JP2018087141A - Compositions for revascularization therapy which contain dedifferentiated fat cells as an active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide revascularization therapeutic cells for which exert excellent bloodstream improving effect as compared with bone marrow MSC, ASC and the like which are therapeutic cells of the prior art, the cells being able to easily acquire a sufficient amount required for transplantation and having stable quality.SOLUTION: We found that human DFAT has not only high angiogenic ability but also has high proliferation ability after subculture and can easily acquire the cell number required for transplantation, and also causes no transformation, thereby clarifying for the first time that human DFAT is also effective for angiogenic therapy, and completing the present invention. That is, the invention provides a bloodstream improving agent containing human dedifferentiated fat cells (human DFAT) as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a composition for revascularization therapy. In particular, the present invention relates to a composition for revascularization therapy comprising a dedifferentiated fat cell (DFAT) as an active ingredient.

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 must be collected 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).
Moreover, 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 as in (vii). Therefore, both (vii) and (viii) cells have the merit that they can be prepared in large quantities from a relatively small amount of tissue. However, this is a method of culturing and proliferating stem cells that exist 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.
What can be said in common to the cells (i) to (viii) is that there are individual differences in performance and treatment-refractory cases (non-responders) despite the high treatment costs. It exists, and is usually limited to one treatment in consideration of invasiveness to the patient. 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 revealed that mouse and rat DFAT has angiogenic ability (Non-Patent Documents 1 to 3). However, DFAT in mice and rats has a low growth ability after subculture, so it was difficult to increase to the number necessary for transplantation, and transformation (immortalization) occurred by subculture. When this is applied to humans in the same way because of the possibility of tumorigenicity and safe transplantation, it has been considered that the same phenomenon occurs in human DFAT and is not suitable for treatment.

WO2006 / 112390 pamphlet JP 2012-205927 A Japanese Patent No. 5055611 Japanese Patent No. 5055613

Jumabay M, et al. Journal of Molecular and Cellular Cardiology 47: 565-575, 2009 Soejima K et al. Journal of Plastic Surgery and Hand Surgery 49: 25-31, 2015 Nobusue H et al. Cell and Tissue Research 332: 435-446, 2008

  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.

Even though mouse and rat DFAT has angiogenic potential, for the reasons described above, it has been considered that human DFAT may be the same because it is not suitable for actual treatment. Human DFAT not only has high angiogenic ability, but unlike DFAT of mouse and rat, it has high growth ability after subculture and can easily acquire the number of cells necessary for transplantation, and does not cause transformation. This was the first time that it was found to be effective for human blood vessel regeneration treatment.
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 adipocytes (human DFAT) are subcultured human dedifferentiated adipocytes (human DFAT).

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.

The karyotype analysis result of human DFAT is shown. (A) Photograph of chromosome structure by Giemsa staining. (B) A table showing the measurement results of the number of chromosomes. The cancer related gene list which examined promoter region DNA methylation is shown. 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) in human bone marrow MSC (hBM-MSC) culture supernatant 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. (B) Total length of blood vessel lumens formed in the well (total lumen length), total area (total lumen area), total number of branches (total lumen branch number), junction The graph shown about each of the total of the number of parts (total number of luminal junction parts). The effect of human DFAT transplantation in an immunodeficient mouse lower limb ischemia model is shown. (A) Photographs showing the degree of blood flow in the lower limbs of mice on the 0th and 28th days of transplantation using a laser Doppler blood flow meter. A portion surrounded by a red line labeled “1” indicates an ischemic limb, and a portion surrounded by a white line labeled “2” indicates a healthy limb. (B) The graph which shows the ischemic rate of the ischemic limb with respect to a mouse | mouth healthy side limb in the transplant 0th day, 7th day, 14th day, 21st day, and 28th day.

(Human DFAT)
The method for adjusting human DFAT in the present invention may be carried out 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 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 it is human DFAT can be determined by, for example, whether or not it has the following characteristics unique 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 mature adipocyte marker genes such as lipoprotein lipase and GLUT4, while expression of early differentiation marker genes for 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 at 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 10 ⁹ order cells 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.
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)
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 / 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.

As a treatment method related to revascularization using the human DFAT of the present invention, for example, DFAT (autologous 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, a therapeutic model is assumed in which an HLA full-matched cryopreserved DFAT is thawed and injected into the patient's ischemic site for a patient whose autologous DFAT cannot be prepared for some reason.

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.

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 adjusting injections, add pH adjusters, buffers, stabilizers, preservatives, etc., if necessary, and make subcutaneous, intramuscular, and intravenous injections 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 JP-A-2000-83656 made by the present inventors. . That is, after subjecting a human subcutaneous fat tissue as a donor to collagenase treatment, a single fraction consisting only of monocystic adipocytes was collected by filtration with a mesh having a diameter of 100 to 200 μm. 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 microarray (2-1) Test method Genomic DNA was extracted from human mature adipocytes and human DFAT (n = 3) prepared by the method of Example 1 to obtain human mature adipocytes and human DFAT genome. The change in the copy number was analyzed using a CGH microarray (Agilent).
(2-2) Test results Amplification of the copy number of genomic DNA before and after the culture was hardly observed, and the profile of the DFAT genomic copy number was almost identical to that of mature adipocytes (not shown). It was revealed that the number of genome copies hardly changed during the process of producing DFAT by dedifferentiation of mature adipocytes.

(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) (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 the characteristic methylation variation associated with dedifferentiation has been studied (92 genes CpG) It was not confirmed at all.

(4) Telomerase activity measurement (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

[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 subjected to adherent culture in a culture flask for about 2 weeks 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, MCP-1 was confirmed from human DFAT (FIG. 3).

(2) Quantification of expressed cytokine 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 (hPreadipocyte), human bone marrow MSC (hBM-MSC), and human fibroblasts (hFibroblast). It measured using. 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. 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) Comparison by difference in donor age (2-1) Test method The vascularization ability of DFAT culture supernatants 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 became clear 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) 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 The human DFAT culture supernatant showed significantly higher lumen length, lumen area, number of branches, number of junctions and the like than the human ASC culture supernatant (FIG. 7).

[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
In the DFAT transplantation group, blood flow improvement was significantly observed 3 weeks after transplantation compared to the control group. 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).

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 months 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.

  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)

A blood flow improving agent comprising human dedifferentiated fat cells (human DFAT) as an active ingredient. An angiogenesis promoter comprising 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. The ischemic disease therapeutic agent according to claim 3, wherein the ischemic disease is peripheral arterial disease (PAD) or ischemic cardiomyopathy. 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.