Nucleofection is a valuable transfection method for transient and stable transgene expression in adipose tissue-derived stem cells

Adipose tissue-derived stem cells are a powerful tool for in vitro study of adult stem cell biology. So far, they have not been extensively used for gain or loss of function studies since they are resistant to most common transfection methods. Herein, we tested several classic transfection methods on human multipotent adipose tissue-derived stem (hMADS) cells. Our results showed that lipofectants and calcium phosphate were poorly efficient for transgene delivery in hMADS cells. In contrast, nucleofection, an electroporation-based method that is assumed to target plasmid DNA directly to the cell nucleus, led to a significant transient transgene expression in hMADS cells (up to 76% enhanced green fluorescent protein [EGFP]-positive cells were detected). Furthermore, after selection of hMADS cells that were nucleofected with a selectable plasmid coding for EGFP, stable EGFP expressing clones could be propagated in culture and efficiently induced to differentiate into EGFP-positive adipocytes and osteoblasts. Finally, we verified that nucleofected hMADS cells could produce a functional, transgene-encoded, secreted protein. To this aim, hMADS cells were nucleofected with a plasmid coding for leukemia inhibitory factor (LIF). This protein was detected at high concentrations in supernatants from pCAG-LIF transfected hMADS cells. Moreover, supernatants were able to maintain mouse embryonic stem cells' undifferentiated phenotype, indicating that hMADS cells could secrete a functional LIF protein. Taken together, our data demonstrate that nucleofection allows both transient and stable gene expression in adipose tissue-derived stem cells, without impairing their differentiation potential.     

Effects of exercise training on adipogenesis of stromal-vascular fraction cells in rat epididymal white adipose tissue

Aim: Previous studies have shown that exercise training reduced white adipose tissue (WAT) mass compared to that in sedentary controls, and that the smaller mass contained fewer adipocytes. However, the effect of exercise training on adipogenesis is not completely clear. Therefore, we re-examined the effect of exercise training on adipocyte numbers in WAT and, if such an effect was found tested the adipogenic responses of stromal-vascular fraction (SVF) cells containing adipose tissue-derived stem cells (ADSC) in epididymal WAT from exercise-trained (TR) rats. Methods: Wistar male rats were divided into two groups: control (C) and TR. The TR rats were subjected to exercise on a treadmill for 9 weeks. SVF cells containing ADSC were separated from epididymal WAT by centrifugation. Expression of adipocyte differentiation-related genes and adipogenesis of SVF cells were examined. Results: In SVF cells of TR rats, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and that of PPARγ target lipogenic genes was dramatically downregulated, whereas that of preadipocyte factor-1 gene was significantly upregulated. Lipid accumulation in SVF cells of TR rats after the induction of adipocyte differentiation was significantly suppressed in comparison with that of C rats. Moreover, increased expression of hypoxia-inducible factor-1α (HIF-1α) protein was observed in SVF cells of TR rats. Pre-treatment of YC-1, a potent HIF-1α inhibitor, in SVF cells of TR rats restored adipogenesis. Conclusion: These results suggest that exercise training suppresses the ability of SVF cells to differentiate into adipocytes, and that underlying mechanisms involve the upregulation of HIF-1α expression.     

Sprouty1 regulates neural and endothelial differentiation of mouse embryonic stem cells

Fibroblast growth factor (FGF) signaling is implicated in the control of pluripotency and lineage differentiation of both human and mouse embryonic stem cells (mESCs). FGF4 dependent stimulation of ERK1/2 signaling triggers transition of pluripotent ESCs from self-renewal and lineage commitment. In this study, Sprouty 1 (Spry1) expression was observed in undifferentiated mESCs, where it modulated ERK1/2 activity. Spry1 was confirmed as dispensable for the maintenance of self-renewal. However, suppression of Spry1 expression and subsequent activation of ERK1/2 signaling promoted neural differentiation and inhibited endothelial differentiation of mESCs. Moreover, evidence is presented which indicates that SHP2, a major determinant of balance between mESC self-renewal and differentiation, directly regulates Spry1 activity to modulate ERK1/2 signaling and lineage-specific differentiation in mESCs. Our results show that Spry1 has an essential role in the lineage specific differentiation of mESCs.     

