Insights into the molecular changes of adipocyte dedifferentiation and its future research opportunities.

Recent studies have challenged the traditional belief that mature fat cells are irreversibly differentiated and revealed they can dedifferentiate into fibroblast-like cells known as dedifferentiated fat (DFAT) cells. Resembling pluripotent stem cells, DFAT cells hold great potential as a cell source for stem cell therapy. However, there is limited understanding of the specific changes that occur following adipocyte dedifferentiation and the detailed regulation of this process. This review explores the epigenetic, genetic, and phenotypic alterations associated with DFAT cell dedifferentiation, identifies potential targets for clinical regulation and discusses the current applications and challenges in the field of DFAT cell research.

Muscle regeneration therapy using dedifferentiated fat cell (DFAT) for anal sphincter dysfunction.

PURPOSE: We investigated the effects of mouse-derived DFAT on the myogenic differentiation of a mouse-derived myoblast cell line (C2C12) and examined the therapeutic effects of rat-derived DFAT on anal sphincter injury using a rat model. METHODS: C2C12 cells were cultured using DMEM and DFAT-conditioned medium (DFAT-CM), evaluating MyoD and Myogenin gene expression via RT-PCR. DFAT was locally administered to model rats with anorectal sphincter dysfunction 3 days post-CTX injection. Therapeutic effects were assessed through functional assessment, including anal pressure measurement using solid-state manometry pre/post-CTX, and on days 1, 3, 7, 10, 14, 17, and 21 post-DFAT administration. Histological evaluation involved anal canal excision on days 1, 3, 7, 14, and 21 after CTX administration, followed by hematoxylin-eosin staining. RESULTS: C2C12 cells cultured with DFAT-CM exhibited increased MyoD and Myogenin gene expression compared to control. Anal pressure measurements revealed early recovery of resting pressure in the DFAT-treated group. Histologically, DFAT-treated rats demonstrated an increase in mature muscle cells within newly formed muscle fibers on days 14 and 21 after CTX administration, indicating enhanced muscle tissue repair. CONCLUSION: DFAT demonstrated the potential to enhance histological and functional muscle tissue repair. These findings propose DFAT as a novel therapeutic approach for anorectal sphincter dysfunction treatment.

Effects of dedifferentiated fat cells on neurogenic differentiation and cell proliferation in neuroblastoma cells.

PURPOSE: Mesenchymal stem cells (MSCs) can induce differentiation of neuroblastoma (NB) cells. Properties of dedifferentiated fat cells (DFATs) are similar to those of MSCs. Here, we investigated whether DFATs can induce NB cell differentiation and suppress cell proliferation. METHODS: DFATs were obtained from mature adipocytes isolated from adipose tissue from a ceiling culture. NB cells were cultured in a medium with or without DFATs and, subsequently, cultured in a DFAT-conditioned medium (CM) with or without phosphatidylinositol 3-kinase (PI3K) inhibitor. The neurite lengths were measured, and mRNA expression levels of the neurofilament (NF) and tubulin beta III (TUBß3) were assessed using quantitative real-time RT-PCR. Cell viability was assessed using the WST-1 assay. RESULTS: NB cells cultured with DFATs caused elongation of the neurites and upregulated the expression of NF and Tubß3. NB cells cultured in DFAT-CM demonstrated increased cell viability. NB cells cultured with DFAT-CM and PI3K inhibitors suppressed cell viability. NB cells cultured with DFAT-CM and PI3K inhibitor demonstrated increased neurite length and expression, and upregulation of Tubß3. CONCLUSION: The combined use of DFAT-CM and PI3K inhibitors suppresses the proliferation of NB cells and induces their differentiation. Thus, DFAT may offer new insights into therapeutic approaches in neuroblastoma.

Early histological and ultrastructural changes in expanded murine scalp.

Tissue expansion has been widely used for plastic, reconstructive, and esthetic surgeries. A mouse scalp expansion model can effectively mimic the characteristics of human skin expansion. However, a detailed study of the histological features and ultrastructural characteristics of expanded scalp is lacking, especially early ultrastructural changes. Here, a mouse scalp expansion model was established and the expanded scalp samples were obtained on day 2 (group I) and 4 (group II) post final injection. Histological analysis revealed epidermal thickening, dermal thinning, subcutaneous fat thinning, and capsule formation in the expanded samples. Ultrastructural evaluation showed the presence of keratinocytes with numerous tonofibrils and damaged mitochondria, and several ruptured collagen fibers and increased number of active fibroblasts and myofibroblasts were observed in the dermis and capsules. Adipocyte dedifferentiation was detected in the expanded samples of both groups, but formation of autophagosomes was only detected in the dermal fibroblasts of group I. Thus, early changes in expanded tissue should be carefully monitored, as it may help avoid dermal thinning and promote expanded tissue regeneration.

Osteogenesis of Multipotent Progenitor Cells using the Epigallocatechin Gallate-Modified Gelatin Sponge Scaffold in the Rat Congenital Cleft-Jaw Model.

Cost-effective and functionalized scaffolds are in high demand for stem-cell-based regenerative medicine to treat refractory bone defects in craniofacial abnormalities and injuries. One potential strategy is to utilize pharmacological and cost-effective plant polyphenols and biocompatible proteins, such as gelatin. Nevertheless, the use of chemically modified proteins with plant polyphenols in this strategy has not been standardized. Here, we demonstrated that gelatin chemically modified with epigallocatechin gallate (EGCG), the major catechin isolated from green tea, can be a useful material to induce bone regeneration in a rat congenial cleft-jaw model in vivo when used with/without adipose-derived stem cells or dedifferentiated fat cells. Vacuum-heated gelatin sponges modified with EGCG (vhEGCG-GS) induced superior osteogenesis from these two cell types compared with vacuum-heated gelatin sponges (vhGS). The EGCG-modification converted the water wettability of vhGS to a hydrophilic property (contact angle: 110° to 3.8°) and the zeta potential to a negative surface charge; the modification enhanced the cell adhesion property and promoted calcium phosphate precipitation. These results suggest that the EGCG-modification with chemical synthesis can be a useful platform to modify the physicochemical property of gelatin. This alteration is likely to provide a preferable microenvironment for multipotent progenitor cells, inducing superior bone formation in vivo.

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.

Characteristics and multipotency of equine dedifferentiated fat cells.

Dedifferentiated fat (DFAT) cells have been shown to be multipotent, similar to mesenchymal stem cells (MSCs). In this study, we aimed to establish and characterize equine DFAT cells. Equine adipocytes were ceiling cultured, and then dedifferentiated into DFAT cells by the seventh day of culture. The number of DFAT cells was increased to over 10 million by the fourth passage. Flow cytometry of DFAT cells showed that the cells were strongly positive for CD44, CD90, and major histocompatibility complex (MHC) class I; moderately positive for CD11a/18, CD105, and MHC class II; and negative for CD34 and CD45. Moreover, DFAT cells were positive for the expression of sex determining region Y-box 2 as a marker of multipotency. Finally, we found that DFAT cells could differentiate into osteogenic, chondrogenic, and adipogenic lineages under specific nutrient conditions. Thus, DFAT cells could have clinical applications in tissue regeneration, similar to MSCs derived from adipose tissue.

The perivascular phenotype and behaviors of dedifferentiated cells derived from human mature adipocytes.

Derived from mature adipocytes, dedifferentiated fat (DFAT) cells represent a special group of multipotent cells. However, their phenotype and cellular nature remain unclear. Our study found that human DFAT cells adopted perivascular characteristics and behaviors. Flow cytometry and immunofluorescent staining revealed that human DFAT cells positively expressed markers highly related to perivascular cell lineages, such as CD140b, NG2 and desmin, but were negative for common endothelial markers, including CD31, CD34, and CD309. Furthermore, DFAT cells displayed vascular network formation ability in Matrigel, and they noticeably promoted and stabilized the vessel structures formed by human umbilical vascular endothelial cells (HUVECs) in vitro. These results provide novel evidence on the pericyte nature of human DFAT cells, further supporting the recent model for the perivascular origin of adult stem cells, in which tissue-specific progenitor cells in mesenchymal tissues associate with blood vessels, exhibiting perivascular characteristics and functions.

Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro.

Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT) cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG), the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25-10 µM) in osteogenic medium (OM) with or without 100 nM dexamethasone (Dex) for 12 days (hereafter two osteogenic media were designated as OM(Dex) and OM). Supplementation of 1.25 µM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1) and runt-related transcription factor 2 (RUNX2) and also increased proliferation and mineralization. Compared to OM(Dex) with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex) with 10 µM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex) with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy.

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.

Senescence-associated secretory phenotypes in rat-derived dedifferentiated fat cells with replicative senescence.

Senescence-associated secretory phenotype (SASPs) secreted from senescent cells often cause the deleterious damages to the surrounding tissues. Although dedifferentiated fat (DFAT) cells prepared are considered a promising cell source for regenerative therapies, SASPs from DFAT cells undergoing long-term cell culture, which latently induce replicative senescence, have barely been explored. The present study was designed to investigate senescent behaviors in rat-derived DFAT cells at high passage numbers and to analyze the possible types of SASPs. Our data show that DFAT cells undergo senescence during replicative passaging, as determined by multiple senescent hallmarks including morphological changes in cell shape and nucleus. Moreover, RT2 PCR array analysis indicated that senescent DFAT cells expressed higher levels of 16 inflammatory cytokines (Ccl11, Ccl12, Ccl21, Ccl5, Csf2, Cxcl1, Cxcl12, Ifna2, IL11, IL12a, IL13, IL1a, IL1rn, IL6, Mif, and Tnf) associated with SASPs than non-senescent cells. This study implicates that rat DFAT cells undergo cellular senescence after long-term cell culture; cautious consideration should be paid to treat SASP secretion when senescent DFAT cells are used in regenerative medicine.

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.

Comparative Study of the Osteogenic Differentiation Potential of Adipose Tissue-Derived Stromal Cells and Dedifferentiated Adipose Cells of the Same Tissue Origin under Pro and Antioxidant Conditions.

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.

Enhancement of early cardiac differentiation of dedifferentiated fat cells by dimethyloxalylglycine via notch signaling pathway.

Background: Hypoxia has been reported to possess the ability to induce mature lipid-filled adipocytes to differentiate into fibroblast-like multipotent dedifferentiated fat (DFAT) cells and stem cells such as iPSCs (interstitial pluripotent stem cells) and ESCs (embryonic stem cells) and then to differentiate into cardiomyocytes. However, the effect of hypoxia on cardiac differentiation of DFAT cells and its underlying molecular mechanism remains to be investigated. Objective: To investigate the role of hypoxia in early cardiac differentiation of DFAT cells and the underlying molecular mechanism. Methods: DFAT cells were prepared from 4 to 6 week-age mice and cultured under hypoxic conditions by adding Prolyl hydroxylase inhibitor and dimethyloxalylglycine (DMOG) into the culture media. To inhibit or block Notch signaling, γ-secretase inhibitor-II (GSI-II) and Notch1 siRNA (si-Notch1) were used. DFAT cell viability was detected using MTT assay. qRT-PCR, immunofluorescence microscopy and western blotting were used to evaluate the cardiac differentiation of DFAT cells and co-immunoprecipitation was used to study the interaction between HIF-1α and Notch signaling. Results: 0.6-mM DMOG failed to affect the viability of DFAT cells, but stimulated the cells to express early cardiac transcription factors including Islet1, Nkx2.5 and Gata4 in a time-dependent manner and increase the number of cTnT+ cardiomyocytes (detected at the 28th day after stimulation). It was also demonstrated that DMOG was involved in HIF-1α and Notch signaling as well as HIF-1α-NICD complex formation. Conclusion: Hypoxia enhanced early cardiac differentiation of DFAT cells through HIF-1α and Notch signaling pathway.