Adipogenic differentiation of human adipose-derived stem cells grown as spheroids

Understanding the process of adipogenesis is critical if suitable therapeutics for obesity and related metabolic diseases are to be found. The current study presents proof of feasibility of creating a 3-D spheroid model using human adipose-derived stem cells (hASCs) and their subsequent adipogenic differentiation. hASC spheroids were formed atop an elastin-like polypeptide-polyethyleneimine (ELP-PEI) surface and differentiated using an adipogenic cocktail. Spheroids were matured in the presence of dietary fatty acids (linoleic or oleic acid) and evaluated based on functional markers including intracellular protein, CD36 expression, triglyceride accumulation, and PPAR-γ gene expression. Spheroid size was found to increase as the hASCs matured in the adipocyte maintenance medium, though the fatty acid treatment generally resulted in smaller spheroids compared to control. A stable protein content over the 10-day maturation period indicated contact-inhibited proliferation as well as minimal loss of spheroids during culture. Spheroids treated with fatty acids showed greater amounts of intracellular triglyceride content and greater expression of the key adipogenic gene, PPAR-γ. We also demonstrated that 3-D spheroids outperformed 2-D monolayer cultures in adipogenesis. We then compared the adipogenesis of hASC spheroids to that in 3T3-L1 spheroids and found that the triglyceride accumulation was less profound in hASC spheroids than that in 3T3-L1 adipocytes, correlated with smaller average spheroids, suggesting a relatively slower differentiation process. Taken together, we have shown the feasibility of adipogenic differentiation of patient-derived hASC spheroids, which with further development, may help elucidate key features in the adipogenesis process.     

Analysis of matrix metalloproteinase activity during differentiation of mesenchymal stem cells isolated from different tissues of one donor

Mesenchymal stem cells established from bone marrow (FetMSC) and limb bud (M-FetMSC) of early human embryo, as well as spheroids derived these cells, were induced to undergo osteogenic and adipogenic differentiation. Differentiated cells exhibited the activity of metalloproteinase (MMP)-9, -2, and -1. Its activity was different in osteogenic and adipogenic cells, as well as in monolayer cultures (2D) and cell spheroids (3D). The direct correlation between the level of adipogenic differentiation and gelatinases MMP-9 and MMP-2 activities in both cell lines in 2D and 3D culture was shown. M-FetMSC cells in 2D culture 12 days in culture during showed low potential for adipogenesis and reduced activity of MMP-2 and MMP-9. The low level of adipogenic differentiation in 2D M-FetMSC culture was accompanied with increased MMP-1 activity and enhanced differentiation (3D culture) resulted in a significant increase of both MMP activities. MMP-1 activity varied oppositely. MMP-1 activity declined in 3D cultures with a higher level of adipogenic differentiation. The level of osteogenic differentiation was similar in both cell lines during 2D and 3D cultivation. MMP-1 and -9 activities in both cell lines were not associated with osteogenic differentiation. MMP-2 and MMP-2 activity in these cells remained unchanged. The results suggest MMP implication in FetMSC and М-FetMSC differentiation. The difference in MMP activities during the cell differentiation may be caused by variations in the microenvironment or ECM properties in 2D and 3D cultures.     

Zfp423 Promotes Adipogenic Differentiation of Bovine Stromal Vascular Cells

Intramuscular fat or marbling is critical for the palatability of beef. In mice, very recent studies show that adipocytes and fibroblasts share a common pool of progenitor cells, with Zinc finger protein 423 (Zfp423) as a key initiator of adipogenic differentiation. To evaluate the role of Zfp423 in intramuscular adipogenesis and marbling in beef cattle, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with pCI neo-hEST2 and individual clones were selected by G418. A total of 288 clones (3×96 well plates) were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. In addition, fibro/adipogenic progenitor cells were selected using a surface marker, platelet derived growth factor receptor (PDGFR) α. The expression of Zfp423 was much higher (307.4±61.9%, P<0.05) in high adipogenic cells, while transforming growth factor (TGF)-β was higher (156.1±48.7%, P<0.05) in low adipogenic cells. Following adipogenic differentiation, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were much higher (239.4±84.1% and 310.7±138.4%, respectively, P<0.05) in high adipogenic cells. Over-expression of Zfp423 in stromal vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation, accompanied with the inhibition of TGF-β expression. Zfp423 knockdown by shRNA in high adipogenic cells largely prevented their adipogenic differentiation. The differential regulation of Zfp423 and TGF-β between low and high adipogenic cells is associated with the DNA methylation in their promoters. In conclusion, data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular adipogenesis and marbling in beef cattle.     

