BLT Stages

Abstract Bachelor Project FBT 2020-2021: Synthesis and characterization of extracellular matrix based hydrogels

When a skeletal muscle trauma is too extensive, satellite cells are no longer able to regenerate the tissue. A scaffold, more specifically an extracellular matrix (ECM) based hydrogel can be a solution for this. The benefit of a hydrogel is that it is liquid at room temperature and forms a gel at body temperature. In addition, a hydrogel can take on the irregular shape of the defect. In this investigation, the aim was to compare two decellularization protocols for the synthesis of an ECM-hydrogel. The first decellularization protocol was with sodium dodecyl sulfate (SDS) (Ungerleider et al.), the second with triton X-100 combined with DNase I and trypsin (Roosens et al., 2016). These protocols were evaluated with hematoxylin eosin staining, 4′,6-diamidino-2-phenylindole staining, alcian blue staining and via electron microscopy. In addition, a thorough biochemical analysis was performed via deoxyribonucleic acid (DNA) and glycosaminoglycan (GAG) determination. After this, a hydrogel was synthesized via enzymatic digestion with pepsin. The hydrogels were again histologically evaluated. In addition, myoblasts and fibroblasts were encapsulated in the ECM-hydrogels and evaluated with a live/dead staining, alamar blue assay and tropomyosin staining. After decellularization, less DNA was compered to native tissue. In addition, the GAG’s were significantly reduced at the decellularization of Roosens and cytoplasm was still present in the tissue. After the synthesis of a hydrogel, this was again confirmed by the alcian blue staining. The hydrogel of Roosens was homogenous, which was not the case for the hydrogel of Ungerleider. When cells were encapsulated in the hydrogels, the cells in the gel of Ungerleider died, which was not the case in the gel of Roosens. In conclusion, the decellularization protocols were not successful as too much DNA was still present. Cells were able to proliferate in the hydrogel of Roosens, while the hydrogel of Ungerleider appeared to be toxic. Also, the gel of Ungerleider was not homogenous. Further testing is needed to obtain an optimal hydrogel, namely by improving the decellularization and making the hydrogel more homogenous.

Abstract Bachelor Project FBT 2019-2020: Optimization of isolation, maintenance and phalloidin staining of skeletal muscle cell populations

Tissue engineering is a promising alternative to address the unmet medical need related to volumetric muscle loss. The ultimate aim is to use skeletal muscle constructs built in the lab instead of using an autologous muscle flap to repair these large muscle defects. A lot of research has already been done but clinical translation is still challenging. In the tissue engineering lab of Kulak, research is conducted to overcome these challenges. Most of the research thus far has been conducted with human cells to create a human bio-artificial skeletal muscle (BAM). However, to test the regenerative capacity of these BAMs in a murine model, the focus has been expanded to creating murine BAMs. And thus, myogenic stem cells, satellite cells, from mice need to be obtained to build a murine BAM. In this research, we searched for a working isolation protocol, medium and cultureware coating for obtaining a murine myogenic cell pool. These were scored based on the following parameters: i) cell yield, ii) cell viability, iii) myoblast percentage and iv) fusion capacity of the myoblasts. Three isolation protocols were compared of which one resulted in a low number of cells. As for the media tested, the mouse myoblast growth medium resulted overall in a better cell growth and maintenance compared to the C2C12 growth medium. Next, five different coatings (collagen, gelatin, laminin, poly-L-ornithin/laminin and poly-L-lysine) were compared. The laminin and poly-L-ornithin/laminin coating resulted in the highest adherence and cell yield compared to the other coatings. To further evaluate the obtained cell pool, cells were further subcultured and stained with a desmin antibody to determine the percentage of myoblasts in the cell pool, and a tropomyosin staining to evaluate the fusion capacity of the myoblasts to myotubes. Again, the laminin and poly-L-ornithin/laminin resulted in the highest number of myoblasts and fusion extent, which further strengthens the assumption that poly-L-orthinin/laminin and laminin coating are the best suited coatings to attain (murine) myoblast with sustained fusion capacity. In parallel to the optimization of the cell isolation protocols, a staining to further evaluate the cells when building a 3D construct was tested. For this a phalloidin actin staining, to stain the  cytoskeleton was tested using different brands of phalloidin. Both resulted in a good staining. In conclusion, with this work we were able to select a working isolation protocol, culture media and coating for mouse myoblast isolation and to stain murine cells with an actin staining. 

