Bone marrow-derived dedifferentiated fat cells exhibit similar phenotype as bone marrow mesenchymal stem cells with high osteogenic differentiation and bone regeneration ability.

BACKGROUND: Mesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model. METHODS: BM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice. RESULTS: BM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model. CONCLUSIONS: We showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

Implantation of dedifferentiated fat cells ameliorated antineutrophil cytoplasmic antibody glomerulonephritis by immunosuppression and increases in tumor necrosis factor-stimulated gene-6.

INTRODUCTION: The implantation of dedifferentiated fat (DFAT) cells has been shown to exert immunosuppressive effects. To develop DFAT cell therapy for antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis, the effects of the implantation of DFAT cells on ANCA glomerulonephritis were investigated in mice. METHODS: PKH26-labeled DFAT cells (105) were infused through the posterior orbital venous plexus to investigate delivery of DFAT cells in ICR mice. DFAT cells (105) were also implanted in SCG mice as a model for ANCA glomerulonephritis. Expression of tumor necrosis factor-stimulated gene-6 (TSG-6) mRNA and protein in kidney was evaluated, and the expression of microRNAs associated with TSG-6 in plasma, lung and kidney was analyzed. Expressions of CD44, prostaglandin (PG) E2, interleukin (IL)-10, IL-1ß, tumor necrosis factor (TNF)-α mRNAs, C-C motif chemokine ligand 17 (CCL-17) and monocyte chemoattractant protein (MCP)-1 proteins were measured in kidney from SCG mice implanted with DFAT cells. RESULTS: After their intravenous infusion, almost all DFAT cells were trapped in the lung and not delivered into the kidney. Implantation of DFAT cells in SCG mice suppressed glomerular crescent formation, decreased urinary protein excretions and increased expression of TSG-6 mRNA, protein and immunostaining in kidney from these mice. Increased expression of microRNA 23b-3p in plasma, kidney and lung; decreased expression of CD44 mRNA; and increased expression of PGE2 and IL-10 mRNAs were also observed in kidney from these mice. Implantation of DFAT cells also decreased the expression of TNF-α and MCP-1 proteins and increased that of CCL-17 protein in kidney from the SCG mice. Survival rates were higher in SCG mice implanted with DFAT cells than in SCG mice without implantation. CONCLUSION: Mechanisms underlying the effects of improvement of ANCA glomerulonephritis are associated with immunosuppressive effects by TSG-6 and the transition of M1-M2 macrophages, suggesting that implantation of DFAT cells may become a cell therapy for ANCA glomerulonephritis.

Regulatory roles of fibronectin and integrin α5 in reorganization of the actin cytoskeleton and completion of adipogenesis.

Cellular differentiation is characterized by changes in cell morphology that are largely determined by actin dynamics. We previously showed that depolymerization of the actin cytoskeleton triggers the differentiation of preadipocytes into mature adipocytes as a result of inhibition of the transcriptional coactivator activity of megakaryoblastic leukemia 1 (MKL1). The extracellular matrix (ECM) influences cell morphology via interaction with integrins, and reorganization of the ECM is associated with cell differentiation. Here we show that interaction between actin dynamics and ECM rearrangement plays a key role in adipocyte differentiation. We found that depolymerization of the actin cytoskeleton precedes disruption and degradation of fibrillar fibronectin (FN) structures at the cell surface after the induction of adipogenesis in cultured preadipocytes. A FN matrix suppressed both reorganization of the actin cytoskeleton into the pattern characteristic of adipocytes and terminal adipocyte differentiation, and these inhibitory effects were overcome by knockdown of integrin α5 (ITGα5). Peroxisome proliferator-activated receptor γ was required for down-regulation of FN during adipocyte differentiation, and MKL1 was necessary for the expression of ITGα5. Our findings suggest that cell-autonomous down-regulation of FN-ITGα5 interaction contributes to reorganization of the actin cytoskeleton and completion of adipocyte differentiation.

