Dedifferentiated Adipocytes Research Articles

Hypertonicity induces mitochondrial extracellular vesicles (MEVs) that activate TNF-α and β-catenin signaling to promote adipocyte dedifferentiation

BackgroundRecent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming.MethodsTo induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of β-catenin and applying BML-284, an agonist of β-catenin.ResultsHypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-β-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-β-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment.ConclusionsHypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/β-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/β-catenin signaling using BML-284.

Tumor and peritumoral adipose tissue crosstalk: De-differentiated adipocytes influence spread of colon carcinoma cells

Colorectal cancer is the second leading cause of cancer and often has a fatal course. There are many studies in the literature that have described a close functional relationship between the tumor mass and surrounding tissue, or tumor stroma, which is affected by the continuous metabolic exchange that occurs at the interface between tumor and tissues in contact with it. There is much evidence that the presence of adipose tissue in stroma plays a fundamental role in modulating the tumor microenvironment and promote tumor development, growth, and angiogenesis due to its endocrine characteristics. In this analysis, we have studied the alterations of adipose tissue surrounding colorectal tumors with MRI and optical imaging in vivo techniques to monitor tumor progression and also performed histological and molecular analysis. We detected differences in the principal adipose markers expressed by adipocytes residing around the rectal colon and observed that peritumoral adipose tissue is exposed to a mesenchymal transition process that leads to the acquisition of a less differentiated phenotype of adipocyte that represents the main cellular type present in tumor stroma. The mesenchymal transition correlated with the acquisition of more aggressive tumor phenotype and could represent a valid target for tumor therapy.

Fat transplantation induces dermal adipose regeneration and reverses skin fibrosis through dedifferentiation and redifferentiation of adipocytes

BackgroundLocalized scleroderma causes cosmetic disfigurement, joint contractures, and other functional impairment, but no currently available medications can reverse the resulting skin lesions. Fat grafting is beneficial for reversing skin fibrosis; however, the mechanism by which adipose tissue transplantation contributes to lesion improvement has not been fully clarified. The purpose of our study was to verify the therapeutic effect of fat grafts in reversing skin fibrosis.MethodsInguinal fat pads from AdipoqCreER+;mT/mG mice, which were treated with tamoxifen, were transplanted to the skin lesion in bleomycin-treated wild-type C57 mice. Tdtomato transgenic mice-derived adipocytes, adipose-derived stem cells (ASCs), dedifferentiated adipocytes (DAs) were embedded in matrigel and transplanted beneath the skin lesion of bleomycin-treated wild-type C57 mice. A transwell co‐culture system was used to verify the effect of ASCs, adipocytes or DAs on scleroderma fibroblasts or monocytes.ResultsAdipocytes from the fat grafts could undergo dedifferentiation and redifferentiation for dermal adipose tissue re-accumulation within the skin lesion. Moreover, compared with ASCs and adipocytes, DAs show greater potency of inducing adipogenesis. ASCs and DAs showed comparable effect on inducing angiogenesis and suppressing macrophage infiltration in fibrotic skin. Co-culture assay showed that DAs and ASCs were able to reduce fibrosis-related genes in human scleroderma fibroblasts and drive M2 macrophage polarization.ConclusionOur results indicated that adipocytes would transform into a more functional and dedifferentiated state and reverse dermal fibrosis, by promoting dermal adipose tissue regeneration, improving angiogenesis, suppressing macrophage-mediated inflammation and myofibroblast accumulation.

Adipocyte mesenchymal transition contributes to mammary tumor progression.

SUMMARYObesity is associated with increased cancer incidence and progression. However, the relationship between adiposity and cancer remains poorly understood at the mechanistic level. Here, we report that adipocytes from tumor-invasive mammary fat undergo de-differentiation to fibroblast-like precursor cells during tumor progression and integrate into the tumor microenvironment. Single-cell sequencing reveals that these de-differentiated adipocytes lose their original identities and transform into multiple cell types, including myofibroblast- and macrophage-like cells, with their characteristic features involved in immune response, inflammation, and extracellular matrix remodeling. The de-differentiated cells are metabolically distinct from tumor-associated fibroblasts but exhibit comparable effects on tumor cell proliferation. Inducing de-differentiation by Xbp1s overexpression promotes tumor progression despite lower adiposity. In contrast, promoting lipid-storage capacity in adipocytes through MitoNEET overexpression curbs tumor growth despite greater adiposity. Collectively, the metabolic interplay between tumor cells and adipocytes induces adipocyte mesenchymal transition and contributes to reconfigure the stroma into a more tumor-friendly microenvironment.

