The roles of Wnt antagonists Dkk1 and sFRP4 during adipogenesis of human adipose tissue‐derived mesenchymal stem cells

Abstract. Objectives: The canonical Wnt signalling pathway performs an important function in the control of adipogenesis. However, the mechanisms and mediators underlying these interactions have yet to be defined in detail. Thus, this study was performed in order to elucidate the roles of the Wnt family during adipogenic differentiation of human adipose tissue‐derived mesenchymal stem cells (hAMSCs). Materials and methods: We assessed several members of the Frizzled (FZD) family, the receptors of Wnts, inhibitors including the secreted frizzled‐related protein (sFRP) family and Dickkopfs (Dkks), and the downstream factor, β‐catenin. Expressional levels of adipogenic markers regulated by the small interfering RNA of Dkk1 (siDkk1) and sFRP4 (sisFRP4) were assessed using real‐time quantitative PCR and Western blot analysis. Results: The mRNA level of Dkk1 was expressed abundantly in the early stages of adipogenesis and decreased rapidly during the late stages of adipogenesis. However, sFRP4 mRNA was up‐regulated gradually during adipogenic differentiation in hAMSCs. Expression of FZD1, FZD7 and β‐catenin were reduced during adipogenic differentiation. Transfection of hAMSCs with siDkk1 or sisFRP4 partially inhibited differentiation of hAMSCs into adipocytes and restored levels of β‐catenin. Conclusions: We determined that Dkk1 was up‐regulated transiently in the early stages of adipogenesis, and that sFRP4 levels increased gradually during adipogeneis via inhibition of Wnt signalling. Collectively, these results show that Dkk1 and sFRP4 perform an important function in adipogenesis in hAMSCs.     

Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells

Wedelolactone is an herbal medicine that is used to treat septic shock, hepatitis and venom poisoning. Although in differentiated and cancer cells, wedelolactone has been identified as anti‐inflammatory, growth inhibitory, and pro‐apoptotic, the effects of wedelolactone on stem cell differentiation remain largely unknown. Here, we report that wedelolactone inhibits the adipogenic differentiation of human adipose tissue‐derived mesenchymal stem cells (hAMSCs). Wedelolactone reduced the formation of lipid droplets and the expression of adipogenesis‐related proteins, such as CCAAT enhancer‐binding protein‐α (C/EBP‐α), peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), lipoprotein lipase (LPL), and adipocyte fatty acid‐binding protein aP2 (aP2). Wedelolactone mediated this process by sustaining ERK activity. In addition, inhibition of ERK activity with PD98059 resulted in reversion of the wedelolactone‐mediated inhibition of adipogenic differentiation. Taken together, these results indicate that wedelolactone inhibits adipogenic differentiation through ERK pathway and suggest a novel inhibitory effect of wedelolactone on adipogenic differentiation in hAMSCs. J. Cell. Biochem. 113: 3436–3445, 2012. © 2012 Wiley Periodicals, Inc.     

Propyl Gallate Inhibits Adipogenesis by Stimulating Extracellular Signal-Related Kinases in Human Adipose Tissue-Derived Mesenchymal Stem Cells

Propyl gallate (PG) used as an additive in various foods has antioxidant and anti-inflammatory effects. Although the functional roles of PG in various cell types are well characterized, it is unknown whether PG has effect on stem cell differentiation. In this study, we demonstrated that PG could inhibit adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells (hAMSCs) by decreasing the accumulation of intracellular lipid droplets. In addition, PG significantly reduced the expression of adipocyte-specific markers including peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein 2 (aP2). PG inhibited adipogenesis in hAMSCs through extracellular regulated kinase (ERK) pathway. Decreased adipogenesis following PG treatment was recovered in response to ERK blocking. Taken together, these results suggest a novel effect of PG on adipocyte differentiation in hAMSCs, supporting a negative role of ERK1/2 pathway in adipogenic differentiation.     

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.     

Artemisinic acid is a regulator of adipocyte differentiation and C/EBP δ expression

Adipocyte dysfunction is associated with the development of obesity. In this study, artemisinic acid, which was isolated from Artemisia annua L., inhibited adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs) and its mechanism of action was determined. The mRNA levels of peroxidase proliferation-activated receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α, late adipogenic factors, were reduced by artemisinic acid. Moreover, the mRNA levels of the PPAR γ target genes lipoprotein lipase, CD36, adipocyte protein, and liver X receptor were down-regulated by artemisinic acid. Artemisinic acid reduced expression of the C/EBP δ gene without impacting C/EBP β. In addition, attempts to elucidate a possible mechanism underlying the artemisinic acid-mediated effects revealed that reduced expression of the C/EBP δ gene was mediated by inhibiting Jun N-terminal kinase (JNK). Additionally, artemisinic acid also reduced the expression of the adipogenesis-associated genes glucose transporter-4 and vascular endothelial growth factor. In addition to the interference of artemisinic acid with adipogenesis, artemisinic acid significantly attenuated tumor necrosis factor-α-induced secretion of interleukin-6 by undifferentiated hAMSCs, thus influencing insulin resistance and the inflammatory state characterizing obesity. Taken together, these findings indicate that inhibiting adipogenic differentiation of hAMSCs by artemisinic acid occurs primarily through reduced expression of C/EBP δ, which is mediated by the inhibition of JNK and suggest that aremisinic acid may be used as a complementary treatment option for obesity associated with metabolic syndrome.     

Isorhamnetin-induced anti-adipogenesis is mediated by stabilization of β-catenin protein

AimsPrevious studies have shown that isorhamnetin has anti-adipogenic effects in mouse 3T3-L1 cells. This study was conducted to elucidate the inhibitory mechanisms of isorhamnetin during adipogenic differentiation of human adipose tissue-derived stem cells (hAMSCs).Main methodsThe effect of isorhamnetin on adipogenic differentiation of hAMSCs was quantified by Oil Red O staining and a triglyceride assay. In addition, real-time PCR and Western blot were used to determine the expression of adipogenesis-related genes.Key findingsIsorhamnetin inhibited the adipocyte differentiation of hAMSCs. Additionally, when the effects of Wnt antagonists that promote adipogenesis were evaluated, isorhamnetin was found to down-regulate the mRNA levels of sFRP1 and Dkk1, but had no effect on the mRNA levels of sFRP2, sFRP3, sFRP4 and Dkk3. Isorhamnetin also inhibited the expression of Wnt receptor and co-receptor genes. Furthermore, isorhamnetin increased the protein levels of β-catenin, an effector molecule of Wnt signaling, but had no effect on the mRNA levels of β-catenin. The phosphorylation level of GSK 3β was also increased by isorhamnetin. These results were confirmed by the fact that the expression of c-myc, cyclin D1 and PPARδ, which are target genes of β-catenin, was upregulated by isorhamnetin. Moreover, isorhamnetin reduced the mRNA expression levels of C/EBPα and PPARγ, which are known to be inhibited by c-myc or by cyclin D1 and PPARδ, respectively.SignificanceOur results indicate that isorhamnetin inhibits the adipogenic differentiation of hAMSCs and that its mechanisms are mediated by the stabilization of β-catenin.     

Notch signalling inhibits the adipogenic differentiation of single‐cell‐derived mesenchymal stem cell clones isolated from human adipose tissue

ADSCs (adipose‐derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue‐derived single‐cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose‐derived single‐cell‐clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ‐secretase inhibitor, DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐l ‐alanyl]‐(S)‐phenylglycine t‐butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft‐tissue augmentation application.     

Glioblastoma therapy with cytotoxic mesenchymal stromal cells optimized by bioluminescence imaging of tumor and therapeutic cell response

Genetically modified adipose tissue derived mesenchymal stromal cells (hAMSCs) with tumor homing capacity have been proposed for localized therapy of chemo- and radiotherapy resistant glioblastomas. We demonstrate an effective procedure to optimize glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo non invasive monitoring of tumor and therapeutic cells. Glioblastoma U87 cells expressing Photinus pyralis luciferase (Pluc) were implanted in combination with hAMSCs expressing a trifunctional Renilla reniformis luciferase-red fluorescent protein-thymidine kinase reporter in the brains of SCID mice that were subsequently treated with ganciclovir (GCV). The resulting optimized therapy was effective and monitoring of tumor cells by bioluminescence imaging (BLI) showed that after 49 days GCV treatment reduced significantly the hAMSC treated tumors; by a factor of 10(4) relative to controls. Using a Pluc reporter regulated by an endothelial specific promoter and in vivo BLI to image hAMSC differentiation we gained insight on the therapeutic mechanism. Implanted hAMSCs homed to tumor vessels, where they differentiated to endothelial cells. We propose that the tumor killing efficiency of genetically modified hAMSCs results from their association with the tumor vascular system and should be useful vehicles to deliver localized therapy to glioblastoma surgical borders following tumor resection.     

Human mesenchymal stem cells and renal tubular epithelial cells differentially influence monocyte-derived dendritic cell differentiation and maturation

Mesenchymal stem cells (MSCs) possess immunosuppressive properties. But also fully differentiated human renal tubular epithelial cells (RTECs) are able to modulate T-cell proliferation in vitro. In this study we compared two MSC populations, human adipose derived stem cells (ASCs) and human amniotic mesenchymal stromal cells (hAMSCs), and RTECs regarding their potential to inhibit monocyte-derived dendritic cell (DC) differentiation and maturation in indirect co-culture.In the presence of hAMSCs and RTECs, monocytes stimulated to undergo DC differentiation were inhibited to acquire surface phenotype of immature and mature DCs. In contrast, ASCs showed only limited suppressive capacity. Secretion of IL-12p70 was suppressed in hAMSC co-cultures and high IL-10 levels were detected in all co-cultures. Prostaglandin E₂ was found in ASC and hAMSC co-cultures, whereas soluble human leukocyte antigen-G was highly elevated only in RTEC co-cultures. Thus, inhibition of DC generation by MSCs and RTECs might be mediated by different soluble factors.     

Fat pad-derived mesenchymal stem cells as a potential source for cell-based adipose tissue repair strategies

Background: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem‐cell exhibits tri‐lineage differentiation potential and is able to maintain its proliferation potential and cell‐surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad‐derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. Materials and methods: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell‐surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. Results: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell‐surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad‐derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator‐activated receptor γ2 and lipoprotein lipase, and oil red O staining. Discussion: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem‐cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.     

Epigallocatechin‐3‐gallate suppresses the lipid deposition through the apoptosis during differentiation in bovine bone marrow mesenchymal stem cells

Epigallocatechin gallate (EGCG), a major component of tea, has known effects on obesity, fatty liver, and obesity‐related cancer. We explored the effects of EGCG on the differentiation of bovine mesenchymal stem cells (BMSCs, which are multipotent) in a dose‐ and time‐dependent manner. Differentiating BMSCs were exposed to various concentrations of EGCG (0, 10, 50, 100, and 200 µM) for 2, 4, and 6 days. BMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/high‐glucose medium with adipogenic inducers for 6 days, and the expression levels of various genes involved in adipogenesis were measured using real‐time polymerase chain reaction (PCR) and Western blotting. We assessed apoptosis by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining of control and EGCG‐exposed cells. We found that EGCG significantly suppressed fat deposition and cell viability (P < 0.05). The mRNA and protein levels of various adipogenic factors were measured. Expression of the genes encoding peroxisome proliferator‐activated receptor gamma (PPARG), CCAAT/enhancer‐binding protein alpha (CEBPA), fatty acid‐binding protein 4 (FABP4), and stearoyl‐CoA desaturase (SCD) were diminished by EGCG during adipogenic differentiation (P < 0.05). We also found that EGCG lowered the expression levels of the adipogenic proteins encoded by these genes (P < 0.05). EGCG induced apoptosis during adipogenic differentiation (P < 0.05). Thus, exposure to EGCG potentially inhibits adipogenesis by triggering apoptosis; the data suggest that EGCG inhibits adipogenic differentiation in BMSCs.     

Larger anti-adipogenic effect of angiotensin II on omental preadipose cells of obese humans

Objective: The ability to form new adipose cells is important to adipose tissue physiology; however, the mechanisms controlling the recruitment of adipocyte progenitors are poorly understood. A role for locally generated angiotensin II in this process is currently proposed. Given that visceral adipose tissue reportedly expresses higher levels of angiotensinogen compared with other depots and the strong association of augmented visceral fat mass with the adverse consequences of obesity, we studied the role of angiotensin II in regulating adipogenic differentiation in omental fat of obese and non‐obese humans. Research Methods and Procedures: The angiotensin II effect on adipose cell formation was evaluated in human omental adipocyte progenitor cells that were stimulated to adipogenic differentiation in vitro. The adipogenic response was measured by the activity of the differentiation marker glycerol‐3‐phosphate dehydrogenase. Results: Angiotensin II reduced the adipogenic response of adipocyte progenitor cells, and the extent of the decrease correlated directly with the subjects’ BMI (p = 0.01, R² = 0.30). A 56.3 ± 3.4% and 44.5 ± 2.7% reduction of adipogenesis was found in obese and non‐obese donors’ cells, respectively (p < 0.01). The effect of angiotensin II was reversed by type 1 angiotensin receptor antagonist losartan. Discussion: A greater anti‐adipogenic response to angiotensin II in omental adipose progenitor cells from obese subjects opens a venue to understand the deregulation of visceral fat tissue cellularity that has been associated with severe functional abnormalities of the obese condition.