Transplantation of Mature Adipocyte-Derived Dedifferentiated Fat Cells Facilitates Periodontal Tissue Regeneration of Class II Furcation Defects in Miniature Pigs.

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.

Urethral injection of dedifferentiated fat cells ameliorates sphincter damage and voiding dysfunction in a rat model of persistence stress urinary incontinence.

PURPOSE: Dedifferentiated fat (DFAT) cells are mature adipocyte-derived multipotent cells that can be applicable to cell-based therapy for stress urinary incontinence (SUI). This study developed a persistence SUI model that allows long-term evaluation using a combination of vaginal distention (VD) and bilateral ovariectomy (OVX) in rats. Then, the therapeutic effects of DFAT cell transplantation in the persistence SUI model was examined. METHODS: In total, 48 Sprague-Dawley rats were divided into four groups and underwent VD (VD group), bilateral OVX (OVX group), VD and bilateral OVX (VD + OVX group), or sham operation (Control group). At 2, 4, and 6 weeks after injury, leak point pressure (LPP) and histological changes of the urethral sphincter were evaluated. Next, 14 rats undergoing VD and bilateral OVX were divided into two groups and administered urethral injection of DFAT cells (DFAT group) or fibroblasts (Fibroblast group). At 6 weeks after the injection, LPP and histology of the urethral sphincter were evaluated. RESULTS: The VD + OVX group retained a decrease in LPP with sphincter muscle atrophy at least until 6 weeks after injury. The LPP and urethral sphincter muscle atrophy in the DFAT group recovered better than those in the fibroblast group. CONCLUSIONS: The persistence SUI model was created by a combination of VD and bilateral OVX in rats. Urethral injection of DFAT cells inhibited sphincter muscle atrophy and improved LPP in the persistence SUI model. These findings suggest that the DFAT cells may be an attractive cell source for cell-based therapy to treat SUI.

Phenotypic and functional properties of dedifferentiated fat cells derived from infrapatellar fat pad.

INTRODUCTION: Mature adipocyte-derived dedifferentiated fat cells (DFATs) are mesenchymal stem cell (MSC)-like cells with high proliferative ability and multilineage differentiation potential. In this study, we first examined whether DFATs can be prepared from infrapatellar fat pad (IFP) and then compared phenotypic and functional properties of IFP-derived DFATs (IFP-DFATs) with those of subcutaneous adipose tissue (SC)-derived DFATs (SC-DFATs). METHODS: Mature adipocytes isolated from IFP and SC in osteoarthritis patients (n = 7) were cultured by ceiling culture method to generate DFATs. Obtained IFP-DFATs and SC-DFATs were subjected to flow cytometric and microarray analysis to compare their immunophenotypes and gene expression profiles. Cell proliferation assay and adipogenic, osteogenic, and chondrogenic differentiation assays were performed to evaluate their functional properties. RESULTS: DFATs could be prepared from IFP and SC with similar efficiency. IFP-DFATs and SC-DFATs exhibited similar immunophenotypes (CD73+, CD90+, CD105+, CD31-, CD45-, HLA-DR-) and tri-lineage (adipogenic, osteogenic, and chondrogenic) differentiation potential, consistent with the minimal criteria for defining MSCs. Microarray analysis revealed that the gene expression profiles in IFP-DFATs were very similar to those in SC-DFATs, although there were certain number of genes that showed different levels of expression. The proliferative activity in IFP-DFATs was significantly (p < 0.05) higher than that in the SC-DFATs. IFP-DFATs showed higher chondrogenic differentiation potential than SC-DFATs in regard to production of soluble galactosaminogalactan and gene expression of type II collagen. CONCLUSIONS: IFP-DFATs showed higher cellular proliferative potential and higher chondrogenic differentiation capacity than SC-DFATs. IFP-DFAT cells may be an attractive cell source for chondrogenic regeneration.

Effect of volatile fatty acids on adipocyte differentiation in bovine dedifferentiated fat (DFAT) cells in vitro.