Pro-tumoral behavior of omental adipocyte-derived fibroblasts in tumor microenvironment at the metastatic site of ovarian cancer.

Adipocyte-rich omentum offers "good soil" for disseminating ovarian cancer (OvCa), contributing to therapeutic difficulty. However, little is understood about the association between adipocytes and tumor growth at peritoneal dissemination site. Herein, we report the induction of adipocyte dedifferentiation by OvCa cells and pro-tumorigenic effects of resulted adipocyte-derived fibroblasts. We confirmed that malignant ascites promoted the dedifferentiation of the primary human adipocytes obtained from surgical omental specimen into omental adipocyte-derived fibroblast (O-ADF) that possess both mesenchymal stem cell and myofibroblast-like features. This promotion of dedifferentiation by malignant ascites was blocked by addition of Wnt signaling inhibitor. The effects of dedifferentiated adipocytes in proliferation and migration of OvCa cells were analyzed with in vitro coculturing experimental models and in vivo mice model, and we demonstrated that OvCa cell lines showed enhanced proliferative characteristics, as well as increased migratory abilities upon coculturing with O-ADF. Additionally, exogenous transforming growth factor-β1 augmented desmoplastic morphological change of O-ADF, leading to higher proliferative ability. Our results suggest that OvCa cells promote dedifferentiation of peritoneal adipocytes by activating Wnt/β-catenin signaling, and generated O-ADFs exhibit pro-tumoral hallmarks.

Epstein-Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment.

The most frequent location of metastatic EBV+ nasopharyngeal carcinoma (NPC) is the bone marrow, an adipocyte-dominant region. Several EBV-associated lymphoepithelioma-like carcinoma (LELC) types also grow in the anatomical vicinity of fat tissues. Here we show that in an adipose tissue-rich tumor setting, EBV targets adipocytes and remodels the tumor microenvironment. Positive immunoreactivity for EBV-encoded early antigen D was detected in adipose tissue near tumor beds of bone marrow metastatic NPC. EBV was capable of infecting primary human adipocytes in vitro, triggering expression of multiple EBV-encoded mRNA and proteins. In infected adipocytes, lipolysis was stimulated through enhanced expression of lipases and the AMPK metabolic pathway. The EBV-mediated imbalance in energy homeostasis was further confirmed by increased release of free fatty acids, glycerol, and expression of proinflammatory adipokines. Clinically, enhanced serum levels of free fatty acids in patients with NPC correlated with poorer recurrence-free survival. EBV-induced delipidation stimulated dedifferentiation of adipocytes into fibroblast-like cells expressing higher levels of S100A4, a marker protein of cancer-associated fibroblasts (CAF). IHC analyses of bone marrow metastatic NPC and salivary LELC revealed similar structural changes of dedifferentiated adipocytes located at the boundaries of EBV+ tumors. S100A4 expression in adipose tissues near tumor beds correlated with fibrotic response, implying that CAFs in the tumor microenvironment are partially derived from EBV-induced dedifferentiated adipocytes. Our data suggest that adipose tissue serves as an EBV reservoir, where EBV orchestrates the interactions between adipose tissues and tumor cells by rearranging metabolic pathways to benefit virus persistence and to promote a protumorigenic microenvironment. SIGNIFICANCE: This study suggests that Epstein-Barr virus hijacks adipocyte lipid metabolism to create a tumor-promoting microenvironment from which reactivation and relapse of infection could potentially occur.

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.

