Adipose Tissue-derived Multi-lineage Progenitor Cells Research Articles

Periodontal Tissue Regeneration by Transplantation of Autologous Adipose Tissue-Derived Multi-Lineage Progenitor Cells With Carbonate Apatite.

We have developed an autologous transplantation method using adipose tissue-derived multi-lineage progenitor cells (ADMPCs) as a method of periodontal tissue regeneration that can be adapted to severe periodontal disease. Our previous clinical study confirmed the safety of autologous transplantation of ADMPCs and demonstrated its usefulness in the treatment of severe periodontal disease. However, in the same clinical study, we found that the fibrin gel used as the scaffold material might have caused gingival recession and impaired tissue regeneration in some patients. Carbonate apatite has a high space-making capacity and has been approved in Japan for periodontal tissue regeneration. In this study, we selected carbonate apatite as a candidate scaffold material for ADMPCs and conducted an in vitro examination of its effect on the cellular function of ADMPCs. We further performed autologous ADMPC transplantation with carbonate apatite as the scaffold material in a model of one-wall bone defects in beagles and then analyzed the effect on periodontal tissue regeneration. The findings showed that carbonate apatite did not affect the cell morphology of ADMPCs and that it promoted proliferation. Moreover, no effect on secretor factor transcription was found. The results of the in vivo analysis confirmed the space-making capacity of carbonate apatite, and the acquisition of significant new attachment was observed in the group involving ADMPC transplantation with carbonate apatite compared with the group involving carbonate apatite application alone. Our results demonstrate the usefulness of carbonate apatite as a scaffold material for ADMPC transplantation.

Periodontal tissue regeneration by transplantation of autologous adipose tissue-derived multi-lineage progenitor cells

Periodontitis is a chronic inflammatory disease that destroys tooth-supporting periodontal tissue. Current periodontal regenerative therapies have unsatisfactory efficacy; therefore, periodontal tissue engineering might be established by developing new cell-based therapies. In this study, we evaluated the safety and efficacy of adipose tissue-derived multi-lineage progenitor cells (ADMPC) autologous transplantation for periodontal tissue regeneration in humans. We conducted an open-label, single-arm exploratory phase I clinical study in which 12 periodontitis patients were transplanted with autologous ADMPCs isolated from subcutaneous adipose tissue. Each patient underwent flap surgery during which autologous ADMPCs were transplanted into the bone defect with a fibrin carrier material. Up to 36 weeks after transplantation, we performed a variety of clinical examinations including periodontal tissue inspection and standardized dental radiographic analysis. A 36-week follow-up demonstrated no severe transplantation-related adverse events in any cases. ADMPC transplantation reduced the probing pocket depth, improved the clinical attachment level, and induced neogenesis of alveolar bone. Therapeutic efficiency was observed in 2- or 3-walled vertical bone defects as well as more severe periodontal bone defects. These results suggest that autologous ADMPC transplantation might be an applicable therapy for severe periodontitis by inducing periodontal regeneration.

Current status-phase I trial of cell-transplantation therapy with allogeneic adipose tissue-derived multilineage progenitor cells in severe familial hypercholesterolemia patients undergoing LDL-apheresis

Current status-phase I trial of cell-transplantation therapy with allogeneic adipose tissue-derived multilineage progenitor cells in severe familial hypercholesterolemia patients undergoing LDL-apheresis

Abstract 602: Development of Cell Therapy in Severe Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) patients are characterized by high LDL cholesterol levels in the blood and premature cardiovascular disease. Although most of heterozygous FH patients are able to be treated with statin, ezetimibe and bile acid sequestrants and anti-PCSK9 antibodies, homozygous FH patients are resistant to drug therapy. Therefore, many of homozygous FH patients used to be treated by LDL-apheresis. However, the effect of removing LDL cholesterol is still not sufficient and definitive therapies need to be developed. With the increase of the evidence about the safety of mesenchymal stem cells and percutaneous transhepatic portal approach in islet transplantation, we have developed a cell transplantation therapy with allogeneic adipose tissue-derived multilineage progenitor cells, as an alternative treatment instead of liver transplantation. We have generated a protocol for the phase 1 clinical study. The primary endpoint should be safety evaluation including allergic reaction, rejection reaction, thrombosis and complications in the blood vessel by cell-transplantation. The secondary endpoint should be the efficacy including changes in serum lipid such as LDL-C, TC, HDL-C, TG and Lp(a), and Secession possibility of LDL-apheresis. We transplanted the first case in February, 2016. Then we are collecting clinical data. We would demonstrate the current status and the issues to be solved for the following study.