Here, we established dedifferentiated fat (DFAT) cells from mature bovine adipocytes and then examined the effects of volatile fatty acids on the differentiation of these DFAT cells into adipocytes in vitro. When mature adipocytes were isolated from bovine adipose tissue and cultured using the ceiling culture method, they were dedifferentiated into fibroblast-like cells without lipid droplets. These fibroblast-like cells, termed bovine DFAT (b-DFAT) cells, actively proliferated. After adipogenic induction, increased expression of adipocyte-specific genes occurred in b-DFAT cells and they redifferentiated into adipocytes with an accumulation of lipid droplets in their cytoplasm. The effects of volatile fatty acids on adipocyte differentiation in b-DFAT cells were also examined. Specifically, acetate, butyrate, and propionate added to adipogenic induction medium significantly enhanced the adipogenesis of b-DFAT cells compared with that observed in control cells; the addition of 10-3  mol of acetate enhanced adipogenesis of b-DFAT cells to the greatest extent. These results suggest that b-DFAT cells derived from bovine mature adipocytes are appropriate for the study of bovine adipocyte differentiation and that the optimum concentration treatment of acetate, a major energy source for ruminants, promotes adipogenesis of b-DFAT cells in vitro.

Generation of metabolically functional hepatocyte-like cells from dedifferentiated fat cells by Foxa2, Hnf4a and Sall1 transduction.

Mature adipocyte-derived dedifferentiated fat (DFAT) cells have been identified to possess similar multipotency to mesenchymal stem cells, but a method for converting DFAT cells into hepatocytes was previously unknown. Here, using comprehensive analysis of gene expression profiles, we have extracted three transcription factors, namely Foxa2, Hnf4a and Sall1 (FHS), that can convert DFAT cells into hepatocytes. Hepatogenic induction has converted FHS-infected DFAT cells into an epithelial-like morphological state and promoted the expression of hepatocyte-specific features. Furthermore, the DFAT-derived hepatocyte-like (D-Hep) cells catalyzed the detoxification of several compounds. These results indicate that the transduction of DFAT cells with three genes, which were extracted by comprehensive gene expression analysis, efficiently generated D-Hep cells with detoxification abilities similar to those of primary hepatocytes. Thus, D-Hep cells may be useful as a new cell source for surrogate hepatocytes and may be applied to drug discovery studies, such as hepatotoxicity screening and drug metabolism tests.

Therapeutic potential of mature adipocyte-derived dedifferentiated fat cells for inflammatory bowel disease.

PURPOSE: Our previous studies demonstrated that mature adipocyte-derived dedifferentiated fat (DFAT) cells possess similar multipotency as mesenchymal stem cells. Here, we examined the immunoregulatory potential of DFAT cells in vitro and the therapeutic effect of DFAT cell transplantation in a mouse inflammatory bowel disease (IBD) model. METHODS: The effect of DFAT cell co-culture on T cell proliferation and expression of immunosuppression-related genes in DFAT cells were evaluated. To create IBD, CD4+CD45RBhigh T cells were intraperitoneally injected into SCID mice. One week later, DFAT cells (1 × 105, DFAT group) or saline (Control group) were intraperitoneally injected. Subsequently bodyweight was measured every week and IBD clinical and histological scores were evaluated at 5 weeks after T cell administration. RESULTS: The T cell proliferation was inhibited by co-cultured DFAT cells in a cell density-dependent manner. Gene expression of TRAIL, IDO1, and NOS2 in DFAT cells was upregulated by TNFα stimulation. DFAT group improved IBD-associated weight loss, IBD clinical and histological scores compared to Control group. CONCLUSION: DFAT cells possess immunoregulatory potential and the cell transplantation promoted recovery from colon damage and improved clinical symptoms in the IBD model. DFAT cells could play an important role in the treatment of IBD.

All-trans retinoic acid induces reprogramming of canine dedifferentiated cells into neuron-like cells.