Culture on Tissue‐Specific Coatings Derived from α‐Amylase‐Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

While extracellular matrix (ECM)‐derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose‐derived stem/stromal cells (ASCs) on a range of ECM‐derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α‐amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α‐amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue‐like ultrastructure that is lost in the pepsin‐digested thin films. ASCs cultured on the α‐amylase‐digested ECM have a more spindle‐shaped morphology, and proliferation is significantly enhanced on the α‐amylase‐digested DAT coatings. Further, the α‐amylase‐digested DAT provides a more pro‐adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol‐3‐phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α‐amylase digestion as a new approach for generating bioactive ECM‐derived coatings, and demonstrates tissue‐specific bioactivity using adipose‐derived ECM to enhance ASC proliferation and adipogenic differentiation.     

Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro

The development of vessel-like structures in vitro to mimic as well as to realize the possibility of tissue-engineered small vascular networks presents a major challenge to cell biologists and biotechnologists. We aimed to establish a three-dimensional (3-D) culture system with an endothelial network that does not require artificial substrates or ECM compounds. By using human skin fibroblasts and endothelial cells (ECs) from the human umbilical vein (HUVECs) in diverse spheroid coculture strategies, we verified that fibroblast support and modulate EC migration, viability, and network formation in a 3-D tissue-like stromal environment. In mixed spheroid cultures consisting of human ECs and fibroblasts, a complex 3-D network with EC tubular structures, lumen formation, pinocytotic activity, and tight junction complexes has been identified on the basis of immunohistochemical and transmission electron microscopic imaging. Tubular networks with extensions up to 400 µm were achieved. When EC suspensions were used, EC migration and network formation were critically affected by the status of the fibroblast. However, the absence of EC migration into the center of 14-day, but not 3-day, precultured fibroblast spheroids could not be attributed to loss of F viability. In parallel, it was also confirmed that migrated ECs that entered cluster-like formations became apoptotic, whereas the majority of those forming vessel-like structures remained viable for >8 days. Our protocols allow us to study the nature of tubule formation in a manner more closely related to the in vivo situation as well as to understand the basis for the integration of capillary networks in tissue grafts and develop methods of quantifying the amount of angiogenesis in spheroids using fibroblast and other cells isolated from the same patient, along with ECs. endothelium; angiogenesis; human umbilical vein endothelial cell; multicellular spheroid; coculture; tubular structures     

Proteomic and electron microscopy study of myogenic differentiation of alveolar mucosa multipotent mesenchymal stromal cells in three-dimensional culture

Myocyte differentiation is featured by adaptation processes, including mitochondria repopulation and cytoskeleton re-organization. The difference between monolayer and spheroid cultured cells at the proteomic level is uncertain. We cultivated alveolar mucosa multipotent mesenchymal stromal cells in spheroids in a myogenic way for the proper conditioning of ECM architecture and cell morphology, which induced spontaneous myogenic differentiation of cells within spheroids. Electron microscopy analysis was used for the morphometry of mitochondria biogenesis, and proteomic was used complementary to unveil events underlying differences between two-dimensional/three-dimensional myoblasts differentiation. The prevalence of elongated mitochondria with an average area of 0.097 μm² was attributed to monolayer cells 7 days after the passage. The population of small mitochondria with a round shape and area of 0.049 μm² (p < 0.05) was observed in spheroid cells cultured under three-dimensional conditions. Cells in spheroids were quantitatively enriched in proteins of mitochondria biogenesis (DNM1L, IDH2, SSBP1), respiratory chain (ACO2, ATP5I, COX5A), extracellular proteins (COL12A1, COL6A1, COL6A2), and cytoskeleton (MYL6, MYL12B, MYH10). Most of the Rab-related transducers were inhibited in spheroid culture. The proteomic assay demonstrated delicate mechanisms of mitochondria autophagy and repopulation, cytoskeleton assembling, and biogenesis. Differences in the ultrastructure of mitochondria indicate active biogenesis under three-dimensional conditions.     