2491-PUB: Proinsulin C-Peptide Attenuates Vascular Dysfunctions by Activating Nrf2

We and others demonstrated that insulin C-peptide has biological activities and may prevent tissue damages by diabetes. Our research using STZ diabetic rats and other models suggested that diabetic vascular dysfunctions, e.g., increased vascular permeability and blood flows are caused by oxidative and nitrosative stress initiated from increased NADH redox (reductive stress). However, C-peptide prevented dysfunctions without normalizing redox state and worked in nondiabetic rats. In this study, we further examined modes of actions and investigated its mechanisms. Male Wistar rats were used in animal study. Human de-differentiated adipocytes (DFAT cells) were used for cell culture study. Lentivirus vector expressing LC3-GFP was created to assess autophagy. Regional blood flow was assessed using microsphere. Acute hyperglycemia induced by glucose infusion for 5 hours resulted in increased regional blood flows in retina, sciatic nerve and kidney. Simultaneous infusion of C-peptide up to 16 mg/kg did not prevent the increases, while twice daily subcutaneous injection of 400 µg/kg for 2-days prior to infusion prevented completely. One-day injection was only slightly effective. Bolus i.v. injection of L-lactate 1 mol/kg but not NaCl caused reductive stress and blood flow increase, and this was also prevented by twice daily 2-days injection of C-peptide. Thus, C-peptide appeared to induce some factors counteracting oxidative stress during 2 days. In DFAT cells, LC3-GFP puncta was increased 3-fold by one time 100 nM C-peptide treatment in 1 hour. This was followed by nuclear localization of Nrf2 transcription factor at 2 hours. At 4 hour, 4-fold increase in Nrf2 target genes, IDH and SRXN1, expression were observed (p<0.05). Similar 35-62% increase in Nrf2 target genes (Idh, Srxn1, Pgd and Hmox) were observed in rat aorta after twice daily 2-days injection of C-peptide. These results suggest that injections of pharmacological dose of C-peptide increase Nrf2 antioxidants expression through activating autophagy. Disclosure F. Lan: None. Y. Lin: None. Z. Gao: None. P. Zhao: None. X. Zhang: None. Y. Shiraishi: None. Y. Ido: None. Funding Japan Society for the Promotion of Science

Interaction between human mature adipocytes and lymphocytes induces T-cell proliferation

Background aimsAdipose tissue is a critical organ that plays a major role in energy balance regulation and the immune response through intricate signals. MethodsWe report on the inter-relation between mature adipocytes and lymphocytes in terms of adipocyte-derived T-cell chemo-attractants and adipocyte metabolic effects on lymphocytes. ResultsDuring the culture time, mature adipocytes changed their structural and functional properties into de-differentiated cells. Isolated mature adipocytes expressed significantly higher levels of CIITA, major histocompatibility complex II (human leukocyte antigen [HLA]-DR) and costimulatory signal molecule CD80 compared with adipocytes after the de-differentiation process. Moreover, human leukocyte antigen–G, which may prevent the immune responses of mesenchymal stromal cells, was expressed at lower level in mature adipocytes compared with de-differentiated adipocytes. In line with these molecular data, functional results showed different immunoregulatory properties between adipocytes before and after the de-differentiation process. Mature adipocytes stimulated the proliferation of total lymphocytes and immunoselected cell populations CD3+, CD4+ and CD8+ in a direct contact-dependent way that involved the major histocompatibility complex I and II pathways. Moreover, adipocytes secreted potential chemo-attractant factors, but data showed that adipocyte-derived culture medium was not sufficient to activate lymphocyte proliferation, suggesting that a direct contact between adipocytes and immune cells was needed. However, specific mature adipocyte cytokines enhanced lymphocyte proliferation in a mixed lymphocyte reaction. ConclusionsIn conclusion, cross-talk occurs between adipocytes and lymphocytes within adipose tissue involving T-cell chemo-attraction by mature adipocytes. Our findings, together with current observations in the field, provide a rationale to identify adipocyte-lymphocyte cross-talk that instigates adipose inflammation.

Biosafety evidence for human dedifferentiated adipocytes.