Spermine Treated-Adipose Tissue-Derived Multi-Lineage Progenitor Cells Improve Left Ventricular Dysfunction in a Swine Model of Chronic Myocardial Infarction

Background: The polyamine spermine enhances differentiation of mouse embryonic stem cells into cardiac lineage. The aim of this study was to determine the effects of spermine on the differentiation of human adipose tissuederived multi-lineage progenitor cells (hADMPCs) into cardiomyocytes both in vitro and in vivo and any subsequent functional effect in a swine model of chronic myocardial infarction. Methods and results: Spermine increased the expression of cardiac markers nkx2.5, islet-1, α-cardiac actin and cardiac troponin I (to 11.2-, 27.5-, 43.6- and 19.1-fold, relative to baseline, respectively) in hADMPCs. Chronic myocardial infarction model with left ventricular dysfunction was induced by balloon occlusion of the diagonal coronary artery followed by reperfusion, with subsequent similar procedure conducted one week later in the left ascending coronary artery (#6). Four weeks later, the immunosuppressed animals (with CyA 5.0 mg/kg intramuscularly (i.m) body weight/day) were transplanted with spermine-treated hADMPC (1×105 , 3×105 , 1×106 or 3×106 cells/kg body weight) via the coronary artery (#6). Cardiac function was assessed by echocardiography at 0, 4, 8 and 12 weeks post-transplantation. Transplantation of these cells improved cardiac function and the most effective dose was 3x105 cells/kg (ejection fraction; 33.4%, 47.0%, 51.5% and 52.9% at 0, 4, 8 and 12 weeks post-transplantation, respectively). At 12-week post-transplantation, spermine-treated hADMPCs differentiated into human-specific troponin I- and α-cardiac actin-positive cells in vivo. Conclusion: Spermine induced differentiation of hADMPCs into cardiomyocytes both in vitro and in vivo and cellular cardiomyoplasty improved cardiac function. Cellular cardiomyoplasty using hADMPC could be potentially effective cell-based therapy.

Trophic factors from adipose tissue-derived multi-lineage progenitor cells promote cytodifferentiation of periodontal ligament cells

Stem and progenitor cells are currently being investigated for their applicability in cell-based therapy for periodontal tissue regeneration. We recently demonstrated that the transplantation of adipose tissue-derived multi-lineage progenitor cells (ADMPCs) enhances periodontal tissue regeneration in beagle dogs. However, the molecular mechanisms by which transplanted ADMPCs induce periodontal tissue regeneration remain to be elucidated. In this study, trophic factors released by ADMPCs were examined for their paracrine effects on human periodontal ligament cell (HPDL) function. ADMPC conditioned medium (ADMPC-CM) up-regulated osteoblastic gene expression, alkaline phosphatase activity and calcified nodule formation in HPDLs, but did not significantly affect their proliferative response. ADMPCs secreted a number of growth factors, including insulin-like growth factor binding protein 6 (IGFBP6), hepatocyte growth factor and vascular endothelial growth factor. Among these, IGFBP6 was most highly expressed. Interestingly, the positive effects of ADMPC-CM on HPDL differentiation were significantly suppressed by transfecting ADMPCs with IGFBP6 siRNA. Our results suggest that ADMPCs transplanted into a defect in periodontal tissue release trophic factors that can stimulate the differentiation of HPDLs to mineralized tissue-forming cells, such as osteoblasts and cementoblasts. IGFBP6 may play crucial roles in ADMPC-induced periodontal regeneration.

An open-label, phase I trial of transplantation therapy with allogeneic adipose tissue-derived multilineage progenitor cells in homozygous familial hypercholesterolemia patients

An open-label, phase I trial of transplantation therapy with allogeneic adipose tissue-derived multilineage progenitor cells in homozygous familial hypercholesterolemia patients

Abstract 50: A Phase I Clinical Trial Evaluating the Safety of With Allogeneic Adipose Tissue-derived Multilineage Progenitor Cells-transplantation Therapy in Homozygous Familial Hypercholesterolemia Patients