The specification of cell identity depends on the exposure of cells to sequences of bioactive ligands. All-trans retinoic acid (ATRA) affects neuronal development in the early stage, and it is involved in neuronal lineage reprogramming. We previously established a fibroblast-like dedifferentiated fat cells (DFATs) derived from highly homogeneous mature adipocytes, which are more suitable for the study of cellular reprogramming. Canine cognitive dysfunction is similar to human cognitive dysfunction, suggesting that dogs could be a pathological and pharmacological model for human neuronal diseases. However, the effect of ATRA on neuronal reprogramming in dogs has remained unclear. Therefore, in this study, we investigated the effect of ATRA on the neuronal reprogramming of canine DFATs. ATRA induced the expression of neuronal marker mRNA/protein. The neuron-like cells showed Ca2+ influx with depolarization (50 mM KCl; 84.75 ± 4.05%) and Na+ channel activation (50 µM veratridine; 96.02 ± 2.02%). Optical imaging of presynaptic terminal activity and detection of neurotransmitter release showed that the neuron-like cells exhibited the GABAergic neuronal property. Genome-wide RNA-sequencing analysis shows that the transcriptome profile of canine DFATs is effectively reprogrammed towards that of cortical interneuron lineage. Collectively, ATRA can produce functional GABAergic cortical interneuron-like cells from canine DFATs, exhibiting neuronal function with > 80% efficiency. We further demonstrated the contribution of JNK3 to ATRA-induced neuronal reprogramming in canine DFATs. In conclusion, the neuron-like cells from canine DFATs could be a powerful tool for translational research in cell transplantation therapy, in vitro disease modeling, and drug screening for neuronal diseases.

The insulin-PI3K-Rac1 axis contributes to terminal adipocyte differentiation through regulation of actin cytoskeleton dynamics.

Adipocyte differentiation is accompanied by a pronounced change in the actin cytoskeleton characterized by the reorganization of filamentous (F)-actin stress fibers into cortical F-actin structures. We previously showed that depolymerization of F-actin stress fibers induced by inactivation of RhoA-ROCK (Rho-associated kinase) signaling acts as a trigger for adipocyte differentiation. The relevance and underlying mechanism of the formation of cortical F-actin structures from depolymerized actin during adipocyte differentiation have remained unclear, however. We have now examined the mechanistic relation between actin dynamics and adipogenic induction. Transient exposure to the actin-depolymerizing agent latrunculin A (LatA) supported the formation of adipocyte-associated cortical actin structures and the completion of terminal adipocyte differentiation in the presence of insulin, whereas long-term exposure to LatA prevented such actin reorganization as well as terminal adipogenesis. Moreover, these effects of insulin were prevented by inhibition of phosphatidylinositol 3-kinase (PI3K)-Rac1 signaling and the actin-related protein 2/3 (Arp2/3) complex which is a critical component of the cortical actin networks. Our findings thus suggest that the insulin-PI3K-Rac1 axis leads to the formation of adipocyte-associated cortical actin structures which is essential for the completion of adipocyte differentiation.

Transplantation of dedifferentiated fat cells combined with a biodegradable type I collagen-recombinant peptide scaffold for critical-size bone defects in rats.

Tissue engineering is a promising approach to supplement existing treatment strategies for craniofacial bone regeneration. In this study, a type I collagen scaffold made from a recombinant peptide (RCP) with an Arg-Gly-Asp motif was developed, and its effect on regeneration in critical-size mandibular bone defects was evaluated. Additionally, the combined effect of the scaffold and lipid-free dedifferentiated fat (DFAT) cells was assessed. Briefly, DFAT cells were separated from mature adipocytes by using a ceiling culture technique based on buoyancy. A 3 cm × 4 cm critical-size bone defect was created in the rat mandible, and regeneration was evaluated by using RCP with DFAT cells. Then, cultured DFAT cells and adipose-derived stem cells (ASCs) were seeded onto RCP scaffolds (DFAT/RCP and ASC/RCP) and implanted into the bone defects. Micro-computed tomography imaging at 8 weeks after implantation showed significantly greater bone regeneration in the DFAT/RCP group than in the ASC/RCP and RCP-alone groups. Similarly, histological analysis showed significantly greater bone width in the DFAT/RCP group than in the ASC/RCP and RCP-alone groups. These findings suggest that DFAT/RCP is effective for bone formation in critical-size bone defects and that DFAT cells are a promising source for bone regeneration.

