Dedifferentiation Of Mature Adipocytes Research Articles

Dynamic interplay between IL-1 and WNT pathways in regulating dermal adipocyte lineage cells during skin development and wound regeneration

Dermal adipocyte lineage cells are highly plastic and can undergo reversible differentiation and dedifferentiation in response to various stimuli. Using single-cell RNA sequencing of developing or wounded mouse skin, we classify dermal fibroblasts (dFBs) into distinct non-adipogenic and adipogenic cell states. Cell differentiation trajectory analyses identify IL-1-NF-κB and WNT-β-catenin as top signaling pathways that positively and negatively associate with adipogenesis, respectively. Upon wounding, activation of adipocyte progenitors and wound-induced adipogenesis are mediated in part by neutrophils through the IL-1R-NF-κB-CREB signaling axis. In contrast, WNT activation, by WNT ligand and/or ablation of Gsk3, inhibits the adipogenic potential of dFBs but promotes lipolysis and dedifferentiation of mature adipocytes, contributing to myofibroblast formation. Finally, sustained WNT activation and inhibition of adipogenesis is seen in human keloids. These data reveal molecular mechanisms underlying the plasticity of dermal adipocyte lineage cells, defining potential therapeutic targets for defective wound healing and scar formation.

Uncoupling Lipid Synthesis from Adipocyte Development.

Obesity results from the expansion of adipose tissue, a versatile tissue regulating energy homeostasis, adipokine secretion, thermogenesis, and inflammation. The primary function of adipocytes is thought to be lipid storage through lipid synthesis, which is presumably intertwined with adipogenesis. However, during prolonged fasting, adipocytes are depleted of lipid droplets yet retain endocrine function and an instant response to nutrients. This observation led us to question whether lipid synthesis and storage can be uncoupled from adipogenesis and adipocyte function. By inhibiting key enzymes in the lipid synthesis pathway during adipocyte development, we demonstrated that a basal level of lipid synthesis is essential for adipogenesis initiation but not for maturation and maintenance of adipocyte identity. Furthermore, inducing dedifferentiation of mature adipocytes abrogated adipocyte identity but not lipid storage. These findings suggest that lipid synthesis and storage are not the defining features of adipocytes and raise the possibility of uncoupling lipid synthesis from adipocyte development to achieve smaller and healthier adipocytes for the treatment of obesity and related disorders.

ECM proteins and cationic polymers coating promote dedifferentiation of patient-derived mature adipocytes to stem cells.

Reprogramming of mature adipocytes is an attractive research area due to the plasticity of these cells. Mature adipocytes can be reprogrammed in vitro, transforming them into dedifferentiated fat cells (DFATs), which are considered a new type of stem cell, and thereby have a high potential for use in tissue engineering and regenerative medicine. However, there are still no reports or findings on in vitro controlling the dedifferentiation. Although ceiling culture performed in related studies is a relatively simple method, its yield is low and does not allow manipulation of mature adipocytes to increase or decrease the dedifferentiation. In this study, to understand the role of physicochemical surface effects on the dedifferentiation of patient-derived mature adipocytes, the surfaces of cell culture flasks were coated with extracellular matrix, basement membrane proteins, and cationic/anionic polymers. Extracellular matrix such as fibronectin and collagen type I, and basement membrane proteins such as collagen type IV and laminin strongly promoted dedifferentiation of mature adipocytes, with laminin showing the highest effect with a DFAT ratio of 2.98 (±0.84). Interestingly, cationic polymers also showed a high dedifferentiation effect, but anionic polymers did not, and poly(diallyl dimethylammonium chloride) showed the highest DFAT ratio of 2.27 (±2.8) among the cationic polymers. Protein assay results revealed that serum proteins were strongly adsorbed on the surfaces of the cationic polymer coating, including inducing high mature adipocyte adhesion. This study demonstrates for the first time the possibility of regulating the transformation of mature adipocytes to DFAT stem cells by controlling the physicochemical properties of the surface of conventional cell culture flasks.

Increased Fat Graft Survival by Promoting Adipocyte Dedifferentiation.