Commitment of mouse embryonic stem cells to the adipocyte lineage requires retinoic acid receptor beta and active GSK3

Key events leading to terminal differentiation of preadipocytes into adipocytes have been identified in recent years. However, signaling pathways involved in the decision of stem cells to follow the adipogenic lineage have not yet been characterized. We have previously shown that differentiating mouse embryonic stem (mES) cells give rise to functional adipocytes upon an early treatment with retinoic acid (RA). The goal of this work was to identify regulators of RA-induced commitment of mES cells to the adipocyte lineage. First, we investigated the role of RA receptor (RAR) isotypes in the induction of mES cell adipogenesis. Using synthetic retinoids selective of RAR isotypes, we show that RARbeta activation is both sufficient and necessary to trigger commitment of mES cells to adipocytes. Then, we performed a small-scale drug screening to find signaling pathways involved in RARbeta-induced mES cell adipogenesis. We show that pharmacological inhibitors of glycogen synthase kinase (GSK) 3, completely inhibit RARbeta-induced adipogenesis in mES cells. This finding uncovers the requirement of active GSK3 in RARbeta-induced commitment of mES cells toward the adipocyte lineage. Finally, we investigated the role of the Wnt pathway, in which GSK3 is a critical negative regulator, in adipocyte commitment by analyzing Wnt pathway activity in RA- and RARbeta-induced mES cell adipogenesis. Our results suggest that although RARbeta and active GSK3 are required for RA-induced adipogenesis, they might be acting through a Wnt pathway-independent mechanism.     

Thy-1 signals through PPARγ to promote lipofibroblast differentiation in the developing lung

Thy-1 is a glycosylphosphytidylinositol-linked cell-surface glycoprotein present on a subset of lung fibroblasts, which plays an important role in postnatal alveolarization. In the present study, we define the role of Thy-1 in pulmonary lipofibroblast differentiation and in the regulation of lipid homeostasis via peroxisome proliferator-activated receptor-γ (PPARγ). Thy-1 was associated with interstitial cells containing lipid droplets in vivo. The transfection of Thy-1 into Thy-1 (-) fibroblasts increased triglyceride content, fatty-acid uptake, and the expression of the lipofibroblast marker adipocyte differentiation-related protein. Thy-1 (+) fibroblasts exhibited 2.4-fold higher PPARγ activity, and the inhibition or activation of PPARγ reduced and increased triglyceride content, respectively. Thy-1 (-) fibroblasts were not responsive to either of the PPARγ agonists ciglitazone or prostaglandin J(2), supporting the importance of Thy-1 in signaling via PPARγ. Thy-1 (+) fibroblasts expressed significantly higher concentrations of fatty-acid transporter protein-3 mRNA, and demonstrated higher rates of fatty-acid uptake and increased triglyceride content. The inhibition of fatty-acid transporter protein function reduced Thy-1 (+) fibroblast lipid content. The expression of Thy-1 in C57BL/6 lung fibroblasts increased during the neonatal period, coinciding with the onset of alveolarization. Thy-1 promoted lipofibroblast differentiation via the expression of PPARγ, stimulated lipid accumulation via fatty-acid esterification, and enhanced the fatty-acid uptake mediated by fatty-acid transporter proteins. Thy-1 is important in the regulation of lipofibroblast differentiation in the developing lung.     

