Mature Adipocyte-derived Dedifferentiated Fat Research Articles

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.

The neovascularization effect of dedifferentiated fat cells

Mature adipocyte-derived dedifferentiated fat (DFAT) cells can be prepared efficiently and with minimal invasiveness to the donor. They can be utilized as a source of transplanted cells during therapy. Although the transplantation of DFAT cells into an ischemic tissue enhances angiogenesis and increases vascular flow, there is little information regarding the mechanism of the therapeutic angiogenesis. To further study this, mice ischemic hindlimb model was used. It was confirmed that in comparison with the adipose derived stem cells and fibroblasts, the transplantation of DFAT cells led to a significant improvement in the blood flow and increased mature blood vessel density. The ability of DFAT cells to secrete angiogenic factors in hypoxic conditions and upon co-culture with vascular endothelial cells was then examined. Furthermore, we examined the possibility that DFAT cells differentiating into pericytes. The therapeutic angiogenic effects of DFAT cells were observed by the secretion of angiogenic factors and pericyte differentiation by transforming growth factor β1 signalling via Smad2/3. DFAT cells can be prepared with minimal invasiveness and high efficiency and are expected to become a source of transplanted cells in the future of angiogenic cell therapy.

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

PurposeOur 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.MethodsThe 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.ResultsThe 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.ConclusionDFAT 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.

Effect of Mature Adipocyte-Derived Dedifferentiated Fat Cells on Formation of Basement Membrane after Cultured Epithelial Autograft on Artificial Dermis.

Artificial dermis is an important option for preparing full-thickness wounds for cultured epithelial autografting. Long-term fragility after cultured epithelial autografting remains a problem, probably because of the lack of basement membrane proteins. The authors hypothesized that treating artificial dermis with mesenchymal stem cells would promote basement membrane protein production. The authors tested this using dedifferentiated fat cells in a porcine experimental model. This study used four male crossbred (Landrace, Large White, and Duroc) swine. Cultured epithelium and dedifferentiated fat cells were prepared from skin and subcutaneous fat tissue harvested from the cervical region. Full-thickness open dorsal wounds were created and treated with artificial dermis to prepare a graft bed for cultured epithelial autograft. Two groups were established: the control group (artificial dermis treated with 0.5 ml of normal saline solution applied to the wounds) and the dedifferentiated fat group (artificial dermis treated with 0.5 × 10 dedifferentiated fat cells suspended in 0.5 ml of normal saline solution sprayed onto the wounds). On postoperative day 10, the prepared cultured epithelium was grafted onto the generated dermis-like tissue. Fourteen days later, tissue specimens were harvested and evaluated histologically. Light microscopy of hematoxylin and eosin-stained sections revealed the beginning of rete ridge formation in the dedifferentiated fat group. Synthesis of both collagen IV and laminin-5 was significantly enhanced in the dedifferentiated fat group. Transmission electron microscopy revealed a nearly mature basement membrane, including anchoring fibrils in the dedifferentiated fat group. Combined use of artificial dermis and dedifferentiated fat cells promotes post-cultured epithelial autograft production and deposition of basement membrane proteins at the dermal-epidermal junction and basement membrane development, including anchoring fibrils.

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

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. 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. DFAT cells formed micromass pellets expressing characteristics of immature cartilage invitro. 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. 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.

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.

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

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. 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. 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. 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.

The Effect of Mature Adipocyte-Derived Dedifferentiated Fat (DFAT) Cells on a Dorsal Skin Flap Model

ABSTRACTBackground: Dedifferentiated fat (DFAT) cells, isolated from mature adipose cell, have high proliferative potential and pluripotency. We report on the expansion of flap survival areas on the back of rats administrating DFAT cells. Materials and Methods: Intraperitoneal adipose tissue was collected from a male Sprague-Dawley (SD) rat. The mature fat cells were cultured on the ceiling surface of culture flask to isolate DFAT cells. On day 7 of the culture, the flask was inverted to allow normal adherent culture. A dorsal caudal-based random pattern flap measuring 2 × 9 cm was raised on each SD rat. We prepared a control group (n = 10) and a flap base injection group in which DFAT cells were injected 2 cm from the flap base (n = 10) and a flap center DFAT injection group (n = 10). In which DFAT cells at 1 × 106 cells/0.1 ml were injected beneath the skin muscle layers of the flap. The flap survival areas were assessed on day 14 after surgery. Results: The mean flap survival rates of the control group, flap center injection group and flap base injection group were 53.6 ± 6.1%, 50.6 ± 6.4% and 65.8 ± 2.4%, respectively. The flap survival areas significantly expanded in the flap base injection group (p < .05). In H-E staining beneath the skin muscle layer connective tissue thickened in the flap base injection group. In the India ink staining, abundant neovascularization was observed inside the thickened parts. Conclusion: The injection of DFAT cells into the flap base promoted the expansion of survival areas.