Mature adipocytes have shown dynamic plasticity to be converted into fibroblast-like and lipid-free cells. After the dedifferentiation process, these cells re-entered the cell cycle and acquired a high proliferation potential, becoming a valid source of stem cells. However, many aspects of the cellular biosafety about dedifferentiated fat cells remained unclear. This study aimed to elucidate their potential susceptibility to malignant transformation and to ascertain the safety of these cells for clinical use. To evaluate the genomic stability of dedifferentiated adipocytes, telomere length, hTERT gene transcription, the capacity of these cells to grow in an anchorage-independent manner and the presence of DNA damage by single cell gel electrophoresis assay were studied. Spontaneous chromosomal alterations were excluded by cytogenetic analysis and the expression level of c-myc and p53, tumor associated genes, were assessed, evaluating also p53 loss of function mutations. Despite the high proliferation capacity of dedifferentiated adipocytes, these cells showed stable telomere length compared with mature adipocytes, no hTERT transcriptions and consequently no telomerase activity, suggesting that both transformation and senescence were avoided. A constant expression level of c-myc and p53, the inability of dedifferentiated adipocytes to grow in an anchorage-independent manner, the absence of DNA damage suggested the safety of these cells. Moreover, a normal karyotype was preserved throughout the dedifferentiation process. Data in vivo showed that dedifferentiated adipocytes analyzed for tumorigenicity did not develop tumors. In conclusion, our data indicated that dedifferentiated adipocytes could be a relatively easily accessible resource for cell therapy and regenerative medicine.

Antiphotoaging effect of conditioned medium of dedifferentiated adipocytes on skin in vivo and in vitro: a mechanistic study.

Photoaging of skin occurs partially due to decreased synthesis and increased degradation of dermal collagen. Antiphotoaging therapy aims to counteract these effects. This study aimed to investigate whether secretory factors from dedifferentiated adipocytes (DAs) could alleviate photoaging in human dermal fibroblasts (HDFs) and in mice and to clarify the underlying mechanism. DAs were acquired and verified based on cellular biomarkers and multilineage differentiation potential. The concentrations of several cytokines in conditioned medium from DAs (DA-CM) were determined. In vivo pathological changes, collagen types I and III, and matrix metalloproteinase (MMP)-1 and -3 were evaluated following the injection of 10-fold concentrated DA-CM into photoaged mice. In vitro, the effect of DA-CM on stress-induced premature senescence in HDFs was investigated by 5-ethynyl-2'-deoxyuridine (EdU) staining and β-galactosidase staining. The influence of DA-CM and transforming growth factor-β1 (TGF-β1) on the secretion of collagen types I and III, MMP-1, and MMP-3 in HDFs was evaluated by ELISA. In vivo, we found that subcutaneously injected 10-fold concentrated DA-CM increased the expression of collagen types I and III. In vitro, DA-CM clearly mitigated the decreased cell proliferation and delayed the senescence status in HDFs induced by ultraviolet B (UVB). HDFs treated with DA-CM exhibited higher collagen types I and III secretion and significantly lower MMP-1 and MMP-3 secretion. The TGF-β1-neutralizing antibody could partially reduce the recovery effect. Our results suggest that DAs may be useful for aging skin and their effects are mainly due to secreted factors, especially TGF-β1, which stimulate collagen synthesis and alleviate collagen degradation in HDFs.

Plasticity of human dedifferentiated adipocytes toward endothelial cells

The process of cellular differentiation in terminally differentiated cells is thought to be irreversible, and these cells are thought to be incapable of differentiating into distinct cell lineages. Our previous study showed that mature adipocytes represent an alternative source of mesenchymal stem cells. Here, results showed the capacity of mature adipocytes to differentiate into endothelial-like cells, using the ability of these cells to revert into an immature phase without any relievable chromosomal alterations. Mature adipocytes were isolated from human omental and subcutaneous fat and were dedifferentiated in vitro. The resulting cells were subcultivated for endothelial differentiation and were analyzed for their expression of specific genes and proteins. Endothelial-like cells were harvested from the differentiation medium and were traditionally cultured to evaluate the endothelial markers and the karyotype. Cells cultured in specific medium formed tube-like structures and expressed several endothelial marker genes and proteins. The endothelial-like cells expressed significantly higher levels of vascular endothelium growth factor receptor 2, vascular endothelial cadherin, Von Willebrand factor, and CD133 than the untreated cells. These cells were positively stained for CD31 and vascular endothelial cadherin, markers of mature endothelial cells. Moreover, the low-density lipoprotein-uptake assay demonstrated a functionally endothelial differentiation of these cells. When these cells were harvested and reseeded in basal medium, they lost the endothelial markers and reacquired the typical mesenchymal stem cell markers and the ability to expand in a short time period. Moreover, karyotype analysis showed that these cells reverted into an immature phase without any karyotype alterations. In conclusion, the results showed that adipocytes exhibited a great plasticity toward the endothelial lineage, suggesting their possible use in cell therapy applications for vascular disease.

