Ceiling culture of human mature white adipocytes with a browning agent: A novel approach to induce transdifferentiation into beige adipocytes.

Excess and dysfunctional adipose tissue plays an important role in metabolic diseases, including obesity, atherosclerosis and type 2 diabetes mellitus. In mammals, adipose tissue is categorized into two types: white and brown. Adult brown tissue is mainly composed of beige adipocytes, which dispose of stored energy as heat and have become increasingly popular as a therapeutic target for obesity. However, there is still a paucity of cell models that allow transdifferentiation of mature white adipocytes into beige adipocytes, as seen in vivo. Here, we describe a novel, ceiling culture-based model of human mature white adipocytes, which transdifferentiate into beige adipocytes under the mechanical force and hypoxia of ceiling culture. We also show that the use of rosiglitazone and rapamycin can modulate transdifferentiation, up and down regulating expression of beige adipocyte-specific genes, respectively. Rosiglitazone additionally facilitated the upregulation of fatty acid lipolysis and oxidation genes. Finally, these beige adipocytes derived from dedifferentiated adipocytes exhibited a progenitor-specific phenotype, with higher expression of mature adipocyte-specific genes than adipocyte-derived stem cells. Overall, we report a novel approach to conveniently cultivate beige adipocytes from white adipocytes in vitro, suitable for mechanistic studies of adipose biology and development of cell and drug therapies in the future.

Focus on dedifferentiated adipocytes: characteristics, mechanisms, and possible applications.

It is largely believed that after undergoing differentiation, adipocytes can no longer divide. Yet, using ceiling culture, it was demonstrated in vitro that some adipocytes are able to regain proliferative abilities by becoming fibroblast-like cells called dedifferentiated adipocytes. Mature adipocytes are abundant, can be easily isolated, and represent a homogenous cell population. Because of these advantageous characteristics, dedifferentiated adipocytes are clinically attractive in tissue engineering as a potential treatment resource for conditions such as type 2 diabetes, cardiac and kidney diseases, as well as autoimmune diseases. The aim of this review article is to summarize current knowledge on adipocyte dedifferentiation by accurately describing dedifferentiated adipocyte characteristics such as morphological appearance, gene expression, antigen signature, pluripotency, and functionality. Current hypotheses possibly explaining the biological mechanisms and cellular reprogramming of the dedifferentiation process are summarized. Dedifferentiated adipocytes show a stem cell-like antigen profile and genome signature which add to their proliferative capacities and their ability to re-differentiate into diverse cell lineages. The dedifferentiation process likely involves liposecretion, that is, the rapid secretion of the cell's lipid droplet. Dedifferentiated adipocytes may allow development of new uses in tissue engineering.

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

OBJECTIVE: 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. METHODS: 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 ß-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. RESULTS: 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. CONCLUSION: 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.

Dedifferentiated Fat (DFAT) cells: A cell source for oral and maxillofacial tissue engineering.

Tissue engineering is a promising method for the regeneration of oral and maxillofacial tissues. Proper selection of a cell source is important for the desired application. This review describes the discovery and usefulness of dedifferentiated fat (DFAT) cells as a cell source for tissue engineering. Dedifferentiated Fat cells are a highly homogeneous cell population (high purity), highly proliferative, and possess a multilineage potential for differentiation into various cell types under proper in vitro inducing conditions and in vivo. Moreover, DFAT cells have a higher differentiation capability of becoming osteoblasts, chondrocytes, and adipocytes than do bone marrow-derived mesenchymal stem cells and/or adipose tissue-derived stem cells. The usefulness of DFAT cells in vivo for periodontal tissue, bone, peripheral nerve, muscle, cartilage, and fat tissue regeneration was reported. Dedifferentiated Fat cells obtained from the human buccal fat pad (BFP) are a minimally invasive procedure with limited esthetic complications for patients. The BFP is a convenient and accessible anatomical site to harvest DFAT cells for dentists and oral surgeons, and thus is a promising cell source for oral and maxillofacial tissue engineering.

Role of the TGF-ß 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-ß 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-ß receptor ALK5) or human TGF-ß1 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-ß1 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-ß1, 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-ß1 or SB431542, respectively, stimulated and inhibited the TGF-ß pathway as shown by increased TGF-ß1, TGF-ß2, COL1A1, and COL6A3 gene expression and decreased expression of TGF-ß1, COL1A1, COL1A2, and COL6A3, respectively. Treatment of DFAT cells with TGF-ß1 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-ß in modulating collagen gene expression during dedifferentiation of mature adipocytes.

Isolation and Characterization of Multipotent Mesenchymal Stem Cells Adhering to Adipocytes in Canine Bone Marrow.