4E-BP1 regulates the differentiation of white adipose tissue

4E Binding protein 1 (4E-BP1) suppresses translation initiation. The absence of 4E-BP1 drastically reduces the amount of adipose tissue in mice. To address the role of 4E-BP1 in adipocyte differentiation, we characterized 4E-BP1^−/− mice in this study. The lack of 4E-BP1 decreased the amount of white adipose tissue and increased the amount of brown adipose tissue. In 4E-BP1^−/− MEF cells, PPARγ coactivator 1 alpha (PGC-1α) expression increased and exogenous 4E-BP1 expression suppressed PGC-1α expression. The level of 4E-BP1 expression was higher in white adipocytes than in brown adipocytes and showed significantly greater up-regulation in white adipocytes than in brown adipocytes during preadipocyte differentiation into mature adipocytes. The amount of PGC-1α was consistently higher in HB cells (a brown preadipocyte cell line) than in HW cells (a white preadipocyte cell line) during differentiation. Moreover, the ectopic over-expression of 4E-BP1 suppressed PGC-1α expression in white adipocytes, but not in brown adipocytes. Thus, the results of our study indicate that 4E-BP1 may suppress brown adipocyte differentiation and PGC-1α expression in white adipose tissues.     

配对盒基因6/小鼠胚胎干细胞细胞系的建立及干细胞生物学特性分析

目的 建立配对盒基因6（Pax6）/小鼠胚胎干细胞（mESCs）细胞系并鉴定其干细胞生物学特性。 方法 体外培养mESCs,将重组载体pEF1α-Pax6-IRES-AcGFP和空载体pEF1α-IRES-AcGFP分别用脂质体法转染mESCs,经G418梯度及荧光蛋白双筛选后,使用细胞免疫荧光染色、免疫印迹法及RT-PCR技术检测Pax6的表达情况,流式细胞术检测Pax6/mESCs阳性细胞的比例。将获得正确的细胞系分别采用细胞免疫荧光染色对其干细胞标志物阶段特异性胚胎抗原1（SSEA1）、八聚体结合转录因子4（OCT4）进行检测,碱性磷酸酶（AP）染色法对其多能性进行检测,流式细胞术检测增殖指数Ki67。将Pax6/mESCs进行肾背囊下移植,移植物行HE染色观察其分化能力。 结果 Pax6成功在mESCs内表达,经G418筛选后,获得Pax6/mESCs细胞系,流式结果显示,Pax6阳性率为90%,免疫荧光显示,干细胞标志物SSEA1、OCT4表达阳性且AP染色阳性,并且在体内移植后能向3个胚层分化。 结论 Pax6成功在mESCs内表达,经G418筛选后,获得细胞系Pax6/mESCs并维持良好的干细胞特性。     

5-Aminoimidazole-4-carboxyamide ribonucleoside induces G(1)/S arrest and Nanog downregulation via p53 and enhances erythroid differentiation

Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK), a key regulator for controlling energy metabolism, is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest, without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers, Nanog and stage-specific embryonic antigen-1, in the presence of leukemia inhibitory factor, without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently, an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation, an effect inhibited by MG132, a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs, it increased expression levels of erythroid cell lineage markers (Ter119, GATA1, Klf1, Hbb-b, and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR, endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs.     

Tyrphostin AG490 Agent Modestly but Significantly Prevents Onset of Type 1 in NOD Mouse; Implication of Immunologic and Metabolic Effects of a Jak-Stat Pathway Inhibitor

Previously, we have reported that the Jak-Stat signaling pathway is defective in NOD mice. In this study, prediabetic female NOD mice (4?weeks) were treated by intraperitoneal injection either with AG490 or DMSO three times per week for 4 consecutive weeks, followed by once a week for an additional 6?weeks. The onset of diabetes was attenuated in NOD mice treated with AG490 relative to DMSO treated control mice (p?相似文献   

Embryonic stem cell-derived adipogenesis

Key events leading to terminal differentiation of preadipocytes into adipocytes have been characterized in the recent years. However, master genes that commit progression from multipotent mesenchymal stem cell to the adipoblast stage of development have not yet been identified. The use of embryonic stem (ES) cells as a route to study early events in adipogenesis and to characterize factors involved in the decision of stem cells to follow the adipogenic pathway is described in this paper. The capacity of lif-/- and lifr-/- ES cells to undergo adipocyte differentiation is reported as an application of mutant ES cells to study gene function during the development of adipose cells.     

