Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.     

去分化脂肪细胞的研究进展

去分化脂肪(Dedifferentiated fat,DFAT)细胞是由人体内含量最丰富的成熟脂肪细胞经体外天花板法培养去分化而来。研究发现:DFAT细胞具有均一性高、对供者年龄要求较低等脂肪来源干细胞(Adipose-derived stem cells,ASCs)和骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)所不具有的优势。此外,它还具有体内外成脂、成软骨、成骨、成肌、成神经等多向分化能力以及免疫调节能力。作为具有潜力的组织工程及同种异体干细胞移植的优秀种子细胞,DFAT细胞在治疗骨缺损、神经性疾病、局部缺血性心脏病及肾脏疾病等方面均具有较好的应用前景,对其开展深入的研究具有重要的理论和实践意义。文中从免疫学性质、多向分化能力及临床应用潜力等方面对DFAT细胞的研究进展作一综述。     

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling,on adipocyte differentiation

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer–binding protein δ expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activator of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator–activator receptor γ expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-α did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.     

Analysis of gene expression profile during 3T3-L1 preadipocyte differentiation

Cellular differentiation is a process in which a group of differentiation specific genes is programmatically induced. This gene expression program leads to changes in both cellular morphological and physiological phenotypes. Using an 18,376-member cDNA/EST microarray, we analyzed the difference in gene expression profiles between differentiated 3T3-L1 adipocyte and non-differentiated 3T3-L1 preadipocyte. From our study a large number of genes and ESTs were identified as differentially induced or suppressed. In this paper we describe the changes of gene expression profile during 3T3-L1 cell differentiation.     

Coupling of proadipocyte growth arrest and differentiation. I. Induction by heparinized medium containing human plasma

The differentiation of proadipocytes in vitro typically required prolonged culture of cells as a high density in high concentrations of serum and added hormones. With such culture conditions it is difficult to design experiments to determine the mechanisms that control the differentiation process. We now describe the rapid and parasynchronous growth arrest and differentiation of low density murine proadipocytes in heparinized medium containing only human plasma. When low density cells are cultured under these conditions, growth arrest at a distinct state in the G1 phase of the cell cycle occurs within 2 d and the differentiation of 80-100% of the cell population occurs within 4 d thereafter. The factors in human plasma which promote growth arrest and differentiation are heat labile and can be separated by barium adsorption. In the following paper we have used these methods to show that there are five separate phases which regulate the coupling of proadipocyte growth arrest and differentiation. The data reported in this paper establish that: (a) high cell density and extensive cell-to- cell contact are not required for adipocyte differentiation, (b) prolonged culture is not required for adipocyte differentiation, and (c) high concentrations of serum and/or added hormones are not required for adipocyte differentiation.     

The novel gene fad158, having a transmembrane domain and leucine-rich repeat, stimulates adipocyte differentiation

Adipocyte differentiation is known to be regulated by a complex array of genes known as master regulators. Using a subtraction method, we previously isolated 102 genes that are expressed in the early stage of adipocyte differentiation. One of these genes named fad158 (factor for adipocyte differentiation 158) seems to be a novel gene, since there is no significantly similar gene listed in databases. Both mouse and human fad158 encode 803 amino acids and contain 4 transmembrane regions and 8 leucine-rich repeat motifs. Expression of fad158 was induced at an early stage in differentiating 3T3-L1 cells and was observed in the skeletal muscle. When the expression was knocked down with an antisense method in 3T3-L1 cells, the accumulation of oil droplets was reduced. Moreover, on overexpression of fad158 in NIH-3T3 cells, which are fibroblasts and do not usually differentiate into adipocytes, stable transformants accumulated oil droplets and showed an elevated expression of adipocyte marker genes, indicating that these cells had differentiated into mature adipocytes. fad158 has the ability to regulate adipocyte differentiation positively, especially at an early stage.     