Impaired Adipogenesis and Insulin Resistance in Epicardial Fat-Mesenchymal Cells From Patients With Cardiovascular Disease

The thickness of epicardial adipose tissue (EAT), which is an inflammatory source for coronary artery disease (CAD), correlates with insulin resistance. One trigger factor is impaired adipogenesis. Here, our aim was to clarify the underlying mechanisms of insulin resistance on EAT-mesenchymal cells (MC). EAT and subcutaneous adipose tissue (SAT) were collected from 19 patients who were undergoing heart surgery. Their dedifferentiated adipocytes (DAs) and/or MCs were cultured. After the induction of adipogenesis or stimulation with insulin, the expression of adipokines was analyzed using real-time polymerase chain reaction (PCR). Colorimetric assays were performed to measure glucose levels and proliferation rate. Proteins modifications were detected via the proteomic approach and Western blot. Our results showed lower adipogenic ability in EAT-MCs than in SAT-MCs. Maximum adiponectin levels were reached within 28-35 days of exposure to adipogenic inducers. Moreover, the adipogenesis profile in EAT-MCs was dependent on the patients' clinical characteristics. The low adipogenic ability of EAT-MCs might be associated with an insulin-resistant state because chronic insulin treatment reduced the inflammatory cytokine expression levels, improved the glucose consumption, and increased the post-translational modifications (PTMs) of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1). We found lower adipogenic ability in EAT-MCs than in SAT-MCs. This lower ability level was dependent on gender and the presence of diabetes, obesity, and CAD. Low adipogenesis ability and insulin resistance in EAT-MCs might shed light on the association between EAT dysfunction and cardiovascular disease.

Glial-like differentiation potential of human mature adipocytes.

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here, we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes, suggesting that these cells could be committed to the neural lineage. After neural induction, NeuroD1, Sox1, Double Cortin, and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties, indicating the inability of these cells to differentiate into mature neurons. In contrast, the differentiated cells expressed a high level of CLDN11, as demonstrated using molecular method, and stained positively for the glial cell markers CLDN11 and GFAP, as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production, which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion, our data demonstrate morphological, molecular, and immunocytochemical evidence of initial neural differentiation of mature adipocytes, committing to a glial lineage.

Reasearch of transdifferentiation between osteoblasts and adipocytes in rabbits

Objective To investigate the possibility of transdifferentiation between osteoblasts and adipocytes in rabbits.Methods Adipocytes were isolated from the subcutaneous adipose tissue in the abdomen of 3-month-old New Zealand white rabbits.Then mature adipocytes were cultured and induced to dedifferentiated adipocytes (DA) by ceiling adherent culture method.DA were culutured in osteogenic medium for 3 weeks,and then calcium nodules of the cells were stained by the alizarin red and by immunocytochemical staining for collagen I and alkaline phosphatase (ALP).Osteoblasts were isolated from the skull of one-week-old rabbits by mechanical digestion and enzyme digestion and cultured and amplified in vitro.Then osteoblasta were culutured in adipogenic medium for 3 weeks.Oil Red O staining was used to visualize lipid-rich vacuoles.The expression of peroxisome proliferator activated receptor γ (PPARγ) mRNA was detected by using reverse transcription-polymerase chain reaction (RT-PCR).Results The extracted mature adipocytes turned into fibroblast-like cells (spindle-shaped) after dedifferentiation.After osteogenic induction,the experiment group expressed type Ⅰ collagen,and there was significant difference as compared with control group (P ＜0.05).The ALP activity in experimental group at 3rd,7th and 14th day (0.165± 0.007,0.253 ± 0.005,0.345 ± 0.007 respectively) was higher than that in control group (0.067 ±0.004,0.076 ± 0.006,0.082 ± 0.003 respectively) ( P ＜ 0.05 ).The osteoblasts obtained from the skull were shot-spindle-shaped and positive for Oil Red O staining after culture with adipogenic medium for 3 weeks.The expression of PPARγmRNA in experimental group was positive,that in control group was negative (P ＜0.05).Conclusion Mature adipocytes can turn into DA by in vitroculture.Adipocytes and osteoblasts can differentiate into each other under some particular conditions. Key words: Adipocyte; Dedifferentiation; Osteoblast