Transplantation of dedifferentiated fat cell-derived micromass pellets contributed to cartilage repair in the rat osteochondral defect model.

BACKGROUND: Mature adipocyte-derived dedifferentiated fat (DFAT) cells possesses the ability to proliferate effectively and the potential to differentiate into multiple linages of mesenchymal tissue; similar to adipose-derived stem cells (ASCs). The purpose of this study is to examine the effects of DFAT cell transplantation on cartilage repair in a rat model of osteochondral defects. METHODS: Full-thickness osteochondral defects were created in the knees of Sprague-Dawley rats bilaterally. Cartilage-like micromass pellets were prepared from green fluorescent protein (GFP)-labeled rat DFAT cells and subsequently transplanted into the affected right knee of these rats. Defects in the left knee were used as a control. Macroscopic and microscopic changes of treated and control defects were evaluated up to 12 weeks post-treatment with DFAT cells. To observe the transplanted cells, sectioned femurs were immunostained for GFP and type II collagen. RESULTS: DFAT cells formed micromass pellets expressing characteristics of immature cartilage in vitro. In the DFAT cell-transplanted limbs, the defects were completely filled with white micromass pellets as early as 2 weeks post-treatment. These limbs became smooth at 4 weeks. Conversely, the defects in the control limbs were still not repaired by 4 weeks. Macroscopic ICRS scores at 2 and 4 weeks were significantly higher in the DFAT cells-transplanted limbs compared to those of the control limbs. The modified O'Driscol histological scores for the DFAT cell-transplanted limbs were significantly higher than those of the control limbs at corresponding time points. GFP-positive DAFT cells were detected in the transplanted area at 2 weeks but hardly visible at 12 weeks post-operation. CONCLUSIONS: Transplantation of DFAT cell-derived micromass pellets contribute to cartilage repair in a rat osteochondral defect model. DFAT cell transplantation may be a viable therapeutic strategy for the repair of osteochondral injuries.

Effect of collagenase concentration on the isolation of small adipocytes from human buccal fat pad.

Dedifferentiated fat (DFAT) cells were isolated from mature adipocytes using the ceiling culture method. Recently, we successfully isolated DFAT cells from adipocytes with a relatively small size (<40 µm). DFAT cells have a higher osteogenic potential than that of medium adipocytes. Therefore, the objective of this study was to determine the optimal concentration of collagenase solution for isolating small adipocytes from human buccal fat pads (BFPs). Four concentrations of collagenase solution (0.01%, 0.02%, 0.1%, and 0.5%) were used, and their effectiveness was assessed by the number of small adipocytes and DFAT cells isolated. The total number of floating adipocytes that dissociated with 0.02% collagenase was 2.5 times of that dissociated with 0.1% collagenase. The number of floating adipocytes with a diameter of ≤29 µm that dissociated with 0.02% collagenase was thrice of those dissociated with 0.1% and 0.5% collagenase. The number of DFAT cells that dissociated with 0.02% collagenase was 1.5 times of that dissociated with 0.1% collagenase. In addition, DFAT cells that dissociated with 0.02% collagenase had a higher osteogenic differentiation potential than those that dissociated with 0.1% collagenase. These results suggest that 0.02% is the optimal collagenase concentration for isolating small adipocytes from BFPs.

Transplantation of mature adipocyte-derived dedifferentiated fat cells into three-wall defects in the rat periodontium induces tissue regeneration.

The transplantation of dedifferentiated fat (DFAT) cells in combination with poly(d,l-lactic-co-glycolic acid) (PLGA) scaffolds has previously been proven as an effective approach in promoting periodontal tissue regeneration in a rat fenestration defect model. The aim of this study was to assess the regenerative potential of DFAT cells in a rat model of three-wall periodontal bone defect. Three-wall bone defects were created bilaterally on the mesial side of rat maxillary first molars and were either left untreated or treated by implantation of PLGA scaffolds with DFAT cells or PLGA alone. Four weeks after surgery, the tissues were processed for micro-computed tomography (micro-CT) and histomorphometric examination. Micro-CT revealed that the PLGA/DFAT group had significantly higher rates of bone regeneration than the other groups, while histomorphometric analysis showed that the PLGA/DFAT group had significantly higher densities of collagen fiber bundles in acellular and cellular cementum than the PLGA group. Moreover, the results indicate that the placement of the PLGA scaffold prevented the downgrowth of the junctional epithelium. These findings suggest that DFAT cells contribute to tissue regeneration in three-wall periodontal defects, while PLGA provides space necessary for periodontal tissue restoration.

Transplantation of dedifferentiation fat cells promotes intervertebral disc regeneration in a rat intervertebral disc degeneration model.

Our group has reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multilineage differentiation potential similar to that observed in mesenchymal stem cells. In the present study, we examined whether DFAT cell transplantation could contribute to intervertebral disc regeneration using a rat intervertebral disc degeneration (IDD) model. The IDD was created in Sprague-Dawley rats by puncturing at level of caudal intervertebral disc under fluoroscopy. One week after injury, rat DFAT cells (5 × 104, DFAT group, n = 13) or phosphate-buffered saline (PBS, control group, n = 13) were injected into the intervertebral disc. Percent disc height index (%DHI) was measured every week and histology of injured disc was evaluated at 8 weeks after transplantation. Radiographic analysis revealed that the %DHI in the DFAT group significantly higher than that in the control group at 2-3 weeks after transplantation. Histological analysis revealed that ectopic formation of nucleus pulposus (NP)-like tissue at the outer layer of annulus fibrosus was frequently observed in the DFAT group but not in the control group. Transplantation experiments using green fluorescent protein (GFP)-labeled DFAT cells revealed that the ectopic NP-like tissue was positive for GFP, suggesting direct differentiation of DFAT cells into NP-like cells. In conclusion, DFAT cell transplantation promoted the regeneration of intervertebral disc and improved intervertebral disc height in the rat IDD model. Because adipose tissue is abundant and easily accessible, DFAT cell transplantation may be an attractive therapeutic strategy against IDD.

Dedifferentiated Fat Cells as a Novel Source for Cell Therapy to Target Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) remains a major cause of mortality and persistent neurological disabilities in affected individuals. At present, hypothermia is considered to be the only applicable treatment option, although growing evidence suggests that cell-based therapy might achieve better outcomes. Dedifferentiated fat (DFAT) cells are derived from mature adipocytes via a dedifferentiation strategy called ceiling culture. Their abundance and ready availability might make them an ideal therapeutic tool for the treatment of HIE. In the present study, we aimed to determine whether the outcome of HIE can be improved by DFAT cell treatment. HI injury was achieved by ligating the left common carotid artery in 7-day-old rat pups, followed by 1-h exposure to 8% O2. Subsequently, the severity of damage was assessed by diffusion-weighted magnetic resonance imaging to assign animals to equivalent groups. 24 h after hypoxia, DFAT cells were injected at 105 cells/pup into the right external jugular vein. To evaluate brain damage in the acute phase, a group of animals was sacrificed 48 h after the insult, and paraffin sections of the brain were stained to assess several acute injury markers. In the chronic phase, the behavioral outcome was measured by performing a series of behavioral tests. From the 24th day of age, the sensorimotor function was examined by evaluating the initial forepaw placement on a cylinder wall and the latency to falling from a rotarod treadmill. The cognitive function was tested with the novel object recognition (NOR) test. In vitro conditioned medium (CM) prepared from cultured DFAT cells was added at various concentrations to neuronal cell cultures, which were then exposed to oxygen-glucose deprivation (OGD). The number of cells that stained positive for the apoptosis marker active caspase-3 decreased by 73 and 52% in the hippocampus and temporal cortex areas of the brain, respectively, in the DFAT-treated pups. Similarly, the numbers of ED-1-positive cells (activated microglia) decreased by 66 and 44%, respectively, in the same areas in the DFAT-treated group. The number of cells positive for the oxidative stress marker 4-hydroxyl-2-nonenal decreased by 68 and 50% in the hippocampus and the parietal cortex areas, respectively, in the DFAT-treated group. The HI insult led to a motor deficit according to the rotarod treadmill and cylinder test, where it significantly affected the vehicle group, whereas no difference was confirmed between the DFAT and sham groups. However, the NOR test indicated no significant differences between any of the groups. DFAT treatment did not reduce the infarct volume, which was confirmed immunohistochemically. According to in vitro experiments, the cell death rates in the DFAT-CM-treated cells were significantly lower than those in the controls when DFAT-CM was added 48 h prior to OGD. The treatment effect of adding DFAT-CM 24 h prior to OGD was also significant. Our results indicate that intravenous injection with DFAT cells is effective for ameliorating HI brain injury, possibly via paracrine effects.