Dedifferentiated fat cells: A cell source for regenerative medicine

The identification of an ideal cell source for tissue regeneration remains a challenge in the stem cell field. The ability of progeny cells to differentiate into other cell types is important for the processes of tissue reconstruction and tissue engineering and has clinical, biochemical or molecular implications. The adaptation of stem cells from adipose tissue for use in regenerative medicine has created a new role for adipocytes. Mature adipocytes can easily be isolated from adipose cell suspensions and allowed to dedifferentiate into lipid-free multipotent cells, referred to as dedifferentiated fat (DFAT) cells. Compared to other adult stem cells, the DFAT cells have unique advantages in their abundance, ease of isolation and homogeneity. Under proper condition in vitro and in vivo, the DFAT cells have exhibited adipogenic, osteogenic, chondrogenic, cardiomyogenc, angiogenic, myogenic, and neurogenic potentials. In this review, we first discuss the phenomena of dedifferentiation and transdifferentiation of cells, and then dedifferentiation of adipocytes in particular. Understanding the dedifferentiation process itself may contribute to our knowledge of normal growth processes, as well as mechanisms of disease. Second, we highlight new developments in DFAT cell culture and summarize the current understanding of DFAT cell properties. The unique features of DFAT cells are promising for clinical applications such as tissue regeneration.     

Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.     

Expression profiling during adipocyte differentiation of 3T3-L1 fibroblasts

The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. Changes in gene expression were measured by DNA microarrays at three time points (24 h, 4 days, and 1 week) during the course of differentiation from preadipocytes to mature adipocytes. Several functional categories of genes were affected by adipocyte conversion. In addition, seven genes were found to be commonly altered by 5-fold or more by adipocyte conversion at all three time points. Lipocalin 2, haptoglobin, serum amyloid A3, stearoyl-CoA desaturase, and 11beta-hydroxysteroid dehydrogenase 1 were induced while actin alpha2 and procollagen VIII alpha1 were suppressed by adipocyte differentiation. Further study of the regulation of these genes and pathways will lead to an increased understanding of the biochemical pathways involved in adipocyte differentiation and possibly to the identification of new therapeutic targets for treatment of obesity and other metabolic diseases.     

Adipocyte differentiation of 3T3-L1 cells in serum-free hormone-supplemented media: effects of insulin and dihydroteleocidin B

We previously established a serum-free hormone-supplemented medium for the induction of adipocyte differentiation of 3T3-L1 cells (Gamou, S. and N. Shimizu. in "Growth and Differentiation of Cells in Defined Environment", H. Murakami et al., ed., Kodansha/Springer-Verlag, pp. 173-178, 1985). Under those conditions the stage of the cell's commitment to adipocyte differentiation was separated from the stage of expression of the adipocyte phenotype. In the current study, the relationship between cell division of the growth-arrested 3T3-L1 cells and their entry into the differentiation program was examined by autoradiography at the individual cell level. It was found that cells treated with the inducers dexamethasone and 1-methyl-3-isobutylxanthine went through DNA synthesis (S phase) prior to lipid accumulation and that insulin enhanced this differentiation process. Under these serum-free hormone-supplemented conditions, the tumor promoter dihydroteleocidin B was found to be a strong inhibitor of adipocyte differentiation.     

Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO)

Abstract. The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well-defined steps: 1 predifferentiation growth arrest; 2 nonterminal (reversible) differentiation and 3 terminal differentiation associated with the irreversible loss of proliferative potential. To further investigate these processes, the effects of dimethyl sulphoxide (DMSO), an agent that affects differentiation in several other cell systems, was tested. The results show that DMSO modulates two distinct steps of adipocyte differentiation. The first effect is evident when growing 3T3 T cells are cultured in differentiation-inducing medium in the presence of DMSO. Therein the expression of adipocyte phenotype is inhibited because the cells fail to growtharrest at the predifferentiation growth arrest state. Instead in the presence of DMSO, cells growth-arrest at a biological state that does not support differentiation. The second effect is evident if nonterminally differentiated adipocytes are cultured in terminal differentiation-inducing medium containing DMSO. Therein the terminal step in differentiation is inhibited. These inhibitory effects occur in a dosage-dependent manner; maximum inhibition of differentiation requires 2% DMSO. Therefore, whereas DMSO typically promotes differentiation in other cell systems, DMSO inhibits multiple steps in the process of adipocyte differentiation. These observations support the conclusion that a single pharmacological agent can have markedly different effects on specific cell types. Even more important, the data establish that DMSO can now be used as a tool to study the molecular mechanisms involved in the multistep process of adipocyte differentiation.