Some adipocytes undergo dedifferentiation after fat transplantation, and this may affect the survival of fat grafts. However, this effect has not been adequately studied. This study aimed to clarify the effect of promoting the dedifferentiation of mature adipocytes on the survival of fat grafts. Mature adipocytes and adipose stem cells (ASCs) were treated with OSI-906 (a specific inhibitor of insulin receptor and insulin-like growth factor-1 receptor) in vitro, and then the dedifferentiation of mature adipocytes and the proliferation of ASCs were evaluated. In the in vivo experiment, human lipoaspirates mixed with phosphate-buffered saline (Group A) or OSI-906 (Group B) were compared in nude mice. Grafts were harvested at 2, 8, and 12 weeks, and volume retention rate, histologic, and immunohistochemical analyses were conducted. OSI-906 can promote the dedifferentiation of mature adipocytes and inhibit the proliferation of ASCs. At 12 weeks, Group B showed a better volume retention rate (mean [standard deviation, SD], 62.3% [7.61%]) than group A (47.75% [6.11%]) (P < .05). Moreover, viable adipocytes and vascularization showed greater improvement in Group B than in Group A. This study suggests that promoting the dedifferentiation of mature adipocytes can improve the survival rate and quality of fat grafts.

Dedifferentiation of Human Adipocytes After Fat Transplantation.

Fat transplantation is a common method employed to treat soft-tissue defects. The dedifferentiation of mature adipocytes has been well documented, but whether it occurs after fat transplantation remains unclear. The major purpose of this project was to investigate the dedifferentiation of mature adipocytes after fat transplantation. Human lipoaspirate tissue was obtained from 6 female patients who underwent esthetic liposuction. Mature adipocytes were extracted and labeled with PKH26, mixed with lipoaspirate, and injected into nude mice. In addition, PKH26+ adipocytes were subjected to a ceiling culture. Grafted fat was harvested from nude mice, and stromal vascular fragment cells were isolated. The immunophenotype of PKH26+ cells was detected by flow cytometry analysis at 2 days and 1 week. The PKH26+ cells were sorted and counted at 2 and 4 weeks to verify their proliferation and multilineage differentiation abilities. Two days after transplantation, almost no PKH26+ cells were found in the stromal vascular fragment cells. The PKH26+ cells found 1 week after transplantation showed a positive expression of cluster of differentiation (CD) 90 (CD90) and CD105 and a negative expression of CD45. This indicates that the labeled adipocytes were dedifferentiated. Its pluripotency was further demonstrated by fluorescent cell sorting and differentiation culture in vitro. In addition, the number of live PKH26+ cells at week 4 [(6.83 ± 1.67) × 104] was similar with that at week 2 [(7.11 ± 1.82) × 104]. Human mature adipocytes can dedifferentiate into stem cell-like cells in vivo after fat transplantation.

Generation of the Compression-induced Dedifferentiated Adipocytes (CiDAs) Using Hypertonic Medium.

Current methods to obtain mesenchymal stem cells (MSCs) involve sampling, culturing, and expanding of primary MSCs from adipose, bone marrow, and umbilical cord tissues. However, the drawbacks are the limited numbers of total cells in MSC pools, and their decaying stemness during in vitro expansion. As an alternative resource, recent ceiling culture methods allow the generation of dedifferentiated fat cells (DFATs) from mature adipocytes. Nevertheless, this process of spontaneous dedifferentiation of mature adipocytes is laborious and time-consuming. This paper describes a modified protocol for in vitro dedifferentiation of adipocytes by employing an additional physical stimulation, which takes advantage of augmenting the stemness-related Wnt/β-catenin signaling. Specifically, this protocol utilizes a polyethylene glycol (PEG)-containing hypertonic medium to introduce extracellular physical stimulation to obtain higher efficiency and introduce a simpler procedure for adipocyte dedifferentiation.

Discussion: Mechanical Signals Induce Dedifferentiation of Mature Adipocytes and Increase the Retention Rate of Fat Grafts.

Discussion: Mechanical Signals Induce Dedifferentiation of Mature Adipocytes and Increase the Retention Rate of Fat Grafts.

Mechanical Signals Induce Dedifferentiation of Mature Adipocytes and Increase the Retention Rate of Fat Grafts.