Transplantation of mature adipocyte-derived dedifferentiated fat cells for the treatment of vesicoureteral reflux in a rat model.

PURPOSE: Autologous cells potentially provide an ideal injectable substance for management in vesicoureteral reflux (VUR). The aim of this study is to examine the effects of mature adipocyte-derived dedifferentiated fat (DFAT) cell transplantation on VUR in a rat bladder pressurization-induced VUR model. METHODS: To create VUR, Sprague-Dawley rats underwent urethral clamping and placement of cystostomy followed by intravesical pressurization. Rat DFAT cells (1 × 106 cells, DFAT group, n = 5) or saline (control group, n = 5) was then injected into the bilateral vesicoureteral junctions. Two weeks later, VUR grade was evaluated on cystography. The number of apoptotic cells in the renal pelvic urothelium, the ureteral inner/outer diameter ratio and the area of connective tissue in the posterior bladder wall were measured. RESULTS: The reflux grade in the DFAT group was significantly lower than that in the control group. The number of apoptotic cells in the renal pelvic urothelium, ureteral inner/outer diameter ratio and connective tissue area in DFAT group were significantly lower in comparison with the control group. CONCLUSIONS: DFAT cell transplantation improved VUR and exerted nephroprotective effects in a rat VUR model.

Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration.

Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration.

Small Buccal Fat Pad Cells Have High Osteogenic Differentiation Potential.

Dedifferentiated fat (DFAT) cells derived from mature adipocytes have mesenchymal stem cells' (MSCs) characteristics. Generally, mature adipocytes are 60-110 µm in diameter; however, association between adipocyte size and dedifferentiation efficiency is still unknown. This study, therefore, investigated the dedifferentiation efficiency of adipocytes based on cell diameter. Buccal fat pad was harvested from five human donors and dissociated by collagenase digestion. After exclusion of unwanted stromal cells by centrifugation, floating adipocytes were collected and their size distribution was analyzed. The floating adipocytes were then separated into two groups depending on cell size using 40- and 100-µm nylon mesh filters: cell diameters less than 40 µm (small adipocytes: S-adipocytes) and cell diameters of 40-100 µm (large adipocytes: L-adipocytes). Finally, we evaluated the efficiency of adipocyte dedifferentiation and then characterized the resultant DFAT cells. The S-adipocytes showed a higher capacity to dedifferentiate into DFAT cells (S-DFAT cells) compared to the L-adipocytes (L-DFAT cells). The S-DFAT cells also showed a relatively higher proportion of CD146-positive cells than L-DFAT cells, and exhibited more osteogenic differentiation ability based on the alkaline phosphatase activity and amount of calcium deposition. These results suggested that the S- and L-DFAT cells had distinct characteristics, and that the higher dedifferentiation potential of S-adipocytes compared to L-adipocytes gives the former group an advantage in yielding DFAT cells.