Familial hypercholesterolemia (FH) is an inherited disorder, mainly caused by defects in low-density lipoprotein (LDL) receptor gene. The patients are characterized by high LDL cholesterol levels in the blood and premature cardiovascular disease. Although most of heterozygous FH patients are usually treated with statin, ezetimibe and bile acid sequestrants, homozygous FH patients are resistant to drug therapy. Therefore, in Japan, many of homozygous FH patients are treated by LDL-apheresis. LDL-apheresis is a great procedure to remove LDL cholesterol from the blood and contribute to improve prognosis of homozygous FH patients. However, the effect of removing LDL cholesterol is temporary and still not enough. As a definitive therapy, liver transplantation therapy could be one of options to recover LDL receptor, but donor is limited in Japan. Therefore, based on the increase of the evidence about the safety of mesenchymal stem cells and percutaneous transhepatic portal approach in islet transplantation, we have developed a cell transplantation therapy with allogeneic adipose tissue-derived multilineage progenitor cells (ADMPCs), as an alternative treatment instead of liver transplantation. Our group has already proved that xenogenic transplantation of human ADMPCs into Watanabe heritable hyperlipidemic rabbits resulted in significant reductions in total cholesterol, and the reductions were observed within 4 weeks and maintained for 12 weeks. These results suggested that hADMPC transplantation could correct the metabolic defects and be a novel therapy for inherited liver diseases. Here, we report a protocol for the first-in-human clinical trial, which has been approved by the institutional review board and Ministry of Health, Labour and Welfare, Japan.

Therapeutic potential of human adipose tissue-derived multi-lineage progenitor cells in liver fibrosis

IntroductionLiver fibrosis is characterized by excessive accumulation of extracellular matrix. In a mouse model of liver fibrosis, systemic injection of bone marrow mesenchymal stem cells (BM-MSCs) was considered to rescue the diseased phenotype. The aim of this study was to assess the effectiveness of human adipose tissue-derived multi-lineage progenitor cells (hADMPCs) in improving liver fibrosis. Methods and resultshADMPCs were isolated from subcutaneous adipose tissues of healthy volunteers and expanded. Six week-old male nude mice were treated with carbon tetra-chloride (CCl4) by intraperitoneal injection twice a week for 6weeks, followed by a tail vein injection of hADMPCs or placebo control. After 6 more weeks of CCl4 injection (12weeks in all), nude mice with hADMPCs transplants exhibited a significant reduction in liver fibrosis, as evidenced by Sirius Red staining, compared with nude mice treated with CCl4 for 12weeks without hADMPCs transplants. Moreover, serum glutamic pyruvate transaminase and total bilirubin levels in hADMPCs-treated nude mice were lower levels than those in placebo controls. Production of fibrinolytic enzyme MMPs from hADMPCs were examined by ELISA and compared to that from BM-MSCs. MMP-2 levels in the culture media were not significantly different, whereas those of MMP-3 and -9 of hADMPCs were higher than those by BM-MSCs. ConclusionThese results showed the mode of action and proof of concept of systemic injection of hADMPCs, which is a promising therapeutic intervention for the treatment of patients with liver fibrosis.

Role of Notch Signaling in the Maintenance of Human Mesenchymal Stem Cells Under Hypoxic Conditions

Human adipose tissue-derived multilineage progenitor cells (hADMPCs) are attractive for cell therapy and tissue engineering because of their multipotency and ease of isolation without serial ethical issues. However, their limited in vitro lifespan in culture systems hinders their therapeutic application. Some somatic stem cells, including hADMPCs, are known to be localized in hypoxic regions; thus, hypoxia may be beneficial for ex vivo culture of these stem cells. These cells exhibit a high level of glycolytic metabolism in the presence of high oxygen levels and further increase their glycolysis rate under hypoxia. However, the physiological role of glycolytic activation and its regulatory mechanisms are still incompletely understood. Here, we show that Notch signaling is required for glycolysis regulation under hypoxic conditions. Our results demonstrate that 5% O2 dramatically increased the glycolysis rate, improved the proliferation efficiency, prevented senescence, and maintained the multipotency of hADMPCs. Intriguingly, these effects were not mediated by hypoxia-inducible factor (HIF), but rather by the Notch signaling pathway. Five percent O2 significantly increased the level of activated Notch1 and expression of its downstream gene, HES1. Furthermore, 5% O2 markedly increased glucose consumption and lactate production of hADMPCs, which decreased back to normoxic levels on treatment with a γ-secretase inhibitor. We also found that HES1 was involved in induction of GLUT3, TPI, and PGK1 in addition to reduction of TIGAR and SCO2 expression. These results clearly suggest that Notch signaling regulates glycolysis under hypoxic conditions and, thus, likely affects the cell lifespan via glycolysis.