Effects of combination therapy using basic fibroblast growth factor and mature adipocyte-derived dedifferentiated fat (DFAT) cells on skin graft revascularisation

Background. Although the benefits of basic fibroblast growth factor (bFGF) for wound healing and angiogenesis are well known, its effects on the process of skin graft revascularisation have not been clarified. It was hypothesised that bFGF would be beneficial to promote taking of skin grafts, but that the effect might be limited in the case of bFGF monotherapy. Therefore, this study investigated the efficacy of combination therapy using bFGF and dedifferentiated fat (DFAT) cells. DFAT cells have multilineage differentiation potential, including into endothelial cells, similar to the case of mesenchymal stem cells (MSC). Methods. Commercially available human recombinant bFGF was used. DFAT cells were prepared from SD strain rats as an adipocyte progenitor cell line from mature adipocytes. Full-thickness skin was lifted from the back of SD strain rats and then grafted back to the original wound site. Four groups were established prior to skin grafting: control group (skin graft alone), bFGF group (treated with bFGF), DFAT group (treated with DFAT cells), and combination group (treated with both bFGF and DFAT cells). Tissue specimens for histological examination were harvested 48 hours after grafting. Results. The histological findings for the bFGF group showed vascular augmentation in the grafted dermis compared with the control group. However, the difference in the number of revascularised vessels per unit area did not reach statistical significance against the control group. In contrast, in the combination group, skin graft revascularisation was significantly promoted, especially in the upper dermis. Conclusion. The results suggest that replacement of the existing graft vessels was markedly promoted by the combination therapy using bFGF and DFAT cells, which may facilitate skin graft taking.

Transplantation of mature adipocyte-derived dedifferentiated fat cells promotes locomotor functional recovery by remyelination and glial scar reduction after spinal cord injury in mice

Mature adipocyte-derived dedifferentiated fat cells (DFAT) have a potential to be useful as new cell-source for cell-based therapy for spinal cord injury (SCI), but the mechanisms remain unclear. The objective of this study was to examine whether DFAT-induced functional recovery is achieved through remyelination and/or glial scar reduction in a mice model of SCI. To accomplish this we subjected adult female mice (n=22) to SCI. On the 8th day post-injury locomotor tests were performed, and the mice were randomly divided into two groups (control and DFAT). The DFAT group received stereotaxic injection of DFAT, while the controls received DMEM medium. Functional tests were conducted at repeated intervals, until the 36th day, and immunohistochemistry or staining was performed on the spinal cord sections. DFAT transplantation significantly improved locomotor function of their hindlimbs, and promoted remyelination and glial scar reduction, when compared to the controls. There were significant and positive correlations between promotion of remyelination or/and reduction of glial scar, and recovery of locomotor function. Furthermore, transplanted DFAT expressed markers for neuron, astrocyte, and oligodendrocyte, along with neurotrophic factors, within the injured spinal cord. In conclusion, DFAT-induced functional recovery in mice after SCI is probably mediated by both cell-autonomous and cell-non-autonomous effects on remyelination of the injured spinal cord.

Effects of mature adipocyte-derived dedifferentiated fat (DFAT) cells on generation and vascularisation of dermis-like tissue after artificial dermis grafting