Human Dedifferentiated Adipocytes Show Similar Properties to Bone Marrow‐Derived Mesenchymal Stem Cells

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. Here, we studied the gene expression and functional properties of mature adipocytes isolated from human omental and subcutaneous fat tissues. We also focused on dedifferentiated adipocytes in culture and their morphologies and functional changes with respect to mature adipocytes, stromal-vascular fraction (SVF)-derived mesenchymal stem cells (MSCs) and bone marrow (BM)-derived MSCs. Isolated mature adipocytes expressed stem cell and reprogramming genes. They replicated in culture after assuming a fibroblast-like shape and expanded similarly to SVF- and BM-derived MSCs. During the dedifferentiation process, mature adipocytes lost their lineage gene expression profile, assumed the typical mesenchymal morphology and immunophenotype, expressed stem cell genes and differentiated into multilineage cells. Moreover, during the dedifferentiation process, we showed changes in the epigenetic status of mature adipocytes, which led dedifferentiated adipocytes to display a similar DNA methylation condition to BM-derived MSCs. Like SVF- and BM-derived MSCs, dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in coculture while mature adipocytes stimulated their growth. Furthermore, dedifferentiated adipocytes maintained the survival and complete differentiation characteristic of hematopoietic stem cells. This is the first study that in addition to characterizing isolated and dedifferentiated adipocytes also reports on the immunoregulatory and hematopoietic supporting functions of these cells. This structural and functional characterization might have clinical applications of both mature and dedifferentiated adipocytes in such fields, as regenerative medicine.

The Cellular Plasticity of Human Adipocytes

Little is known regarding the biology of fat considering its extensive use clinically in soft tissue implantation. Free-fat transfer is problematic the result of graft site volume loss, appearing histologically as the replacement of mature adipocytes with a fibroblast-like infiltrate. We hypothesize that these histologic changes reflect a dedifferentiation of ischemic mature adipocytes instead of, or in addition to, a more traditional response. To explore this hypothesis, we studied the in vitro morphologic changes of cultured mature human adipocytes isolated from liposuctioned adipose tissue. Most adipocytes over time lost significant amounts of intracellular lipid. Ultimately, these cells lost all lipid, appeared fibroblastic, and proliferated to confluence. Adipogenic induction of such dedifferentiated adipocytes resulted in reaccumulation of intracellular lipid. This study demonstrates that mature adipocytes can be cultured from human liposuctioned fat, they can dedifferentiate into fibroblastic cells, and the fibroblast-like cells can be expanded and turned into lipid-synthesizing adipocytes. Exploration of this cellular plasticity might ultimately yield important insights into free-fat transfer and novel tissue-engineering strategies.

TNF -Mediated Inhibition and Reversal of Adipocyte Differentiation Is Accompanied by Suppressed Expression of PPAR without Effects on Pref-1 Expression

Tumor necrosis factor α (TNFα) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNFα inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor γ (PPARγ) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARγ and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARγ occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNFα-mediated inhibition of adipocyte differentiation, PPARγ messenger RNA (mRNA) expression stays at low levels. In contrast, TNFα treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors[ such as TNFα, basic fibroblast growth factor, transforming growth factor β, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARγ mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNFα to fully differentiated adipocytes results in the rapid disappearance of PPARγ protein expression and the rapid loss of PPARγ DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNFα-generated signals, whereas PPARγ expression remains sensitive to TNFα at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.

TNF alpha-mediated inhibition and reversal of adipocyte differentiation is accompanied by suppressed expression of PPARgamma without effects on Pref-1 expression.

Tumor necrosis factor alpha (TNF alpha) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNF alpha inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARgamma and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARgamma occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNF alpha-mediated inhibition of adipocyte differentiation, PPARgamma messenger RNA (mRNA) expression stays at low levels. In contrast, TNF alpha treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors [such as TNF alpha, basic fibroblast growth factor, transforming growth factor beta, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARgamma mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNF alpha to fully differentiated adipocytes results in the rapid disappearance of PPARgamma protein expression and the rapid loss of PPARgamma DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNF alpha-generated signals, whereas PPARgamma expression remains sensitive to TNF alpha at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.