Large-Volume Vascularized Muscle Grafts Engineered From Groin Adipose Tissue in Perfusion Bioreactor Culture.

BACKGROUND: Muscle tissue engineering still remains a major challenge. An axial vascular pedicle and a perfusion bioreactor are necessary for the development and maintenance of a large-volume engineered muscle tissue to provide circulation within the construct. This study aimed to determine whether large-volume vascularized muscle-like constructs could be made from rat groin adipose tissue in a perfusion bioreactor. METHODS: Epigastric adipofascial flaps based on the inferior superficial epigastric vessels were elevated bilaterally in male Lewis rats and connected to the bioreactor. The system was run using a cable pump and filled with myogenic differentiation medium in the perfusion bioreactor for 1, 3, 5, or 7 weeks. The resulting tissue constructs were characterized with respect to the morphology and muscle-related expression of genes and proteins. RESULTS: The histological examination demonstrated intact muscle-like tissue fibers; myogenesis was verified by the expression of myosin, MADS box transcription enhancer factor 2 D, desmin-a disintegrin and metalloproteinase domain (ADAM) 12-and M-cadherin using reverse transcription-polymerase chain reaction. Western blot analysis for desmin, MyoD1, N-cadherin, and ADAM12 was performed to verify the myogenic phenotype of the extracted differentiated tissue and prove the formation of muscle-like constructs. CONCLUSIONS: A large-volume vascularized muscle tissue could be engineered in a perfusion bioreactor. The resulting tissue had muscle-like histological features and expressed muscle-related genes and proteins, indicating that the trans-differentiation of adipose tissue into muscle tissue occurred.

LEF-1 regulates proliferation and MMP-7 transcription in breast cancer cells.

Matrix metalloproteinase-7 (MMP-7) is a small secreted proteolytic enzyme with broad substrate specificity. Its expression is associated with tumor invasion, metastasis, and survival in a variety of cancers including breast cancer. Using bioinformatics analysis, a conserved LEF-1 binding site became obvious that is mapped at the promoter region of the genomic MMP-7 locus. Consequently, electrophoretic mobility shift assay demonstrated in vitro binding of LEF-1 to the predicted MMP-7 promoter binding site. Here, we demonstrate that lymphoid enhancer binding factor-1 (LEF-1) is associated with regulation of the proliferation-associated cyclin D1 and a gene encoding MMP-7 in breast cancer cells. Thus, a decrease of LEF-1 expression using LEF-1 siRNA resulted in down-regulation of cyclin D and MMP-7 expression, respectively. Moreover, cell cycle analysis of LEF-1 siRNA-transfected human breast cancer cells revealed a significant arrest in G2/M phase. Taken together, our results indicate a pivotal role of LEF-1 in the regulation of proliferation and MMP-7 transcription in breast cancer cells.

Validation of differential gene expression in muscle engineered from rat groin adipose tissue by quantitative real-time PCR.

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a highly sensitive tool that can be used for accurate and reliable gene expression analysis; however, a critical factor for creating reliable data in relative quantification is the normalization of the expression data of the genes of interest. In this study, we demonstrate the important process of validating four muscle-specific genes (myosin, desmin, MEF2D and ADAM12) and 10 common potential reference genes (ß-2-microglobulin, RPL32, RPL17, α-tubulin, CYC, ET1A, ß-actin, HSPCB, SDHA and GAPDH) in engineered muscle tissues. Tissue samples were generated out of rat groin adipose tissues by myogenic induction in a perfusion bioreactor for 7, 21 and 49 days. Results of analyzed muscle-specific genes suggested that the gene expression pattern corresponding to myogenic induction observed in adequately treated rat adipose tissue was time-dependent, making the length of time in culture in myogenic medium an important factor. Our data suggest that the reference genes were expressed variably in the different samples. During engineered muscle development, ß-2-microglobulin, RPL32 and RPL17 were the most stably expressed genes. The commonly used reference genes ß-actin and GAPDH appeared to be too unstable for normalization of qRT-PCR expression in engineered muscle tissue. The use of ß-2-microglobulin, RPL32 and RPL17 as internal standards may improve the accuracy of gene expression studies aimed at muscle tissue engineering under the proposed settings.

An Identification and Characterization of the Axolotl (Ambystoma mexicanum, Amex) Telomerase Reverse Transcriptase (Amex TERT).

The Mexican axolotl is one of the few vertebrates that is able to replace its lost body parts during lifespan. Due to its remarkable regenerative abilities, the axolotl emerged as a model organism especially for limb regeneration. Telomeres and the telomerase enzyme are crucial for regeneration and protection against aging processes and degenerating diseases. Despite its relevance for regeneration, the axolotl telomerase and telomere length have not yet been investigated. Therefore, in the present paper, we reveal the sequence of the axolotl telomerase reverse transcriptase gene (Tert) and protein (TERT). Multiple sequence alignment (MSA) showed the known conserved RT- and TERT-specific motifs and residues found in other TERTs. In addition, we establish methods to determine the Tert expression (RT-PCR) and telomerase activity (Q-TRAP) of adult axolotl and blastema tissues. We found that both differentiated forelimb tissue and regenerating blastema tissue express Tert and show telomerase activity. Furthermore, blastema tissue appears to exhibit a higher Tert expression and telomerase activity. The presence of active telomerase in adult somatic cells is a decisive difference to somatic cells of non-regenerating vertebrates, such as humans. These findings indicate that telomere biology may play a key role in the regenerative abilities of cells.

Influence of glucose and insulin in human adipogenic differentiation models with adipose-derived stem cells.