Periodontal tissue regeneration by transplantation of adipose tissue-derived multi-lineage progenitor cells

Periodontal tissue regeneration by transplantation of adipose tissue-derived multi-lineage progenitor cells

Tightly Regulated and Homogeneous Transgene Expression in Human Adipose-Derived Mesenchymal Stem Cells by Lentivirus with Tet-Off System

Genetic modification of human adipose tissue–derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.

Human adipose tissue-derived multilineage progenitor cells exposed to oxidative stress induce neurite outgrowth in PC12 cells through p38 MAPK signaling

BackgroundAdipose tissues contain populations of pluripotent mesenchymal stem cells that also secrete various cytokines and growth factors to support repair of damaged tissues. In this study, we examined the role of oxidative stress on human adipose-derived multilineage progenitor cells (hADMPCs) in neurite outgrowth in cells of the rat pheochromocytoma cell line (PC12).ResultsWe found that glutathione depletion in hADMPCs, caused by treatment with buthionine sulfoximine (BSO), resulted in the promotion of neurite outgrowth in PC12 cells through upregulation of bone morphogenetic protein 2 (BMP2) and fibroblast growth factor 2 (FGF2) transcription in, and secretion from, hADMPCs. Addition of N-acetylcysteine, a precursor of the intracellular antioxidant glutathione, suppressed the BSO-mediated upregulation of BMP2 and FGF2. Moreover, BSO treatment caused phosphorylation of p38 MAPK in hADMPCs. Inhibition of p38 MAPK was sufficient to suppress BMP2 and FGF2 expression, while this expression was significantly upregulated by overexpression of a constitutively active form of MKK6, which is an upstream molecule from p38 MAPK.ConclusionsOur results clearly suggest that glutathione depletion, followed by accumulation of reactive oxygen species, stimulates the activation of p38 MAPK and subsequent expression of BMP2 and FGF2 in hADMPCs. Thus, transplantation of hADMPCs into neurodegenerative lesions such as stroke and Parkinson’s disease, in which the transplanted hADMPCs are exposed to oxidative stress, can be the basis for simple and safe therapies.

Intracoronary artery transplantation of cardiomyoblast-like cells from human adipose tissue-derived multi-lineage progenitor cells improve left ventricular dysfunction and survival in a swine model of chronic myocardial infarction

Transplantation of human cardiomyoblast-like cells (hCLCs) from human adipose tissue-derived multi-lineage progenitor cells improved left ventricular function and survival of rats with myocardial infarction. Here we examined the effect of intracoronary artery transplantation of human CLCs in a swine model of chronic heart failure. Twenty-four pigs underwent balloon-occlusion of the first diagonal branch followed by reperfusion, with a second balloon-occlusion of the left ascending coronary artery 1week later followed by reperfusion. Four weeks after the second occlusion/reperfusion, 17 of the 18 surviving animals with severe chronic MI (ejection fraction <35% by echocardiography) were immunosuppressed then randomly assigned to receive either intracoronary artery transplantation of hCLCs hADMPCs or placebo lactic Ringer’s solution with heparin. Intracoronary artery transplantation was followed by the distribution of DiI-stained hCLCs into the scarred myocardial milieu. Echocardiography at post-transplant days 4 and 8weeks showed rescue and maintenance of cardiac function in the hCLCs transplanted group, but not in the control animals, indicating myocardial functional recovery by hCLCs intracoronary transplantation. At 8week post-transplantation, 7 of 8 hCLCs transplanted animals were still alive compared with only 1 of the 5 control (p=0.0147). Histological studies at week 12 post-transplantation demonstrated engraftment of the pre DiI-stained hCLCs into the scarred myocardium and their expression of human specific alpha-cardiac actin. Human alpha cardiac actin-positive cells also expressed cardiac nuclear factors; nkx2.5 and GATA-4. Our results suggest that intracoronary artery transplantation of hCLCs is a potentially effective therapeutic strategy for future cardiac tissue regeneration.

Adipose Tissue-Derived Multi-lineage Progenitor Cells as a Promising Tool for In Situ Stem Cell Therapy

Adipose tissue-derived cell sources are attractive for regenerative medicine due to the easy and safe accessibility of adipose tissue, lack of ethical issues, and their availability in large quantities. We have developed an isolation method for distinct stem cells, and named the cells adipose tissue-derived multilineage progenitor cells (ADMPC). ADMPC have higher potential for differentiation into adipocytic, osteocytic, and chondrocytic progeny than the widely reported adipose tissue-derived stromal/stem cells (ADSC). ADMPC can also differentiate into hepatocyte-like clusters in vitro by induction with the hepatogenic cytokines, hepatocyte growth factor, oncostatin M, and basic fibroblast growth factor. In vivo, ADMPC were reprogrammed in situ into hepatocyte-like cells that corrected the metabolic defect in hyperlipidemic Watanabe rabbits after transplantation via the portal vein. Such cells are potentially useful in regenerative medicine as sources for in situ stem cell therapy. Keywords: adipose tissue, ADMPC, in situ stem cell therapy, in situ reprogramming, in situ differentiation, ADSC, ADMSC, ASC, hepatocyte, umbilical cord.

