ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）を有効成分とする虚血性疾患治療剤。 A therapeutic agent for ischemic disease comprising human dedifferentiated fat cells (human DFAT) as an active ingredient.
- 虚血性疾患が末梢動脈疾患（ＰＡＤ）または虚血性心筋症である、請求項３に記載の虚血性疾患治療剤。 The ischemic disease therapeutic agent according to claim 3, wherein the ischemic disease is peripheral arterial disease (PAD) or ischemic cardiomyopathy.
- ヒト脱分化脂肪細胞（ヒトＤＦＡＴ）が、１～４代継代培養されたヒト脱分化脂肪細胞（ヒトＤＦＡＴ）である、請求項１～４のいずれかに記載の剤。 The agent according to any one of claims 1 to 4, wherein the human dedifferentiated adipocytes (human DFAT) are human dedifferentiated adipocytes (human DFAT) subcultured for 1 to 4 passages.
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| JP2020066624A (en) * | 2018-10-18 | 2020-04-30 | 学校法人日本大学 | Composition for treating necrotizing enteritis |
| JP7348612B2 (en) | 2018-10-18 | 2023-09-21 | 学校法人日本大学 | Composition for treating necrotizing enterocolitis |
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