Type I collagen inhibits adipogenic differentiation via YAP activation in vitro

Extracellular matrix (ECM) has a marked influence on adipose tissue development. Adipose tissue formation is initiated with proliferation of preadipocytes and migration before undergoing further differentiation into mature adipocytes. Previous studies showed that collagen I (col I) provides a good substratum for 3T3-L1 preadipocytes to grow and migrate. However, it remains unclear whether and how col I regulates adipogenic differentiation of preadipocytes. This study reports that lipid accumulation, representing in vitro adipogenesis of the 3T3-L1 preadipocytes or the mouse primary adipocyte precursor cells derived from subcutaneous adipose tissue in the inguinal region is inhibited by the culture on col I, owing to downregulation of adipogenic factors. Previous study shows that col I enhances 3T3-L1 cell migration via stimulating the nuclear translocation of yes-associated protein (YAP). In this study, we report that downregulation of YAP is associated with in vitro adipogenesis of preadipocytes as well as with in vivo adipose tissue of high-fat diet fed mice. Increased expression of YAP in the cells cultured on col I-coated dishes is correlated with repression of adipogenic differentiation processes. The inactivation of YAP using YAP inhibitor, verteporfin, or YAP small-interfering RNA enhanced adipogenic differentiation and reversed the inhibitory effect of col I. Activation of YAP either by the transfection of YAP plasmid or the silence of large tumor suppressor 1 (LATS1), an inhibitory kinase of YAP, inhibited adipogenic differentiation. The results indicate that col I inhibits adipogenic differentiation via YAP activation in vitro.     

Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation

We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31⁺, KDR⁺, and CD34⁺. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.     

Adipogenic potential of perivascular adipose tissue preadipocytes is improved by coculture with primary adipocytes

Perivascular adipose tissue (PVAT) has the capacity to secrete vasoactive mediators with the potential to regulate vascular function. Given its location adjacent to the vasculature, PVAT dysfunction may be part of the pathophysiology of cardiovascular diseases. To study the mechanisms of PVAT dysfunction, several adipogenic models have been proposed. However, these approaches do not adequately reflect PVAT adipocyte phenotypes variability that depends on their anatomical location. Despite PVAT importance in modulating vascular function, to date, there is not a depot-specific adipogenic model for PVAT adipocytes. We present a model that uses coculturing of PVAT stromal vascular fraction derived preadipocytes with primary adipocytes isolated from the same PVAT. Preadipocytes were isolated from thoracic aorta PVAT and mesenteric resistance artery PVAT (mPVAT). Upon confluency, cells were induced to differentiate for 7 and 14 days using a standard protocol (SP) or standard protocol cocultured with primary adipocytes isolated from the same adipose depots (SPA) for 96, 120, and 144 h. SPA reduced the time for differentiation of stromal vascular fraction derived preadipocytes and increased their capacity to store lipids compared with SP as indicated by lipid accumulation, lipolytic responses, gene marker profile expression, and adiponectin secretion. The coculture system improved adipogenesis efficiency by enhancing lipid accumulation and reducing the time of induction, therefore, is a more efficient method compared to SP alone.     

Defective Differentiation of Adipose Precursor Cells from Lipodystrophic Mice Lacking Perilipin 1

Perilipin 1 (Plin1) localizes at the surface of lipid droplets to regulate triglyceride storage and hydrolysis in adipocytes. Plin1 defect leads to low adiposity in mice and partial lipodystrophy in human. This study investigated the roles of Plin1 in adipocyte differentiation. Plin1 null (-/-) mice showed plenty of multilocular adipocytes and small unilocular adipocytes in adipose tissue, along with lack of a subpopulation of adipose progenitor cells capable of in vivo adipogenesis and along with downregulation of adipogenic pathway. Before initiation of differentiation, adipose stromal-vascular cells (SVCs) from Plin1-/- mice already accumulated numerous tiny lipid droplets, which increased in number and size during the first 12-h induction but thereafter became disappeared at day 1 of differentiation. The adipogenic signaling was dysregulated despite protein level of PPARγ was near normal in Plin1-/- SVCs like in Plin1-/- adipose tissue. Heterozygous Plin1+/- SVCs were able to develop lipid droplets, with both the number and size more than in Plin1-/- SVCs but less than in Plin1+/+ SVCs, indicating that Plin1 haploinsufficiency accounts for attenuated adipogenesis. Aberrant lipid droplet growth and differentiation of Plin1-/- SVCs were rescued by adenoviral Plin1 expression and were ameliorated by enhanced or prolonged adipogenic stimulation. Our finding suggests that Plin1 plays an important role in adipocyte differentiation and provides an insight into the pathology of partial lipodystrophy in patients with Plin1 mutation.     

Human bone marrow-derived stromal cells show highly efficient stress-resistant adipogenesis on denatured collagen IV matrix but not on its native counterpart: Implications for obesity

Collagen IV is the major matrix component associated with differentiating adipocytes in adipose tissues, and the understanding of its contribution in adipogenic differentiation could be important for elucidation of mechanisms and processes driving the obesity. Therefore, in the light of our previous findings of differential effects of structural conformation of collagen I matrix on differentiation of bone marrow stromal cells, we investigated whether similar phenomenon occurs on collagen IV matrix in native and denatured structural states. The results of the present study show that native collagen IV is unsupportive of adipogenic differentiation and very little if any adipogenesis occurs on this matrix in the presence of adipogenic stimuli. In sharp contrast to native collagen IV, the same matrix in denatured structural state drives highly efficient adipogenic differentiation suggesting that it might be the major driver of adipogenesis in adipose tissues and that the ratio of native to denatured matrix might regulate the intensity of adipogenesis and possibly underlies the obesity. In contrast to observations that adipogenesis on denatured collagen I (collagen I is the major matrix component in musculoskeletal tissues) is suppressed by stress, adipogenesis on denatured collagen IV appears to be stress-resistant suggesting an explanation for the observed ineffectiveness of physical exercise, i.e. mechanical stress, in the reduction of adipose tissues. The obesity was shown to be associated with overproduction of MMPs and decline in levels of TIMPs. Such a shift in MMP/TIMP balance was considered a consequence of the pathology. In the light of the present study, however, this shift might constitute the primary source of the decease. The findings of the present study suggest strategies for the treatment of obesity, raise significant questions and indicate directions for further experimentation.     

Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks

Scaffold-free techniques in the developmental tissue engineering area are designed to mimic in vivo embryonic processes with the aim of biofabricating, in vitro, tissues with more authentic properties. Cell clusters called spheroids are the basis for scaffold-free tissue engineering. In this review, we explore the use of spheroids from adult mesenchymal stem/stromal cells as a model in the developmental engineering area in order to mimic the developmental stages of cartilage and bone tissues. Spheroids from adult mesenchymal stromal/stem cells lineages recapitulate crucial events in bone and cartilage formation during embryogenesis, and are capable of spontaneously fusing to other spheroids, making them ideal building blocks for bone and cartilage tissue engineering. Here, we discuss data from ours and other labs on the use of adipose stromal/stem cell spheroids in chondrogenesis and osteogenesis in vitro. Overall, recent studies support the notion that spheroids are ideal "building blocks" for tissue engineering by “bottom-up” approaches, which are based on tissue assembly by advanced techniques such as three-dimensional bioprinting. Further studies on the cellular and molecular mechanisms that orchestrate spheroid fusion are now crucial to support continued development of bottom-up tissue engineering approaches such as three-dimensional bioprinting.     

Human adipose stromal vascular cell delivery in a fibrin spray

Background aimsAdipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system.MethodsSVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVage system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel fibrin spray system. SVF cell growth and differentiation were documented by immunofluorescence staining and photomicrography.ResultsSVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SVF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD146+ and α-smooth muscle actin+ perivascular/stromal cells.ConclusionsHuman adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.     

Multiple differentiation pathways of rat mammary stromal cells in vitro: acquisition of a fibroblast, adipocyte or endothelial phenotype is dependent on hormonal and extracellular matrix stimulation

It has previously been shown that mammary stromal cells possess the ability to maintain a fibroblast-like phenotype or differentiate in vitro into mature adipocytes in a hormone-dependent manner. This paper reports that rat mammary stromal cells can also differentiate into capillary-like structures in vitro when cultured on a reconstituted basement membrane (RBM). The differentiation potential of mammary stromal cells was compared with that of human umbilical vein endothelial cells (HUVEC) and 3T3-L1 preadipocytes. When cultured on plastic, mammary stromal cells, 3T3-L1 and HUVEC maintained a fibroblast-like phenotype. Mammary stromal cells and 3T3-L1, but not HUVEC, differentiated into mature adipocytes when cultured in adipogenic medium. When plated on reconstituted basement membrane, all three cell types began to migrate and organize themselves into an interconnected capillary network. By 18-20 h, mammary stromal cells organized into complex, highly branched capillary-like tubules whereas 3T3-L1 cells and HUVEC formed more simple structures. Cross-sectional analysis demonstrated the presence of an internal lumen. Mammary stromal cells were unique in their ability to progressively develop into a three-dimensional, highly branched network invading the RBM surface. The network formation was enhanced by the presence of vascular endothelial growth factor (VEGF) and was inhibited by the anti-angiogenic drug suramin. Western blotting analysis demonstrated the presence of the endothelial-specific marker flk-1, as well as the presence of the tight-junction-associated protein ZO-1. Mammary stromal cell differentiation into capillary structures was not a terminal state, since these cells were still able to differentiate into adipocytes when exposed to adipogenic medium. These findings suggest that mammary stromal cells differentiate into fibroblasts, adipocytes or vascular structures in a hormone- and substatum-dependent manner, and may explain the dramatic changes in stromal composition during both normal mammary gland development and tumorigenesis.     

Modulation of adipogenesis-related gene expression by estrogen-related receptor gamma during adipocytic differentiation

Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in oxidative metabolism and mitochondrial biogenesis in brown adipose tissue and heart. However, the physiological role of ERRgamma in adipogenesis and the development of white adipose tissue has not been well studied. Here we show that ERRgamma was up-regulated in murine mesenchyme-derived cells, especially in ST2 and C3H10T1/2 cells, at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. The up-regulation of ERRgamma mRNA was also observed in inguinal white adipose and brown adipose tissues of mice fed a high-fat diet. Gene knockdown by ERRgamma-specific siRNA results in mRNA down-regulation of adipogenic marker genes including fatty acid binding protein 4, PPARgamma, and PGC-1beta in a preadipocyte cell line 3T3-L1 preadipocytes and mesenchymal ST2 and C3H10T1/2 cells in the adipogenesis medium. In contrast, stable expression of ERRgamma in 3T3-L1 cells resulted in up-regulation of these adipogenic marker genes under the adipogenic condition. These results suggest that ERRgamma positively regulate the adipocyte differentiation with modulating the expression of various adipogenesis-related genes.     

脂肪干细胞的诱导分化及其来源的研究

目的:通过组织块培养法得到脂肪干细胞(adipose-derived stem cells,ADSCs),探讨其诱导分化潜能,并初步研究ADSCs的来源。方法:用脂肪组织块培养法培养原代人ADSCs。第三代ADSCs进行成脂和成骨诱导分化,分别用油红O和茜素红S染色进行鉴定。脂肪组织块培养七天后取脂肪组织进行Hematoxylin-eosin Staining(HE)染色观察ADSCs组织分布。结果:用脂肪组织块培养法成功培养出原代人ADSCs。ADSCs传代到第8代,依然保持着良好的增殖能力和细胞形态。ADSCs能成功诱导成脂肪细胞和骨细胞。通过对培养七天后的脂肪组织块进行HE染色,发现ADSCs主要分布在脂肪组织的间质血管和结缔组织周围。结论:用脂肪组织块培养出来的ADSCs具有成脂和成骨分化的潜能。ADSCs主要定位于间质血管和结缔组织周围。     

Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor γ agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-β1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.     

CLA differently regulates adipogenesis in stromal vascular cells from porcine subcutaneous adipose and skeletal muscle

Conjugated linoleic acid (CLA), a mixture of isomers of linoleic acid, has previously been shown to be able to decrease porcine subcutaneous (SC) adipose tissue levels while increasing the count of intramuscular (IM) adipose tissue in vivo. However, the underlying mechanisms through which it acts are poorly understood. The objective of this study was to investigate the different effects of CLA on adipogenesis in cultured SC adipose tissue and IM stromal vascular cells obtained from neonatal pigs. As shown here, trans-10, cis-12 CLA decreased the expression of adipocyte-specific genes as well as adipose precursor cell numbers and the accumulation of lipid in cultured SC adipose tissue stromal vascular cells. However, the cis-9, trans-11 CLA did not alter adipogenesis in SC cultures. On the other hand, both CLA isomers increased the expression of adipocyte-specific genes in IM cultures, together with the increasing accumulation of lipid and Oil Red O-stained cells. Collectively, these data show that CLA decreases SC adipose tissue but increases IM adipose tissue by different regulation of adipocyte-specific gene expression. These results suggest that adipogenesis in IM adipocytes differs from that in SC adipocytes.