Autologous fat grafting represents an attractive source for tissue engineering applications in the field of reconstructive medicine. However, in adipogenic differentiation protocols for human adipose-derived stem cells, the concentration of glucose and insulin varies considerably. With the intent to gain maximum tissue augmentation, we focused on the late phase of adipogenesis. In this study, we modified the differentiation protocol for adipose-derived stem cells by prolongation of the induction period and the application highly concentrated glucose and insulin. Human adipose-derived stem cells were isolated from subcutaneous depots and differentiated in a standard induction medium for the first two weeks, followed by two weeks with varying glucose and insulin concentrations. Morphological changes assessed using Oil-Red-O staining were examined for corresponding alterations in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL). Furthermore, glucose and lactate levels in conditioned media were monitored over the period of differentiation. We found high-glucose media increasing the level of lipid accumulation and the size of single droplets whereas insulin significantly showed a dose-dependent negative effect on fat storage. However, whereas high glucose stimulated PPARγ transcription, expression levels in insulin-treated cells remained constant. Results permit assumptions that a high-glucose medium intensifies the degree of differentiation in mature adipocytes providing conditions to promote graft volume while we have identified highly concentrated insulin treatment as an inhibitor of lipid storage in the late adipogenic differentiation.

Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained.

Role of lifeguard ß-isoform in the development of breast cancer.

In the last century there has been great progress in the treatment of breast cancer by improving drug and radiation therapy as well as surgical techniques. Despite this development, breast cancer remains a major cause of death among women in Europe and the US. The cause of breast cancer at the cellular level is still not fully understood. In the present study, we investigated the expression of the Lifeguard ß-isoform in breast cancer tissues. In contrast to Lifeguard, the ß­isoform has one transmembrane domain less, which is the last of seven (99 bp), and due to this we suspect that the Lifeguard ß-isoform exhibits a different function. We determined the expression and function of the ß-isoform of Lifeguard in breast cancer cell lines (MCF-7 and MDA-MB-231), a human breast epithelial cell line (MCF10A) and in breast tumour tissue sections. Western blotting, PCR arrays and immunofluorescence were used to investigate the expression of Lifeguard and its ß-isoform. Moreover, we investigated the ability of Lifeguard ß-isoform expression to inhibit apoptosis induced by Fas. Our results indicated that Lifeguard ß-isoform is strongly expressed in breast tumour tissues. More notably, we demonstrated that Fas sensitivity was reduced in the MCF10A breast cells expressing the Lifeguard ß-isoform. Taken together, our findings indicate the role of the Lifeguard ß-isoform as an anti­apoptotic protein and provide further evidence of the potential of the Lifeguard ß-isoform as a target for the development of novel therapeutic strategies.

Murine embryonic fibroblast cell lines differentiate into three mesenchymal lineages to different extents: new models to investigate differentiation processes.

Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as "feeder cells." Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation.

Observed changes in the morphology and phenotype of breast cancer cells in direct co-culture with adipose-derived stem cells.

BACKGROUND: Regarding aesthetics and long-term stability, cell-assisted lipotransfer is a promising method for breast reconstruction. Here, autologous fat grafts enriched with autologous adipose-derived stem cells are transferred. However, as adipose-derived stem cells secrete high amounts of growth factors, potential risks of tumor reactivation remain. In this study, influences of adipose-derived stem cells on inflammatory breast cancer cells were evaluated in a direct co-culture system. METHODS: Human adipose-derived stem cells were isolated and cultivated either alone or in a direct co-culture with the inflammatory breast carcinoma cell line T47D. At different time points, cell morphology was observed by scanning electron microscopy, cell membranes were stained by immunofluorescence, and gene expression was analyzed by real-time polymerase chain reaction. RESULTS: In co-cultures, T47D breast carcinoma cells showed tumorsphere-typical growth surrounded by a monolayer of adipose-derived stem cells. Direct cell-to-cell contacts could be observed between the two different cell types. Immunofluorescence revealed vesicular exchange and fusion between carcinoma cells and adipose-derived stem cells. Expression levels of transcriptional genes for typical malignancy markers were substantially higher in co-cultures compared with single cultures. CONCLUSIONS: Direct intercellular contact between carcinoma cells and adipose-derived stem cells by means of exosomal vesicular exchange was revealed. Breast cancer cells displayed a change towards a more malignant phenotype associated with higher rates of metastasis and worsened prognosis. As cell-assisted lipotransfer is often performed after breast cancer surgery, transfer of adipose-derived stem cells might lead to deterioration of prognosis in case of recurrence as it has been described for inflammatory breast cancer.

Silencing of anti-apoptotic transmembrane protein lifeguard sensitizes solid tumor cell lines MCF-7 and SW872 to perifosine-induced cell death activation.

Lifeguard (LFG), an anti-apoptotic protein with high expression rates in breast cancer cells, has been identified as a molecule that inhibits death mediated by Fas. The molecular function of LFG and its regulation in the carcinogenesis of human breast and sarcoma cells, however, remains to be elucidated. In the present study, we investigated the ability of LFG expression to inhibit apoptosis induced by the alkyl-phospholipid perifosine. Results showed that LFG was able to be downregulated in selected sarcoma and breast cancer cell lines characterized by high endogenous LFG expression. A decreased LFG expression led to enhanced sensitivity to treatment with an agonistic Fas antibody or treatment with perifosine. Taken together, our findings indicate the role of LFG as an anti-apoptotic protein and provide further evidence of the potential of LFG as a target for the development of novel therapeutic strategies.