Although artificial dermis (AD) is effective for skin reconstruction, it requires two separate procedures, because the AD must be vascularised before skin grafts. To shorten the period of the dermis-like tissue generation before the secondary skin grafting must be beneficial. Dedifferentiated fat (DFAT) cells are isolated from mature adipose cell suspensions and have potential to differentiate into multiple cell types including endothelial cells. This study aimed to investigate effects of DFAT cells on dermal regeneration after AD grafts in rats. The effects of combination use of DFAT cells and basic fibroblast growth factor (bFGF) were also tested to mimic clinical situations. DFAT cells were isolated from SD rats. Full-thickness wounds were created on the back of rats followed by AD grafting. Five groups were established; Group I: control, Group II: treated with DFAT cells, Group III: treated with bFGF, Group IV: treated with both of DFAT cells and bFGF, and Group V: treated with Green fluorescent protein (GFP)-labelled DFAT cells and bFGF. Histological evaluation was serially performed. Group IV showed markedly promoted vascularisation of dermis-like tissue. In particular, capillary infiltration into the dermis was obtained within 2 days. Immunohistochemical examination revealed that the transplanted DFAT cells had differentiated into endothelial cells and participated in angiogenesis. Group IV also showed a marked increase in the thickness of the dermis like tissue. The present results suggest that the use of DFAT cells under bFGF treatment could be beneficial to shorten the period required for dermal regeneration and vascularisation and contribute to use AD more effectively and safely.

Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells

It has been reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multilineage differentiation potential similar to that observed in mesenchymal stem cells. Since DFAT cells can be prepared from a small quantity of adipose tissue, they could facilitate cell-based therapies in small companion animals such as cats. The present study examined whether multipotent DFAT cells can be generated from feline adipose tissue, and the properties of DFAT cells were compared with those of adipose-derived stem cells (ASCs). DFAT cells and ASCs were prepared from the floating mature adipocyte fraction and the stromal vascular fraction, respectively, of collagenase-digested feline omental adipose tissue. Both cell types were evaluated for growth kinetics, colony-forming unit fibroblast (CFU-F) frequency, immunophenotypic properties, and multilineage differentiation potential.DFAT cells and ASCs could be generated from approximately 1g of adipose tissue and were grown and subcultured on laminin-coated dishes. The frequency of CFU-Fs in DFAT cells (35.8%) was significantly higher than that in ASCs (20.8%) at passage 1 (P1). DFAT cells and ASCs displayed similar immunophenotypes (CD44+, CD90+, CD105+, CD14−, CD34− and CD45−). Alpha-smooth muscle actin-positive cells were readily detected in ASCs (15.2±7.2%) but were rare in DFAT cells (2.2±3.2%) at P1. Both cell types exhibited adipogenic, osteogenic, chondrogenic, and smooth muscle cell differentiation potential in vitro. In conclusion, feline DFAT cells exhibited similar properties to ASCs but displayed higher CFU-F frequency and greater homogeneity. DFAT cells, like ASCs, may be an attractive source for cell-based therapies in cats.

Osteogenic Effects of Dedifferentiated Fat Cell Transplantation in Rabbit Models of Bone Defect and Ovariectomy-Induced Osteoporosis

We have previously reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells have a high proliferative activity and the potential to differentiate into lineages of mesenchymal tissue similar to bone marrow mesenchymal stem cells (MSCs). In the present study, we examined the effects of autologous DFAT cell transplantation on bone regeneration in a rabbit bone defect model and an ovariectomy (OVX)-induced osteoporosis model. The formation of tissue-engineered bone (TEB) was observed when rabbit DFAT cells were loaded onto a β-tricalcium phosphate (TCP)/collagen sponge and cultured in an osteogenic differentiation medium for 3 weeks. Autologous implantation of DFAT cell-mediated TEB constructs promoted bone regeneration in a rabbit tibial defect model. Regenerated bone tissue induced by transplantation of DFAT cell-mediated TEB constructs was histologically well differentiated and exhibited higher bone strength in a three-point bending test compared to that induced by the β-TCP/collagen sponge alone. In OVX-induced osteoporosis model rabbits, DFAT cells were obtained with the osteogenic activity similar to cells from healthy rabbits. Intrabone marrow injection of autologous DFAT cells significantly increased the bone mineral density (BMD) at the injected site in the OVX rabbits. Transplanted DFAT cells remained mainly on the injection side of the bone marrow by at least 28 days after intrabone marrow injection and a part of them expressed osteocalcin. In conclusion, these results demonstrate that autologous implantation of DFAT cells contributed to bone regeneration in a rabbit bone defect model and an OVX-induced osteoporosis model. DFAT cells may be an attractive cell source for cell-based bone tissue engineering to treat nonunion fractures in all patients, including those with osteoporosis.