Abstract Bachelor Project FBT 2018-2019: Analysis of elastomeric scaffolds for skeletal muscle tissue engineering

Current therapies regarding volumetric muscle loss do not meet patients’ needs. As volumetric muscle loss can lead to functional defects, increased stiffness and reduced functional muscle contraction and relaxation, new approaches are warranted. This study aims to evaluate the applicability of porous elastomeric scaffolds in skeletal muscle tissue engineering. These polyester scaffolds include poly glycerol sebacate and poly xylitol sebacate whether or not crosslinked with citric acid, obtaining poly glycerol sebacate citrate and poly xylitol sebacate citrate respectively. Four different sterilization methods were assessed. Treatment with ethanol, peracetic acid, UV irradiation and lyophilization all have shown to be sufficient for microorganism elimination. Additionally, alternative pre-treatments were assessed regarding cell viability.

Results of a live/dead staining have shown that the scaffolds need an ethanol treatment or an incubation period in growth medium prior to cell seeding. Based on live/dead and immunocytochemistry assays, the building blocks that make up the elastomeric scaffolds (citric acid, sebacic acid, glycerol and xylitol) have proven to have no adverse effect on the survival and differentiation of the myoblasts. Visualization of the cells after a propidium iodide staining, demonstrated a minimum penetration extent of the myoblasts in the scaffolds up to a depth of approximately 500 µm. Moreover, myoblasts were distributed in a homogeneous manner and seemed to survive at this depth indicating the scaffolds have the ability to provide for a sufficient transport of nutrients, growth factors and waste products. Furthermore, results of an Alamar blue assay demonstrated no significant differences in metabolism rate of myoblast populations seeded on each scaffold (P < 0,05). In conclusion, myoblasts are able to penetrate, survive and proliferate when seeded on top of these scaffolds, making them highly promising for skeletal muscle tissue engineering applications. However, to make a selection in these scaffolds regarding their suitability as temporary extracellular matrix, further research is needed.

Abstract 2018-2019: muscle tissue gene expression comparison between human and mouse

Background

Countless studies use mice to perform experiments on for fundamental or applied research. This research plays an important role in solving issues that impact the overall well-being of humans. Therefore we have to understand which genes and pathways in Mus musculus are representable for Homo sapiens and perhaps more importantly, which ones are not. Cross-platform cross-species meta-analyses are powerful bioinformatic workflows to find similarities or differences across different studies that use gene expressions from micro arrays. This project analyses muscle tissue of Homo sapiens and Mus musculus in a postexercise context.

Method

R was used as the programming language to create the workflow. The genes that were annotated to the micro array expression values, were orthologues that were found via NCBI HomoloGene. Multiple meta-analyses for differential expression were tested, among which most notably; the REM and rOP methods (e.g. genes have to be differentially expressed in at least +-70% of all studies). For the meta-pathway analysis MAPE (Meta-Analysis Pathway Enrichment) software was used with rOP as the method to calculate the p-values of the genes and Fisher’s exact test for the Pathway Enrichment. Additionally, pathway clustering was performed to make pathway screening easier. Interesting pathways were visualized on gene-level to look for potential differences between species.

Results

Meta-analysis was done cross-platform cross-species (e.g. one study with mouse samples after 2 hours of exercise and 1 under the same conditions for human). Parameters were changed to let the rOP algorithm search for DEG’s1 within every individual study, but also across the species, so that both different and similar DEG’s would be used to search for pathways (this setup created the best pathway results). Pathway analysis resulted into logical pathways that were present in mouse and/or human combined with the fact that the FDR values and amount of relevant genes per pathway were adequate. However, the majority of the pathways found were as to be expected. No significant or new discoveries were found in the similarities between Homo sapiens and Mus musculus. Some of the pathways were visualised at gene-level at a certain time point in order to find differences between mouse and human, resulting in differently activated gene patterns or different subpathways.

Conclusions

More relevant pathways with more differentially expressed genes are found via a metaanalysis workflow compared to the conventional non-meta-method. Timepoints were compared, and even though the metabolism of mice is faster than that of humans, the timepoints that matched best were the same ones (e.g. samples after 2 hours match best in human and mouse). After pathway visualization, it’s clear that most pathways are activated in both organisms within similar timepoints, but are different on a gene-level (overexpression versus under expression or different subpathways within the pathway). 

Abstract traineeship advanced bachelor of bioinformatics 2017-2018:  Comparative transcriptomic analysis between species

During the internship, in the KuLeuven of Kortijk, several projects, that were given by the mentor, were handled by the intern. The first one was the processing and comparison of expression data between data of 2 articles and the data given by a laboratory in Leuven. For this several normalizations were tested before an actual PCA could be done. As for the visualization, there were also numerous packages that were tested to produce 3D plots and to see if they had the ability to export it as a vector (pdf or svg).

In the following assignment, the intern also had to find a way as to how they would visualize the differences between dendrograms created from the hierarchical clustering of species based on GC content, GC landscapes and the tree of life. For this the package dendextend was used to create tanglegrams, which puts two dendrograms next to each other and draws lines to the corresponding species. An entanglement factor was also calculated, this represent the degree of difference between the two dendrograms as a number.