The ceiling culture method has been used to isolate mature adipocytes from adipose tissue that can be dedifferentiated into fibroblastic cells, also known as dedifferentiated fat (DFAT) cells that self-renew and are multipotent, with much higher homogeneity and colony-forming efficiency than those of adipose tissue-derived mesenchymal stem cells. We cultured adipocytes from canine bone marrow using this technique, with the expectation of obtaining DFAT cells. However, contrary to our expectations, continuous monitoring of ceiling cultures by time-lapse microscopy revealed many small cells adhering to adipocytes that proliferated rapidly into cells with a fibroblastic morphology and without any dedifferentiation from adipocytes. We named these cells bone marrow peri-adipocyte cells (BM-PACs) and demonstrated the multipotent properties of BM-PACs compared to that of conventionally cultured canine bone marrow mesenchymal stem cells (BMMSCs). BM-PACs showed significantly greater clonogenicity and proliferation ability than BMMSCs. An in vitro trilineage differentiation assay revealed that BM-PACs possess adipogenic, osteogenic, and chondrogenic capacities superior to those of BMMSCs. Flow cytometric analysis revealed that the expression of CD73, which plays an important role in cell growth and differentiation, was significantly higher in BM-PACs than in BMMSCs. These results indicate that canine BM-PACs have stem cell characteristics that are superior to those of BMMSCs, and that these mesenchymal stem cells (MSCs) appear to be a feasible source for cell-based therapies in dogs.

Progress in cell culture systems for pathological research.

Cell culture is a well-established standard technique and a fundamental tool in biology and medicine. Establishment of a novel culture method by meeting various challenges can sometimes open up new fields of cell biology and medicine. An artificial microenvironment for cultured cells is made up of complicated factors, including cytokines, scaffold material type, cell-cell interactions, and physical stress. To replicate the tissue architecture, cell-cell interactions, and specific physical microenvironment, we previously demonstrated the effectiveness of a three-dimensional culture system, and further established two simple culture systems: air-liquid interface (ALI) and fluid flow stress (FFS). A three-dimensional collagen gel culture system can replicate cell-cell interactions in vitro. As skin is constantly exposed to air, the ALI system closely mimicked the skin microenvironment and maintained the homeostasis of the epidermis and dermis. The ALI culture system also revealed the possibility of skin regeneration through ectopic mesenchymal cell involvement. Fluid streaming and shear stress were recently demonstrated to constitute the critical microenvironment for various cell types. The FFS system demonstrated that fluid streaming induced epithelial-mesenchymal transition of mesothelial cells, leading to peritoneal fibrosis. Our novel culture systems will hopefully open up new fields of regenerative medicine and pathological research.

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.

Bovine dedifferentiated adipose tissue (DFAT) cells: DFAT cell isolation.

Dedifferentiated fat cells (DFAT cells) are derived from lipid-containing (mature) adipocytes, which possess the ability to symmetrically or asymmetrically proliferate, replicate, and redifferentiate/transdifferentiate. Robust cell isolation and downstream culture methods are needed to isolate large numbers of DFAT cells from any (one) adipose depot in order to establish population dynamics and regulation of the cells within and across laboratories. In order to establish more consistent/repeatable methodology here we report on two different methods to establish viable DFAT cell cultures: both traditional cell culture flasks and non-traditional (flat) cell culture plates were used for ceiling culture establishment. Adipocytes (maternal cells of the DFAT cells) were easier to remove from flat culture plates than flasks and the flat plates also allowed cloning rings to be utilized for cell/cell population isolation. While additional aspects of usage of flat-bottomed cell culture plates may yet need to be optimized by definition of optimum bio-coating to enhance cell attachment, utilization of flat plate approaches will allow more efficient study of the dedifferentiation process or the DFAT progeny cells. To extend our preliminary observations, dedifferentiation of Wagyu intramuscular fat (IMF)-derived mature adipocytes and redifferentiation ability of DFAT cells utilizing the aforementioned isolation protocols were examined in traditional basal media/differentiation induction media (DMI) containing adipogenic inducement reagents. In the absence of treatment approximately 10% isolated Wagyu IMF-mature adipocytes dedifferentiated spontaneously and 70% DFAT cells displayed protracted adipogenesis 12 d after confluence in vitro. Lipid-free intracellular vesicles in the cytoplasm (vesicles possessing an intact membrane but with no any observable or stainable lipid inside) were observed during redifferentiation. One to 30% DFAT cells redifferentiated into lipid-assimilating adipocytes in the DMI media, with distinct lipid-droplets in the cytoplasm and with no observable lipid-free vesicles inside. Moreover, a high confluence level promoted the redifferentiation efficiency of DFAT cells. Wagyu IMF dedifferentiated DFAT cells exhibited unique adipogenesis modes in vitro, revealing a useful cell model for studying adipogenesis and lipid metabolism.