Dedifferentiated fat cells differentiate into osteoblasts in titanium fiber mesh

Mature adipocyte-derived dedifferentiated fat (DFAT) cells rapidly differentiate into osteoblasts under three-dimensional culture conditions. However, it has not been demonstrated that DFAT cells can differentiate into osteoblasts in a rigid scaffold consisting of titanium fiber mesh (TFM). We examined the proliferation and osteogenic differentiation ability of DFAT cells using TFM as a scaffold. DFAT cells derived from rabbit subcutaneous fat were seeded into TFM and cultured in osteogenic medium containing dexamethasone, L-ascorbic acid 2-phosphate and β-glycerophosphate for 14days. In scanning electron microscopy (SEM) analysis, well-spread cells covered the titanium fibers on day 3, and appeared to increase in number from day 3 to 7. Numerous globular accretions were found and almost completely covered the fibers on day 14. Cell proliferation, as measured by DNA content in the TFM, was significantly higher on day 7 compared with that of day 1. Osteocalcin and calcium content in the TFM were significantly higher on day 14 compared to those of days 1, 3, and 7, indicating DFAT cells differentiated into osteoblasts. We theorize that globular accretions observed in SEM analysis may be calcified matrix resulting from osteocalcin secreted by osteoblasts binding calcium contained in fetal bovine serum. In this study, we demonstrated that DFAT cells differentiate into osteoblasts and deposit mineralized matrices in TFM. Therefore, the combination of DFAT cells and TFM may be an attractive option for bone tissue engineering.

Transplantation of mature adipocyte‐derived dedifferentiated fat (DFAT) cells improves urethral sphincter contractility in a rat model

To examine the effects of mature adipocyte-derived dedifferentiated fat (DFAT) cell transplantation on urethral tissue regeneration and sphincter function. Sixteen female Sprague-Dawley rats underwent vaginal distension (VD) for 3 h. Subsequently, green fluorescence protein (GFP)-labeled DFAT cells (1×10(6) in 20 µL saline, DFAT group, n=8) or saline (20 µL, control group, n=8) were injected into paraurethral connective tissue. Two weeks following VD, leak point pressure (LPP) was measured and an immunohistochemical analysis of the urethra was performed to evaluate urethral sphincter regeneration. The VD model was characterized by atrophy of the urethral sphincter and showed a decrease in LPP. DFAT cell transplantation resulted in a significant improvement of LPP (DFAT group: 37.3±6.4 vs control group: 21.7±5.7 mmHg, P<0.01). Immunohistochemistry revealed that the striated muscle thickness and smooth muscle α-actin-positive area were significantly (P<0.05) larger in the DFAT group than in the control group. DFAT cell transplantation enhanced macrophage accumulation followed by an increased number of cells in the proliferative state. Transplanted DFAT cells were observed in the damaged smooth muscle layer and showed positive staining for smooth muscle α-actin, suggesting conversion into the smooth muscle cell phenotype. DFAT cell transplantation promotes sphincter muscle regeneration and improves LPP in the rat VD model.

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.

Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro

We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin, followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.

Mature Adipocyte-Derived Dedifferentiated Fat Cells Can Trans-Differentiate into Osteoblasts &lt;i&gt;In Vitro&lt;/i&gt; and &lt;i&gt;In Vivo&lt;/i&gt; only by All-Trans Retinoic Acid

We investigated whether de-differentiated fat (DFAT) cells, a mature adipocyte-derived preadipocyte cell line, can be induced to trans-differentiate into osteoblasts in vitro and in vivo. All-trans retinoic acid (RA) induced expression of osteoblast-specific mRNAs encoding Cbfa1/Runx2, osterix, alkaline phosphatase, osteopontin, parathyroid hormone receptor, and osteocalcin in the DFAT cells, but did not induce the expression of adipocyte-specific mRNAs encoding PPARgamma2, C/EBPalpha, and GLUT4. Moreover, alkaline phosphatase activity was expressed in DFAT cells and the cells underwent mineralization of the bone matrix in vitro. Furthermore, when DFAT cells were transplanted subcutaneously into C57BL/6N mice in diffusion chambers, these cells formed ectopic osteoid tissue without any host cell-invasion of the chambers. These results indicate that DFAT cells derived from mature adipocytes can be converted into fully differentiated osteoblasts in vitro and in vivo using RA. DFAT cells provide a unique model for studying the lineage commitment of the adipocytes and osteoblasts derived from mesenchymal stem cells. Identification of the pathways that regulate these processes could lead to the development of new therapeutic strategies for control of unwarranted growth of bone and adipose tissue.