Mature adipocytes dedifferentiate in vivo on application of a soft-tissue expander. Dedifferentiated adipocytes can proliferate and redifferentiate. This study used tissue expanders to pretreat adipose flaps, to increase the retention rate after fat graft. A soft-tissue expander and silicone sheet were implanted beneath the left and right inguinal fat pads of rats, respectively. After 7 days of expansion, the adipose tissue derived from the pads was transplanted beneath dorsal skin. Samples were harvested at various time points, and histologic, immunohistochemical, and gene expression analyses were conducted. Mature adipocytes were cultured in vitro under a pressure of 520 Pa. Changes in cell morphology, the cytoskeleton, and expression of mechanical signal-related proteins were investigated. Pressure in adipose flaps increased to 25 kPa on expansion. Mature adipocytes dedifferentiated following expansion. At 1 week after transplantation, the expression of vascular endothelial growth factor (p < 0.05) was higher in the expanded group. The retention rate at 12 weeks after transplantation was higher in the expanded group (56 ± 3 percent) than in the control group (32 ± 3 percent) (p < 0.05), and the surviving/regenerating zones (p < 0.01) were wider. The lipid content of mature adipocytes gradually decreased on culture under increased pressure, and these cells regained a proliferative capacity. This was accompanied by increased expression of mechanical signal--related proteins (p < 0.05). Mechanical signals may induce dedifferentiation of mature adipocytes. Dedifferentiated adipocytes increase the retention rate of fat grafts by acting as seed cells.

Discussion: Mechanical Signals Induce Dedifferentiation of Mature Adipocytes and Increase the Retention Rate of Fat Grafts.

Discussion: Mechanical Signals Induce Dedifferentiation of Mature Adipocytes and Increase the Retention Rate of Fat Grafts.

Adipocyte dedifferentiation in health and diseases.

Adipose tissues collectively as an endocrine organ and energy storage are crucial for systemic metabolic homeostasis. The major cell type in the adipose tissue, the adipocytes or fat cells, are remarkably plastic and can increase or decrease their size and number to adapt to changes in systemic or local metabolism. Changes in adipocyte size occur through hypertrophy or atrophy, and changes in cell numbers mainly involve de novo generation of new cells or death of existing cells. Recently, dedifferentiation, whereby a mature adipocyte is reverted to an undifferentiated progenitor-like status, has been reported as a mechanism underlying adipocyte plasticity. Dedifferentiation of mature adipocytes has been observed under both physiological and pathological conditions. This review covers several aspects of adipocyte dedifferentiation, its relevance to adipose tissue function, molecular pathways that drive dedifferentiation, and the potential of therapeutic targeting adipocyte dedifferentiation in human health and metabolic diseases.

Dedifferentiation of mature adipocytes with periodic exposure to cold.

Lipid-containing adipocytes can dedifferentiate into fibroblast-like cells under appropriate culture conditions, which are known as dedifferentiated fat (DFAT) cells. However, the relative low dedifferentiation efficiency with the established protocols limit their widespread applications. In this study, we found that adipocyte dedifferentiation could be promoted via periodic exposure to cold (10°C) in vitro. The lipid droplets in mature adipocytes were reduced by culturing the cells in periodic cooling/heating cycles (10-37°C) for one week. The periodic temperature change led to the down-regulation of the adipogenic genes (FABP4, Leptin) and up-regulation of the mitochondrial uncoupling related genes (UCP1, PGC-1α, and PRDM16). In addition, the enhanced expression of the cell proliferation marker Ki67 was observed in the dedifferentiated fibroblast-like cells after periodic exposure to cold, as compared to the cells cultured in 37°C. Our in vitro model provides a simple and effective approach to promote lipolysis and can be used to improve the dedifferentiation efficiency of adipocytes towards multipotent DFAT cells.

Characterization and visualization of the liposecretion process taking place during ceiling culture of human mature adipocytes.