Maintenance of murine embryonic stem cells' self-renewal and pluripotency with increase in proliferation rate by a bovine granulosa cell line-conditioned medium

Murine embryonic stem cells (mESCs) are pluripotent cells that can be propagated in an undifferentiated state in continuous culture on a feeder layer or without feeders in the presence of leukemia inhibitory factor (LIF). Although there has been a great advance since their establishment, ESC culture is still complex and expensive. Therefore, finding culture conditions that maintain the self-renewal of ESCs, preventing their differentiation and promoting their proliferation, is still an area of great interest. In this work, we studied the effects of the conditioned medium from a bovine granulosa cell line (BGC-CM) on the maintenance of self-renewal and pluripotency of mESCs. We found that this medium is able to maintain mESCs' self-renewal while preserving its critical properties without LIF addition. mESCs cultured in BGC-CM expressed the stem cell markers Oct4, Sox2, Nanog, SSEA-1, Klf4, Rex1, and ECAT1. Moreover, mESCs cultured in BGC-CM gave rise to embryoid bodies and teratomas that differentiated effectively to diverse cell populations from endoderm, mesoderm, and ectoderm. Further, we found that mESCs cultured in BGC-CM have an increased proliferation rate compared with cells grown in the mESC standard culture medium supplemented with LIF. These findings may provide a powerful tool to culture mESCs for long periods of time with high proliferation rate while preserving its basic characteristics, contributing to the application of these cells to assess potential tissue engineering and cellular therapy applications.     

Interferon gamma inhibits adipogenesis in vitro and prevents marrow fat infiltration in oophorectomized mice

Interferon gamma (IFNγ) has been reported to induce osteoblastogenesis from mesenchymal stem cells (MSCs) both in vitro and in vivo. With ageing, adipocytes outnumber osteoblasts within the bone microenvironment leading to a decrease in bone formation. Since both osteoblasts and adipocytes are of mesenchymal origin, we hypothesized that IFNγ treatment might negatively affect adipogenesis while stimulating osteoblastogenesis in human MSC. To test this hypothesis, human MSCs were induced to differentiate into adipocytes in the presence or absence of osteogenic doses of IFNγ (1, 10, and 100 ng/ml). IFNγ-treated MSC showed a decrease in adipocyte differentiation and lipid deposition when compared with vehicle-treated controls. Additionally, adipogenic markers were significantly decreased by IFNγ treatment at the same doses that have been reported to have a strong osteogenic effect in vitro. Furthermore, DNA binding of peroxisome proliferator-activated receptor gamma was significantly lower in IFNγ-treated differentiating MSC. Subsequently, ovariectomized C57BL6 mice were treated with osteogenic doses of IFNγ three times a week for 6 weeks. In distal femur, treated mice showed significantly higher hematopoiesis concomitant with lower levels of fat volume/total volume, adipocyte number, and expression of adipogenic markers when compared with the vehicle-treated mice. Together, these findings demonstrate that, at osteogenic doses, IFNγ also acts as an inhibitor of adipogenesis in vitro and prevents marrow fat infiltration while favors hematopoiesis in ovariectomized mice.     

Hedgehog signaling alters adipocyte maturation of human mesenchymal stem cells

Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling, the dysregulation of which causes several pathologies, such as congenital defects and cancer, is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells, such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis, but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells, the maturation process, but not the early steps of human mesenchymal stem cell differentiation, is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein alpha and peroxisome proliferator-activated receptor (PPAR) gamma2 expression, whereas PPARgamma1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes, adipogenesis appears dramatically impaired, with reduced lipid accumulation, a decrease in adipocyte-specific markers, and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells.