Gene expression patterns of bovine perimuscular preadipocytes during adipogenesis

Bovine perimuscular fat (PMF) preadipocytes were induced to undergo adipogenesis in vitro in our recent study to define the expression patterns of genes involved in the differentiation process. Based on the understanding of the interaction among adipogenic genes, a broad overview of gene expression profile in the differentiating PMF preadipocytes was evaluated using bovine specific DNA microarray from day 2 to 8 post-differentiation induction. A total of 100 significantly differentially expressed genes were detected between differentiated and control cells including those involved in several biochemical pathways and cellular/molecular signaling. In addition, quantitative real-time PCR validated that typical adipogenic genes were up-regulated at early differentiation in the preadipocytes. These results suggest that the PMF preadipocyte system is available as a novel in vitro model for molecular adipogenesis studies in the bovine and that a series of genes are switched on/off during early events associated with adipogenesis.     

Three-dimensional spheroid culture of adipose stromal vascular cells for studying adipogenesis in beef cattle

Protocols designed for the adipogenic differentiation of human and mouse cells are commonly used for inducing the adipogenesis of bovine stromal vascular cells. However, likely due to metabolic differences between ruminant and non-ruminant animals, these methods result in only few cells undergoing complete adipogenesis with minimal lipid droplet accumulation. Here, we discuss the development of an adipogenic differentiation protocol for bovine primary cells through a three-dimensional spheroid culture. Stromal vascular cells derived from bovine intramuscular fat were isolated and stored in liquid nitrogen before culturing. Cells were cultured in hanging drops for 3 days to allow for the formation of spherical structures. The spheroids were then transferred to cell culture plates with endothelial basal medium-2 for 3 days and in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with a standard adipogenic cocktail for 3 additional days, which were then allowed to fully differentiate for 3 days in DMEM supplemented with insulin. Compared with conventional two-dimensional culture, cells in a three-dimensional spheroid culture system had higher adipogenic gene expression and consequently contained more adipocytes with larger lipid droplets. In addition, endothelial induction of spheroids prior to adipogenic differentiation is essential for efficient induction of adipogenesis of bovine stromal vascular cells, mimicking in vivo adipose development. In summary, the newly developed three-dimensional spheroid culture method is an efficient way to induce adipogenic differentiation and study adipose development of cells derived from ruminant animals, which also can be used for studying the role of angiogenesis in adipose development.     

Proliferation and differentiation of progeny of ovine unilocular fat cells (adipofibroblasts)

Summary The responsiveness of progeny of sheep-derived unilocular fat cells (adipofibroblasts) to dexamethasone, insulin, insulinlike growth factor I (IGF-I), growth hormone (GH), and basic fibroblast growth factor (FGF) was determined in a clonal culture system. Primary cultures of mature adipocytes were obtained from intermuscular adipose tissue (semimembranosus/semitendinosus seam depot) of sheep by ceiling culture techniques. Following degeneration of unilocular fat droplets and re-establishment of fibroblasticlike adipofibroblasts, all adipofibroblasts adhering to upper flask surfaces were collected and isolated away from fibroblasts (which had no multilocular vesicles) by Percoll^? gradient centrifugation. Progeny derived from a single adipofibroblast were isolated and tested for the ability to proliferate, differentiate, and accumulate lipids. Stock cultures of adipofibroblasts reached confluence in 5 d and were induced to differentiate from 7 to 9 d with dexamethasone-methyl isobutylxanthine-insulin (DMI). Incubation with insulin, IGF-I, GH, or FGF prior to confluence followed by induction with DMI produced no direct (priming) effect on subsequent differentiation. When substituted individually in place of DMI during the 2 d differentiation/induction period, all factors induced differentiation of cultured adipofibroblasts as determined by lipogenesis (P<.05) and lipoprotein lipase activity (P<.05). Thus, isolated adipofibroblasts from sheep muscle may be induced by hormones and growth factors to display mature adipocyte morphology in cell culture. Further definition of the adipofibroblast culture system may aid in the identification of mechanisms regulating adipocyte development in sheep skeletal muscle, as well as in the study of intercommunication between fat and muscle cells. With technical assistance from B. Mathison.     

Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels

Advances in bioengineering, material chemistry, and developmental biology have led to the design of three-dimensional (3D) culture systems that better resemble the surrounding structure and chemistry of the in situ niches of cells in tissues. This study was designed to characterize and compare porcine adipose-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) induced to differentiate toward osteogenic and adipogenic lineages in vitro by using a 3D alginate hydrogel. The morphology and gene expression of the two cell populations during differentiation were analyzed. Both ADSC and BMSC showed morphological evidence of osteogenic and adipogenic differentiation. Expression patterns of genes characteristic of the onset of osteogenic differentiation (ALP, COL1A1, SPARC, SPP1) were low at the beginning of culture and generally increased during the period of differentiation up to 28 days in culture. Expression of genes associated with adipogenic differentiation (ACSL1, ADFP, ADIPOQ, CD36, DBI, DGAT2, PPARG, SCD) was consistently increased in ADSC cultured in alginate hydrogel relative to the start of differentiation. However, adipogenic gene expression of BMSC cultured in alginate hydrogel was more limited when compared with that of ADSC. Evaluation of cell numbers (via the MTT staining assay) suggested a greater viability of BMSC under osteogenic conditions in alginate hydrogels than under adipogenic conditions, whereas ADSC had greater viability under adipogenic conditions than under osteogenic conditions. This study thus provides an important initial evaluation of ADSC and BMSC seeded and differentiated toward the osteogenic and adipogenic cell lineages in a 3D alginate hydrogel in vitro.     

Rho激酶抑制剂Y-27632对C3H10T1/2细胞增殖与成脂分化的作用

探究Rho激酶抑制剂Y-27632对间充质干细胞（mesenchymal stem cells,MSCs）C3H10T1/2增殖和成脂分化的影响.实验分为对照组、成脂诱导组和Y-27632处理组（Y-27632+成脂诱导）. 利用MTT检测细胞增殖情况,油红O染色,异丙醇萃取法检测细胞成脂分化情况,半定量RT-PCR检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator activiated receptor γ, PPARγ)和CCAAT增强子结合蛋白α (CCAAT enhancer binding protein α, C/EBPα)基因表达. 结果表明,Y-27632能够显著抑制C3H10T1/2细胞的增殖(P<0.05),并呈一定的浓度依赖性;高浓度Y-27632对C3H10T1/2细胞成脂分化具有显著抑制作用（P<0.05）;半定量RT-PCR结果显示,成脂诱导处理组PPARγ和C/EBPα表达量在第3 d、5 d和7 d显著低于成脂诱导组（P<0.05）. 综上所述,Y-27632能够抑制C3H10T1/2细胞增殖与成脂分化.     

Mesenchymal Stromal Cell Proliferation,Gene Expression and Protein Production in Human Platelet-Rich Plasma-Supplemented Media

Background

Platelet-rich plasma (PRP) is increasingly used as a cell culture supplement, in order to reduce the contact of human cells with animal-derived products during in vitro expansion. The effect of supplementation changes on cell growth and protein production is not fully characterized.

Methods

Human mesenchymal stromal cells from bone marrow, adipose tissue and Wharton''s Jelly were isolated and cultured in PRP-supplemented media. Proliferation, in vitro differentiation, expression of cell surface markers, mRNA expression of key genes and protein secretion were quantified.

Results

10% PRP sustained five to tenfold increased cell proliferation as compared to 10% fetal bovine serum. Regarding cell differentiation, PRP reduced adipogenic differentiation and increased calcium deposits in bone marrow and adipose tissue-mesenchymal stromal cells. Wharton''s Jelly derived mesenchymal stromal cells secreted higher concentrations of chemokines and growth factors than other mesenchymal stromal cells when cultured in PRP-supplemented media. Bone marrow derived mesenchymal stromal cells secreted higher concentrations of pro-inflammatory and pro-angiogenic proteins. Mesenchymal stromal cells isolated from adipose tissue secreted higher amounts of extracellular matrix components.

Conclusions

Mesenchymal stromal cells purified from different tissues have distinct properties regarding differentiation, angiogenic, inflammatory and matrix remodeling potential when cultured in PRP supplemented media. These abilities should be further characterized in order to choose the best protocols for their therapeutic use.     

Co-culture of bovine muscle satellite cells with preadipocytes increases PPARγ and C/EBPβ gene expression in differentiated myoblasts and increases GPR43 gene expression in adipocytes

We hypothesized that preadipocyte differentiation would be depressed by differentiating myoblasts, whereas preadipocytes would promote adipogenic gene expression in myoblasts in a co-culture system. We also determined the effects of arginine, a biological precursor of nitric oxide, and/or trans-10, cis-12 conjugated linoleic acid (CLA) on adipogenic gene expression during differentiation of bovine preadipocytes and myoblasts. Bovine semimembranosus satellite cells (BSC) and subcutaneous preadipocytes were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's modified Eagle medium (DMEM) and 1% antibiotics during the 3-day proliferation period. After proliferation, BSC and preadipocytes were treated for 3 days with 3% horse serum/DMEM and 5% FBS/DMEM with antibiotics, respectively. Media also contained 100 μM oleic acid, 10 μg/ml insulin, 1 μg/ml pioglitazone and 1 μg/ml dexamethasone. Subsequently, the differentiating myoblasts and adipocytes were cultured in their respective media with 5 mM arginine and/or 40 μM trans-10, cis-12 CLA for 4 days. Finally, myoblasts and adipocytes were single- or co-cultured for 2 h singly or in combination. Arginine stimulated SCD gene expression, whereas CLA depressed SCD gene expression in adipocytes and myoblasts (P=.002). Co-culture of adipocytes and myoblasts elicited an increase in C/EBPβ and PPARγ gene expression in differentiated myoblasts (P≤.01) and an increase in GPR43 gene expression in adipocytes (P=.01). Expression of AMPKα and CPT1ß was unaffected by co-culture, although SCD gene expression tended (P=.12) to be depressed by co-culture. These experiments demonstrated that co-culture of adipocytes with myoblasts increased adipogenic gene expression in the myoblastic cells.     

The effects of cyclin-dependent kinase inhibitors on adipogenic differentiation of human mesenchymal stem cells

The molecular mechanisms that couple growth arrest and cell differentiation were examined during adipogenesis. Here, to understand the cyclin-dependent kinase inhibitor (CKI) genes involved in the progression of adipogenic differentiation, we examined changes in the protein and mRNA expression levels of CKI genes in vitro. During the onset of growth arrest associated with adipogenic differentiation, two independent families of CKI genes, p27Kip1 and p18INK4c, were significantly increased. The expressions of p27Kip1 and p18INK4c, regulated at the level of protein and mRNA accumulation, were directly coupled to adipogenic differentiation. This finding was supported by the inhibition of adipogenic differentiation caused by short interfering RNA (siRNA). In this study, we investigated the regulatory effects of transforming growth factor beta-1 (TGFβ-1) on CKI genes involved in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Only the up-regulation of p18INK4c during adipogenic differentiation, and not that of the p27Kip1 gene was prevented by treatment with TGFβ-1, one of the factors that inhibit adipogenesis in vitro. This finding indicates a close correlation between adipogenic differentiation and p18INK4c induction in hMSCs. Thus, these data demonstrate a role for the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors in regulating adipogenic differentiation, thereby providing a molecular mechanism that couples growth arrest and differentiation.     

Maintenance of human adipose derived stem cell (hASC) differentiation capabilities using a 3D culture

In this study, 3D culture system for human adipose-derived stem cell (hASC) using a BioLevitator as the bioreactor for microcarrier-based cultures was established. During the culturing period, hASCs preferred to grow in crevices between microcarriers and a high viability was maintained even when reaching confluency. Adipogenic or osteogenic differential medium was used to induce hASCs and differential potentials of these cells were compared between 2D and 3D environments via RT-PCR and staining quantifications. CEBP/α gene expression was significant higher in 3D condition at day 21 (P < 0.05). Staining quantification indicates that cells cultured in 3D condition have significant better differentiation potential from day 14 to 21 for both adipogenic and osteogenic lineages (P < 0.01).     

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.     

Defined serum-free media for in vitro expansion of adipose-derived mesenchymal stem cells

BackgroundThere is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use.MethodsAdipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls.ResultsAd-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS.ConclusionsThe defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes.     

In vitro cardiomyogenic differentiation of adipose‐derived stromal cells using transforming growth factor‐β1

Transplanting stem cells differentiated towards a cardiac lineage can regenerate cardiac muscle tissues to treat myocardial infarction. In this study, we tested the hypothesis that transforming growth factor‐β1 (TGF‐β1) induces cardiomyogenic differentiation of adipose‐ derived stromal cells (ADSCs) in vitro. Rat ADSCs were cultured with TGF‐β1 (10 ng ml^?1) for 2 weeks in vitro. ADSCs cultured without TGF‐β1 served as a control. The mRNA expression of cardiac‐specific gene was induced by TGF‐β1, while the control culture did not show cardiac‐specific gene expression. Immunocytochemical analyses showed that a small fraction of ADSCs cultured with TGF‐β1 for 2 weeks stained positively for cardiac myosin heavy chain (MHC) and α‐sarcomeric actin. Flow cytometric analyses showed that the proportion of cells expressing cardiac MHC increased with TGF‐β1. However, no mesenchymal differentiation (e.g., osteogenic and adipogenic differentiation) was detected other than cardiomyogenic differentiation. These results showed that TGF‐β1 induce ADSC cardiomyogenic differentiation in vitro, which could be useful for myocardial infarction stem cell therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.     

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.     

Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem Cells

Background

Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells.

Methodology/Principal Findings

In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed.

Conclusion/Significance

Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.     

Adipogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice-including relationship of sex differences

We have previously demonstrated that adipose-derived stromal cells (ASCs) as well as bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. Both types are considered to include mesenchymal stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have also previously reported the plasticity of BSCs and ASCs. In this study, we focused on adipogenic differentiation in vitro by ASCs harvested from GFP transgenic mice. Moreover, preadipocytes and mature adipocytes were harvested at the same time, and the cells were cultured to compare them with ASCs. Inguinal fat pads from GFP transgenic mice were used for the isolation of ASCs, preadipocytes, and mature adipocytes. After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks. Adipogenic differentiation of ASCs was assessed by Oil Red O staining and the expression of the adipocyte specific peroxisome proliferative activated receptor gamma2 (PPAR-gamma2) gene. These ASCs stained positively, and expression of PPAR-gamma2 was detected. Moreover, we also tried to characterize the influence of sex differences on the adipogenic differentiation of ASCs harvested from both male and female mice. This was assessed by the expression levels of the PPAR-gamma2 gene using real-time PCR. The results showed that the expression levels of ASCs harvested from female mice were a maximum of 2.89 times greater than those harvested from male mice. This suggests that the adipogenic differentiation of ASCs is closely related to sex differences.