Search Results (23)

Mature adipocyte-derived dedifferentiated fat (DFAT) cells show proliferative capabilities and multipotency. Given that the buccal fat pad (BFP) serves as a readily available resource for DFAT cell isolation, BFP-derived DFAT (BFP-DFAT) cells are a promising candidate in orofacial tissue engineering. In this research, we assessed the regenerative capacity of the periodontium through autologous BFP-DFAT cell transplantation in adult swine (micro-minipigs; MMPs). The BFP-DFAT cells were transplanted into inflammation-inducing two-walled infrabony periodontal defects located on the mesial of the second mandibular premolar (n = 6). Twelve weeks post-transplantation, a remarkable attachment gain was noted in the DFAT group, based on probing depths and clinical attachment levels. Histological and immunohistochemical analyses indicated new continuous cellular cementum and alveolar bone formation within the created infrabony defect. Well-organized periodontal ligament-like fibers were embedded between newly formed cementum and the alveolar bone. Histometric analysis demonstrated that the DFAT group had a 2.2-fold increase in new alveolar bone length and a 2.2-fold enhancement in vascularization than those in the control group. Except for minor inflammation in the lungs, no teratomas were detected in the recipient MMPs. BFP-DFAT cells significantly enhanced periodontal tissue regeneration, thus representing an optimal source for tissue engineering applications in dentistry. Full article

Dedifferentiated fat (DFAT) cells are adipocyte-derived cells that are able to differentiate into multiple cell lineages such as adipocytes, osteoblasts and chondrocytes, similar to mesenchymal stem cells (MSCs). Despite their great potential for developing novel clinical interventions by using their multipotency, the detailed mechanisms of how adipocytes undergo dedifferentiation into DFAT cells are not completely understood, because useful in vitro tools for studying adipocyte dedifferentiation are missing. In this study, we show that mature adipocytes derived from the MSC cell line C3H10T1/2 underwent dedifferentiation into cells with DFAT cell-like characteristics, when they were cultured in an inverted flask. During the dedifferentiation, expression levels of genes and protein specific to adipocytes were continuously decreased, whereas those for MSC, proliferation and WNT/β-catenin signaling were gradually increased. These DFAT-like cells also underwent differentiation into adipocytes, osteoblasts and chondrocytes with their specific cell morphology and gene expression. We also observed that an individually cultured single adipocyte also underwent dedifferentiation into DFAT-like cells that were able to differentiate into the multiple cell lineages. Our results indicate that C3H10T1/2 cells could be a great tool for determining molecular biological and biochemical mechanisms underlying adipocyte dedifferentiation. Full article

Background/Objectives: Rabies is almost invariably fatal once clinical symptoms manifest. Timely and accurate diagnosis is essential for effective treatment and prevention. Dogs are the principal reservoirs of the virus, particularly in developing nations, highlighting the importance of precise diagnostic and control measures to prevent human cases. This systematic review and meta-analysis assessed the accuracy of laboratory tests for diagnosing rabies in humans and dogs. Methods: The PubMed database was searched for published studies on rabies diagnosis between 1990 and 2024. Following PRISMA statement recommendations, we included 60 studies that met the selection criteria. Results: The results demonstrated the effectiveness of immunological tests like the Enzyme-Linked Immunosorbent Assay (ELISA) and molecular tests such as Reverse Transcription Polymerase Chain Reaction (RT-PCR) for both humans and dogs. In this study, the Direct Fluorescent Antibody Test (DFAT) exhibited lower diagnostic performance, with an area under the curve for false positive rates (AUC_FPR = 0.887). In contrast, ELISA (AUC_FPR = 0.909) and RT-PCR (AUC_FPR = 0.905) provided more consistent results. Notably, the Rapid Immunochromatographic Test (RIT) showed the best performance (AUC_FPR = 0.949), highlighting its superior diagnostic capabilities compared to DFAT. Conclusions: These findings underscore the need to modernize rabies diagnostic protocols by incorporating advanced methodologies to improve diagnostic accuracy, reduce transmission, and decrease mortality rates. Full article

Obesity is concurrent with immunological dysregulation, resulting in chronic low-grade inflammation and cellular dysfunction. In pancreatic islets, this loss of function has been correlated with mature β-cells dedifferentiating into a precursor-like state through constant exposure to inflammatory stressors. As mature adipocytes likewise have the capability to dedifferentiate in vitro and in vivo, we wanted to analyze this cellular change in relation to adipose tissue (AT) inflammation and adipose tissue macrophage (ATM) activity. Using our organotypic AT explant culture method combined with a double-reporter mouse model for labeling ATMs and mature adipocytes, we were able to visualize and quantify dedifferentiated fat (DFAT) cells in AT explants. Preliminary testing showed increased dedifferentiation after tamoxifen (TAM) stimulation, making TAM-dependent lineage-tracing models unsuitable for quantification of naturally occurring DFAT cells. The regulatory role of ATMs in adipocyte dedifferentiation was shown through macrophage depletion using Plexxicon 5622 or clodronate liposomes, which significantly increased DFAT cell levels. Subsequent bulk RNA sequencing of macrophage-depleted explants revealed enrichment of the tumor necrosis factor α (TNFα) signaling pathway as well as downregulation of associated genes. Direct stimulation with TNFα decreased adipocyte dedifferentiation, while application of a TNFα-neutralizing antibody did not significantly alter DFAT cell levels. Our findings suggest a regulatory role of resident ATMs in maintaining the mature adipocyte phenotype and preventing excessive adipocyte dedifferentiation. The specific regulatory pathways as well as the impact that DFAT cells might have on ATMs, and vice versa, are subject to further investigation. Full article

The DFO, a special hexadentate chelator with three hydroxamate moieties, is a bifunctional 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)- 6,11,17, 22- tetraazaheptaeicosine] thiourea (p-SCN-Bn-Df), a significant next-generation ligand. The presence of the thiocyanate (-SCN) group makes it capable of hydrolysis and the protonation process. In this study aims to optimize the HPLC protocol for 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(n-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine] thiourea (p-SCN-Bn-Df) via the Reversed-Phase Chromatography (RP-HPLC) method. A variety of mobile phases were tested in various ratios of solvent constituents such as methanol/water, acetonitrile/water, and phosphate buffer along with at variable pH concentrations. However, when employing a mobile phase consisting of water to acetonitrile containing 0.1% TFA (05:95, v/v) in an isocratic manner, satisfactory separation and symmetric peaks were observed. This method utilized an Eclipsed C-18 column (5 μm, 4.6 × 250 mm) column with a flow rate of 0.5 mL/min. The maximum absorption of p-SCN-Bn-Dfat 254 nm wavelength was selected as the detection wavelength. The Retention time (t_R) of p-SCN-Bn-Df was found at 5.205 min. The ICH guideline was used to evaluate the linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), specificity, and system appropriateness criteria to validate the optimized chromatographic and spectrophotometric procedures. For accurate compound separation in pharmaceutical and environmental analyses, this phase is adaptable and often used. This study is useful for the evaluation of p-SCN-Bn-Df QC parameters and chelation rates with different radioisotopes e.g., Zirconuim-89 (Zr-89). Full article

Background: Extracellular vesicles (EVs) derived from stem cells demonstrate significant potential in bone regeneration. Adipose tissue is regarded as a stem cell reservoir with abundant reserves and easy accessibility. Compared to adipose-derived stem cells (ASCs), dedifferentiated fat cells (DFATs) possess similar stem cell characteristics but exhibit greater proliferative capacity, higher homogeneity, and an enhanced osteogenic differentiation potential. This study is the first to examine the effect of DFATs-derived EVs on bone regeneration and elucidate their potential mechanisms of action. Methods: Primary DFATs were cultured using the “ceiling culture” method and EVs were isolated by ultracentrifugation and characterized. Experiments were performed to assess the impact of the EVs on the proliferation, migration, and osteogenesis of bone marrow mesenchymal stem cells (BMSCs). Subsequently, high-throughput miRNA sequencing was conducted on the EVs derived from DFATs that had undergone 0 days (0d-EVs) and 14 days (14d-EVs) of osteogenic differentiation. Results: The results indicated that the EVs derived from DFATs which experienced 14 days of osteogenic induction significantly promoted the proliferation, migration, and osteogenic differentiation of BMSCs. High-throughput sequencing results revealed that up-regulated miRNAs in the 14d-EVs were primarily involved in biological processes such as the Notch signaling pathway and the positive regulation of cell movement and migration. The target genes of these differently expressed miRNAs were enriched in osteogenesis-related signaling pathways. Conclusion: This study innovatively demonstrated that conditioned EVs (14d-EVs) derived from DFATs promoted the osteogenic differentiation of BMSCs via miRNAs, offering a promising cell-free therapeutic option for bone defect. Full article

The active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is a principal regulator of calcium homeostasis through activation of the vitamin D receptor (VDR). Previous studies have shown that 2α-(3-hydroxypropyl)-1,25D₃ (O1C3) and 2α-(3-hydroxypropoxy)-1,25D₃ (O2C3), vitamin D derivatives resistant to inactivation enzymes, can activate VDR, induce leukemic cell differentiation, and increase blood calcium levels in rats more effectively than 1,25(OH)₂D₃. In this study, to further investigate the usefulness of 2α-substituted vitamin D derivatives, we examined the effects of O2C3, O1C3, and their derivatives on VDR activity in cells and mouse tissues and on osteoblast differentiation of dedifferentiated fat (DFAT) cells, a cell type with potential therapeutic application in regenerative medicine. In cell culture experiments using kidney-derived HEK293 cells, intestinal mucosa-derived CaCO₂ cells, and osteoblast-derived MG63 cells, and in mouse experiments, O2C2, O2C3, O1C3, and O1C4 had a weaker effect than or equivalent effect to 1,25(OH)₂D₃ in VDR transactivation and induction of the VDR target gene CYP24A1, but they enhanced osteoblast differentiation in DFAT cells equally to or more effectively than 1,25(OH)₂D₃. In long-term treatment with the compound without the medium change (7 days), the derivatives enhanced osteoblast differentiation more effectively than 1,25(OH)₂D₃. O2C3 and O1C3 were more stable than 1,25(OH)₂D₃ in DFAT cell culture. These results indicate that 2α-substituted vitamin D derivatives, such as inactivation-resistant O2C3 and O1C3, are more effective than 1,25(OH)₂D₃ in osteoblast differentiation of DFAT cells, suggesting potential roles in regenerative medicine with DFAT cells and other multipotent cells. Full article

Introduction: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O₂ concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. Results: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. Conclusion: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state. Full article

Adipose tissue-derived stromal cells (ASCs) play an important role in various therapeutic approaches to bone regeneration. However, such applications become challenging when the obtained cells show a functional disorder, e.g., an impaired osteogenic differentiation potential (ODP). In addition to ASCs, human adipose tissue is also a source for another cell type with therapeutic potential, the dedifferentiated fat cells (DFATs), which can be obtained from mature adipocytes. Here, we for the first time compared the ODPs of each donors ASC and DFAT obtained from the same adipose tissue sample as well as the role of oxidative stress or antioxidative catalase on their osteogenic outcome. Osteogenic potential of ASC and DFAT from nine human donors were compared in vitro. Flow cytometry, staining for calcium accumulation with alizarin red, alkaline phosphatase assay and Western blots were used over an osteogenic induction period of up to 14 days. H₂O₂ was used to induce oxidative stress and catalase was used as an antioxidative measure. We have found that ASC and DFAT cultures’ ODPs are nearly identical. If ASCs from an adipose tissue sample showed good or bad ODP, so did the corresponding DFAT cultures. The inter-individual variability of the donor ODPs was immense with a maximum factor of about 20 and correlated neither with the age nor the sex of the donors of the adipose tissue. Oxidative stress in the form of exogenously added H₂O₂ led to a significant ODP decrease in both cell types, with this ODP decrease being significantly lower in DFAT cultures than in the corresponding ASC cultures. Regardless of the individual cell culture-specific ODP, however, exogenously applied catalase led to an approx. 2.5-fold increase in osteogenesis in the ASC and DFAT cultures. Catalase appears to be a potent pro-osteogenic factor, at least in vitro. A new finding that points to innovative strategies and therapeutic approaches in bone regeneration. Furthermore, our results show that DFATs behave similarly to ASCs of the same adipose tissue sample with respect to ODPs and could therefore be a very attractive and readily available source of multipotent stem cells in bone regenerative therapies. Full article

Focusing on the forgotten lessons and potential legacy of the urban projects proposed by the Japanese avant-garde architectural movement of Metabolism, which emerged in the 1960s, and on the critical and comparative study of large-scale waterfront regeneration and design in Tokyo and Sydney, two education and research projects were initiated at UNSW-School of the Built Environment in 2019 and 2021, with the aim of looking at the future of the city in an age of climate change, global warming and rising sea levels. Structured around a variety of international workshops, joint design studios, collaborative archive and documentary reviews, multi-disciplinary seminars, and international symposia, with the participation of national and international academics and scholars, both research projects are currently in progress, with the support of competitive research grants from the Japan Foundation, the Australia–Japan Foundation (AJF), the Australian Department of Foreign Affairs and Trade (DFAT), and other internal grants from the Faculty of Art, Design and Architecture (ADA) at UNSW Sydney. Full article

Adipose tissue is composed mostly of adipocytes that are in contact with capillaries. By using a ceiling culture method based on buoyancy, lipid-free fibroblast-like cells, also known as dedifferentiated fat (DFAT) cells, can be separated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and transdifferentiate into various cell types under appropriate culture conditions. Herein, we sought to compare the regenerative potential of collagen matrix alone (control) with autologous DFAT cell-loaded collagen matrix transplantation in adult miniature pigs (microminipigs; MMPs). We established and transplanted DFAT cells into inflammation-inducing periodontal class II furcation defects. At 12 weeks after cell transplantation, a marked attachment gain was observed based on the clinical parameters of probing depth (PD) and clinical attachment level (CAL). Additionally, micro computed tomography (CT) revealed hard tissue formation in furcation defects of the second premolar. The cemento-enamel junction and alveolar bone crest distance was significantly shorter following transplantation. Moreover, newly formed cellular cementum, well-oriented periodontal ligament-like fibers, and alveolar bone formation were observed via histological analysis. No teratomas were found in the internal organs of recipient MMPs. Taken together, these findings suggest that DFAT cells can safely enhance periodontal tissue regeneration. Full article

Rabies, a zoonotic encephalitis due to transmission of a lyssavirus, such as rabies virus (RABV), has the highest case fatality of any infectious disease. A global program for the elimination of human rabies caused by dogs is proposed for realization by 2030. Sensitive, specific, and inexpensive diagnostic tests are necessary for enhanced surveillance to detect infection, inform public health and veterinary professionals during risk assessments of exposure, and support overall programmatic goals. Multiple laboratory techniques are used to confirm a suspect case of rabies. One method for the detection of lyssavirus antigens within the brain is the direct rapid immunohistochemical test (dRIT), using light microscopy, and suitable for use under field conditions. Besides dogs, other major RABV reservoirs reside among mammalian mesocarnivores and bats. To date, use of the dRIT has been applied primarily for the diagnosis of RABV in suspect mesocarnivores. The purpose of this study was to assess the usefulness of the dRIT to the diagnosis of rabies in bats, compared to the gold-standard, the direct fluorescent antibody test (DFAT). Brains of 264 suspect bats, consisting of 21 species from Arizona and Texas, were used in the evaluation of the dRIT. The overall sensitivity of the dRIT was 100% (0.969–1.0, 95% CI) and the specificity was 94.6% (0.896–0.976, 95% CI), comparable to the DFAT. This preliminary study demonstrated the utility of the dRIT in the confirmation of RABV infection in bats. Future studies should include additional geographic, lyssavirus, and mammalian species representations for broader application during enhanced rabies surveillance, with incorporation of any potential adjustments to standard protocols, as needed. Full article

Mechanical and resorbable scaffolds are in high demand for stem cell-based regenerative medicine, to treat refractory bone defects in craniofacial abnormalities and injuries. Multipotent progenitor cells, such as dedifferentiated fat (DFAT) cells, are prospective sources for regenerative therapies. Herein, we aimed to demonstrate that a composite gelatin sponge (α-TCP/GS) of alfa-tricalcium phosphate (α-TCP) mixed with gelatin scaffolds (GS), with/without DFATs, induced bone regeneration in a rat calvarial defect model in vivo. α-TCP/GS was prepared by mixing α-TCP and 2% GS using vacuum-heated methods. α-TCP/GS samples with/without DFATs were transplanted into the model. After 4 weeks of implantation, the samples were subjected to micro-computed tomography (μ-CT) and histological analysis. α-TCP/GS possessed adequate mechanical strength; α-TCP did not convert to hydroxyapatite upon contact with water, as determined by X-ray diffraction. Moreover, stable α-TCP/GS was formed by electrostatic interactions, and verified based on the infrared peak shifts. μ-CT analyses showed that bone formation was higher in the α-TCP/GS+ DFAT group than in the α-TCP/GS group. Therefore, the implantation of α-TCP/GS comprising DFAT cells enhanced bone regeneration and vascularization, demonstrating the potential for healing critical-sized bone defects. Full article

(1) Background: The control of angiogenesis is essential in disease treatment. We investigated angiogenesis-promoting or -suppressing factors and their molecular mechanisms. (2) Methods: Angiogenesis from HUVECs was quantitatively analyzed using the Angiogenesis Analysis Kit (Kurabo, Osaka, Japan). Human rAng-1-producing 107-35 CHO cells or mouse DFAT-D1 cells were co-cultured with HUVEC. Antioxidant polyphenols were added to the culture. Gene expression was analyzed by RT-PCR. (3) Results: The addition of rAng-1-producing cells, their culture supernatant, or commercially available rAng-1 showed a promoting effect on angiogenesis. The co-culture of DFAT-D1 cells promoted angiogenesis. Polyphenols showed a dose-dependent inhibitory effect on angiogenesis. Luteolin and quercetin showed remarkable anti-angiogenic effects. The expression of vWF, Flk1, and PECAM-1 was increased by adding rAng-1-producing cell culture supernatant. Polyphenols suppressed these genes. Apigenin and luteolin markedly suppressed α-SMA and Flk1. Resveratrol and quercetin enhanced the expression of PPARγ, and luteolin suppressed the expression of COX-1. The expression of endothelial nitric oxide synthase (eNOS), an oxidative stress-related gene, was slightly increased by luteolin. These results suggest that polyphenols induce ROS reduction. (4) Conclusions: We showed the promoting effect of Ang-1 or DFAT and the suppressing effect of polyphenols on angiogenesis and studied their molecular mechanisms. These results help control angiogenesis in regenerative therapy. Full article

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies. Full article