Abstract 9201: Transplantation of Adipose Tissue-Derived Multi-Lineage Progenitor Cells Reduces Serum Cholesterol in Hyperlipidemic Watanabe Rabbits

Background : Familial hypercholesterolemia (FH) is an autosomal co-dominant disease characterized by high concentrations of pro-atherogenic lipoproteins and premature atherosclerosis. We examined the response to in situ stem cell therapy using adipose tissue-derived multi-lineage progenitor stem cells (hADMPC) in the LDL-receptor deficient Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for homozygous FH. Methods : WHHL rabbits received either normal control rabbit-derived, GFP-rabbit-derived or WHHL rabbit-derived ADMPC (normal-ADMPC, GFP-ADMPC and diseased-ADMPC, respectively) via the portal vein. This was followed by 12-week immunosuppressive therapy to avoid allogenic rejection. In situ survival and differentiation of the ADMPC into hepatocytes was examined by flow cytometry and immunohistochemical analysis, respectively. Lipid profile was examined before-, and 4-, 8- and 12 weeks after transplantation. LDL clearance was examined at the end of the study by125I-LDL turnover. Results : In situ survival of GFP-ADMPC was confirmed after transplantation. The cells integrated into the hepatic parenchyma and co-expressed GFP and hepatocyte markers such as albumin, indicating that the cells were reprogrammed into hepatocytes-like cells in situ . Transplantation of normal-ADMPC but not diseased-ADMPC resulted in a significant reduction of serum total- and LDL- cholesterol after transplantation. 125I-LDL turnover study showed significant improvement in the rate of LDL clearance in the WHHL rabbits with transplanted normal-ADMPC but not in those transplanted with diseased-ADMPC. Conclusion : Transplantation of ADMPC but not diseased ones corrected the metabolic defects in WHHL rabbits, suggesting that ADMPC transplantation is a potentially useful therapy for FH.

HMG-CoA reductase inhibitor augments the serum total cholesterol-lowering effect of human adipose tissue-derived multilineage progenitor cells in hyperlipidemic homozygous Watanabe rabbits

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins secondary to deficiency in low-density lipoprotein (LDL) receptor. We reported recently the use of in situ stem cell therapy of human adipose tissue-derived multilineage progenitor cells (hADMPCs) in lowering serum total cholesterol in the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of homozygous FH. Here we demonstrate that pravastatin, an HMG-CoA reductase inhibitor, augmented the cholesterol-lowering effect of transplanted hADMPCs and enhanced LDL clearance in homozygous WHHL rabbit. The results suggest the potential beneficial effects of in situ stem cell therapy in concert with appropriately selected pharmaceutical agents, in regenerative medicine.

Transplantation of Human Adipose Tissue-Derived Multilineage Progenitor Cells Reduces Serum Cholesterol in Hyperlipidemic Watanabe Rabbits

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins and premature atherosclerosis secondary to low-density lipoprotein (LDL) receptor deficiency. We examined a novel cell therapy strategy for the treatment of FH in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for homozygous FH. We delivered human adipose tissue-derived multilineage progenitor cells (hADMPCs) via portal vein and followed by immunosuppressive regimen to avoid xenogenic rejection. Transplantation of hADMPCs resulted in significant reductions in total cholesterol, and the reductions were observed within 4 weeks and maintained for 12 weeks. (125)I-LDL turnover study showed that the rate of LDL clearance was significantly higher in the WHHL rabbits with transplanted hADMPCs than those without transplanted. After transplantation hADMPCs were localized in the portal triad, subsequently integrated into the hepatic parenchyma. The integrated cells expressed human albumin, human alpha-1-antitrypsin, human Factor IX, human LDL receptors, and human bile salt export pump, indicating that the transplanted hADMPCs resided, survived, and showed hepatocytic differentiation in vivo and lowered serum cholesterol in the WHHL rabbits. These results suggested that hADMPC transplantation could correct the metabolic defects and be a novel therapy for inherited liver diseases.