To investigate and further characterize the process of mature adipocyte dedifferentiation. Our hypothesis was that dedifferentiation does not involve mitosis but rather a phenomenon of liposecretion. Mature adipocytes were isolated by collagenase digestion of human adipose tissue samples. Ceiling cultures were established using our six-well plate model. Cells were treated with cytosine β-d-arabinofuranoside (AraC) or vincristine (VCR), two agents blocking cell division, and were compared with vehicle. Liposecretion events were visualized by time-lapse microscopy, with and without AraC in adipocytes transducted with a baculovirus. Microscopic analyses were performed after labeling phosphorylated histone 3 and cyclin B1 in ceiling cultures. Treatment with AraC almost entirely prevented the formation of fibroblasts up to 12 days of ceiling culture. Similar results were obtained with VCR. The antimitotic effectiveness of the treatment was confirmed in fibroblast cultures from the adipose tissue stromal-vascular fraction by proliferation assays and colony-forming unit experiments. Using time-lapse microscopy, we visualized liposecretion events in which a large lipid droplet was rapidly secreted from isolated mature adipocytes. The same phenomenon was observed with AraC. This was observed in conjunction with histone 3 phosphorylation and cyclin B1 segregation to the nucleus. Our results support the notion that dedifferentiation involves rapid secretion of the lipid droplet by the adipocytes with concomitant generation of fibroblast-like cells that subsequently proliferate to generate the dedifferentiated adipocyte population during ceiling culture. The presence of mitotic markers suggests that this process involves cell cycle progression, although cell division does not occur.

HASC and DFAT, Multipotent Stem Cells for Regenerative Medicine: A Comparison of Their Potential Differentiation In Vitro.

Adipose tissue comprises both adipose and non-adipose cells such as mesenchymal stem cells. These cells show a surface antigenic profile similar to that of bone-marrow-derived MSC. The cells derived from the dedifferentiation of mature adipocytes (DFAT) are another cell population with characteristics of stemness. The aim of this study is to provide evidence of the stemness, proliferation, and differentiation of human adipose stem cells (hASC) and DFAT obtained from human subcutaneous AT and evaluate their potential use in regenerative medicine. Cell populations were studied by histochemical and molecular biology techniques. Both hASC and DFAT were positive for MSC markers. Their proliferative capacity was similar and both populations were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. DFAT were able to accumulate lipids and their lipoprotein lipase and adiponectin gene expression were high. Alkaline phosphatase and RUNX2 gene expression were greater in hASC than in DFAT at 14 days but became similar after three weeks. Both cell populations were able to differentiate into chondrocytes, showing positive staining with Alcian Blue and gene expression of SOX9 and ACAN. In conclusion, both hASC and DFAT populations derived from AT have a high differentiation capacity and thus may have applications in regenerative medicine.

Role of the TGF-β pathway in dedifferentiation of human mature adipocytes.

Dedifferentiation of adipocytes contributes to the generation of a proliferative cell population that could be useful in cellular therapy or tissue engineering. Adipocytes can dedifferentiate into precursor cells to acquire a fibroblast‐like phenotype using ceiling culture, in which the buoyancy of fat cells is exploited to allow them to adhere to the inner surface of a container. Ceiling culture is usually performed in flasks, which limits the ability to test various culture conditions. Using a new six‐well plate ceiling culture approach, we examined the relevance of TGF‐β signaling during dedifferentiation. Adipose tissue samples from patients undergoing bariatric surgery were digested with collagenase, and cell suspensions were used for ceiling cultures. Using the six‐well plate approach, cells were treated with SB431542 (an inhibitor of TGF‐β receptor ALK5) or human TGF‐β1 during dedifferentiation. Gene expression was measured in these cultures and in whole adipose tissue, the stromal–vascular fraction (SVF), mature adipocytes, and dedifferentiated fat (DFAT) cells. TGF‐β1 and collagen type I alpha 1 (COL1A1) gene expression was significantly higher in DFAT cells compared to whole adipose tissue samples and SVF cells. TGF‐β1, COL1A1, and COL6A3 gene expression was significantly higher at day 12 of dedifferentiation compared to day 0. In the six‐well plate model, treatment with TGF‐β1 or SB431542, respectively, stimulated and inhibited the TGF‐β pathway as shown by increased TGF‐β1, TGF‐β2, COL1A1, and COL6A3 gene expression and decreased expression of TGF‐β1, COL1A1, COL1A2, and COL6A3, respectively. Treatment of DFAT cells with TGF‐β1 increased the phosphorylation level of SMAD 2 and SMAD 3. Thus, a new six‐well plate model for ceiling culture allowed us to demonstrate a role for TGF‐β in modulating collagen gene expression during dedifferentiation of mature adipocytes.

Temporal Changes in Gene Expression Profile during Mature Adipocyte Dedifferentiation.

Objective. To characterize changes in gene expression profile during human mature adipocyte dedifferentiation in ceiling culture. Methods. Subcutaneous (SC) and omental (OM) adipose tissue samples were obtained from 4 participants paired for age and BMI. Isolated adipocytes were dedifferentiated in ceiling culture. Gene expression analysis at days 0, 4, 7, and 12 of the cultures was performed using Affymetrix Human Gene 2.0 STvi arrays. Hierarchical clustering according to similarity of expression changes was used to identify overrepresented functions. Results. Four clusters gathered genes with similar expression between day 4 to day 7 but decreasing expression from day 7 to day 12. Most of these genes coded for proteins involved in adipocyte functions (LIPE, PLIN1, DGAT2, PNPLA2, ADIPOQ, CEBPA, LPL, FABP4, SCD, INSR, and LEP). Expression of several genes coding for proteins implicated in cellular proliferation and growth or cell cycle increased significantly from day 7 to day 12 (WNT5A, KITLG, and FGF5). Genes coding for extracellular matrix proteins were differentially expressed between days 0, 4, 7, and 12 (COL1A1, COL1A2, and COL6A3, MMP1, and TGFB1). Conclusion. Dedifferentiation is associated with downregulation of transcripts encoding proteins involved in mature adipocyte functions and upregulation of genes involved in matrix remodeling, cellular development, and cell cycle.

Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.

The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used.

Decelerating Mature Adipocyte Dedifferentiation by Media Composition.

The establishment of adipose tissue test systems is still a major challenge in the investigation of cellular and molecular interactions responsible for the pathogenesis of inflammatory diseases involving adipose tissue. Mature adipocytes are mainly involved in these pathologies, but rarely used in vitro, due to the lack of an appropriate culture medium which inhibits dedifferentiation and maintains adipocyte functionality. In our study, we showed that Dulbecco's Modified Eagle's Medium/Ham's F-12 with 10% fetal calf serum (FCS) reported for the culture of mature adipocytes favors dedifferentiation, which was accompanied by a high glycerol release, a decreasing release of leptin, and a low expression of the adipocyte marker perilipin A, but high expression of CD73 after 21 days. Optimized media containing FCS, biotin, pantothenate, insulin, and dexamethasone decelerated the dedifferentiation process. These cells showed a lower lipolysis rate, a high level of leptin release, as well as a high expression of perilipin A. CD73-positive dedifferentiated fat cells were only found in low quantity. In this work, we showed that mature adipocytes when cultured under optimized conditions could be highly valuable for adipose tissue engineering in vitro.

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Differentiation of 3T3-L1 preadipocytes is inhibited under a modified ceiling culture.

Ceiling culture is an inverted and closed cell culture system which represents a novel method for exploring adipocyte characteristics and function. Although the role of ceiling culture in mature adipocyte dedifferentiation has been extensively studied, its potential effects on preadipocyte differentiation remain unclear. In this study, we established a simplified dish ceiling culture method for 3T3-L1 preadipocytes and showed that our novel ceiling culture method could reproduce the function of the traditional flask ceiling culture. Then, we investigated the effects of ceiling culture on 3T3-L1 preadipocyte differentiation by Oil red O staining and RT-qPCR. The results showed that ceiling culture significantly impaired triglyceride accumulation and adipogenic marker genes expression in 3T3-L1 preadipocytes. These findings suggest that ceiling culture inhibited 3T3-L1 preadipocyte differentiation while inducing mature adipocytes dedifferentiation. Taken together, our data facilitate the understanding of the property of ceiling culture and promote the study of revealing the underlying mechanisms of mature adipocytes dedifferenatiation.