Daniela Grimm

Searching the literature for proteins facilitates the identification of biological processes, if advanced methods of analysis are linked: a case study on microgravity-caused changes in cells

ABSTRACT Background: More than one hundred reports were published about the characterization of cells from malignant and healthy tissues, as well as of endothelial cells and stem cells exposed to microgravity conditions. Methods: We retrieved publications about microgravity related studies on each type of cells, extracted the proteins mentioned therein and analyzed them aiming to identify biological processes affected by microgravity culture conditions. Results: The analysis revealed 66 different biological processes, 19 of them were always detected when papers about the four types of cells were analyzed. Conclusion: Since a response to the removal of gravity is common to the different cell types, some of the 19 biological processes could play a role in cellular adaption to microgravity. Applying computer programs, to extract and analyze proteins and genes mentioned in publications becomes essential for scientists interested to get an overview of the rapidly growing fields of gravitational biology and space medicine.

Changes in Gene Expression, Protein Content and Morphology of Chondrocytes Cultured on a 3D Random Position Machine and 2D Rotating Clinostat

H cartilage is a critical tissue to normal articular function. Natural restoring of this tissue in patients with rheumatoid arthritis, osteoarthritis or traumas is extremely limited considering its low regenerative capacity. These conditions cause an enormous constraint to the daily routine and, consequently, lower quality of life and they are managed, according with severity, through the use of pharmaceutics, surgery, transplant or prosthetic procedures. Tissue Engineering (TE) emerges as a new promising tool to provide long lasting regenerating solutions. In this work we used the combination of 3D extruded-based scaffolds made of poly (e-caprolactone) (PCL) and human bone marrow-derived mesenchymal stem cells (MSC) to provide initial higher cell densities, followed by differentiation in chondrocytes. The aim of this study is to understand the influence in cell behavior of scaffolds manufactured by layer-by-layer extrusion with a pore size (190 – 390 μm) and fiber alignments (045o and 0-90o), as well as of two different atmospheric conditions, normoxia (21% O2) and hypoxia (5% O2). Results obtained showed that pore size and fiber alignment do not pose a limitation for cell adhesion and proliferation within the range of pore sizes studied. However, cell distribution and dimension of chondrocyte aggregates strongly depend on fiber alignment. The results obtained in this study point out that higher chondrocyte population is obtained, when previously to differentiation stage, MSC are expanded in the PCL under hypoxia condition, rather than normoxia.O group at the Translational Centre for Regenerative Medicine in Leipzig developed a method for melanocyte cultivation from the Outer Root Sheath(ORS) by expanding stem cells, precursors and dedicated cells and differentiating them into melanocytes. Based on this methodology, we are developing an autologous, transplantation-based, causal treatment of depigmentation disorders such as Vitiligo. In the scope of cultivation, we are tracking shifts in gene expression profiles, from pluripotency in undifferentiated cells to melanotic status in late differentiation stages of the ORS melanocyte culture.Introduction: Spermidine is a naturally occurring polyamine involved in multiple biological processes, including DNA metabolism, autophagy and aging. Spermidine induces autophagy in cultured yeast and mammalian cells, as well as in nematodes and flies. Genetic inactivation of genes essential for autophagy abolishes the life span-prolonging effect of spermidine in yeast, nematodes and flies. These findings complement expanding evidence that autophagy mediates cytoprotection against a variety of noxious agents and can confer longevity when induced at the whole-organism level. In an acute burn injury the zone of stasis is initially vital but may progress to coagulation necrosis with time. In this experimental study, we hypothesized that apoptosis, autophagy induced by spermidine plays a role in burn wound progression.M clinical trials are underway to evaluate the use of adipose tissue-derived stromal cells (hASC) for regenerative medicine. Nevertheless, prior to its clinical application, hASC need ex vivo expansion in compliance with GMP guidelines in order to obtain a suitable amount of cells. Fetal bovine serum (FBS) is the most widely used serum in standard culture conditions, but the use of animal origin components bears disadvantages and is not an option in clinical cell therapy due to several safety issues. So our aim was to show the effectiveness of pooled allogeneic human serum (aHS) as a supplement for hASC culturing. In this study, the hASC were expanded in DMEM supplemented with aHS and the immunophenotype and differentiation potential of the cells were evaluated. The first difference observed between FBS and aHS cultures was that hASC in aHS proliferated markedly faster than in FBS, which was confirmed by Ki-67 expression, the cumulative population doubling, and by a growth curve. To better understand this hASC expansion pattern, the transcription factors C-myc and C-fos as well as mRNA levels of hTERT, and pluripotency genes (OCT-4 and NANOG) were assessed. No significant expression was observed and the hASC cultured in aHS underwent replicative senescence with normal karyotype. These cells assayed were not able to generate a teratoma. Therefore, this study confirms and extends the benefits of rapid expansion of hASC in culture media supplemented with aHS, with the cells maintaining their phenotype and with no spontaneous cell transformation.A is characterized by an impairment of muscle regenerative potential and a progressive loss of skeletal muscle. This condition, called sarcopenia, has important health care implications for humans, as it contributes to frailty, functional loss and premature death. The ability of skeletal muscle to regenerate is owed to a population of myogenic stem cells called satellite cells (SCs). In previous papers of our research group we demonstrated an age-related decrease in the antioxidant capacity of human SCs that may negatively affect the ageing SCs ability to repair muscle. Despite the involvement of caspases in muscle wasting, the real role of these enzymes is still controversial. To test the possibility of caspases involvement in SCs death in human ageing muscles, we cultured in vitro primary cells derived from vastus lateralis of young and aged subjects. We analysed both initiator caspases and effector caspases in the presence or absence of specific or broad pharmacologic inhibitors. Furthermore, we evaluated the expression of various genes that play a critical role in oxidative stress and cell death. Our findings highlighted an increased rate of spontaneous apoptosis and an up-regulation of CASP2, 6, 7, 9 and other cell death genes in aged SCs, supporting the hypothesis of an intrinsic ageing of SCs and previous reports demonstrating an increased susceptibility to apoptosis of SCs from old animals. These results suggest that a greater proportion of SCs from old subjects might undergo programmed cell death in vivo in response to stressful stimuli, thereby impairing skeletal muscle regeneration.P migration of stem cells, either endogenous or transplanted, is crucially important for embryonic development, homeostasis in adults, and tissue repair after injury. However, the detailed mechanisms of the directed migration of these cells are not clear. During the past few years, our study showed that the differentiating mesenchymal stem cells (MSCs) possess different migratory capacity and the chemotactic responses of these cells correlates closely with their differentiation states. Accordingly, the formation and the asymmetrical distribution of focal adhesions (FAs) between the leading lamella and the cell rear, the phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as the turnover of FAs varied greatly in differentiating MSCs, leading to the most effective chemotactic responses of MSCs in certain differentiation states. Further, we demonstrated that signaling through PI3K/Akt and MAPKs are involved in regulating the directed migration of MSCs. More importantly, we found that beta-catenin signaling is prerequisite for the chemotactic migration of MSCs. In this talk, I will summarize our data regarding the regulatory effects of beta-catenin signaling on MSCs that undergo chemotaxis.Chondrocytes are the only cell type found in human cartilage consisting of proteoglycans and type II collagen. Several studies on chondrocytes cultured either in Space or on a ground-based facility for simulation of microgravity revealed that these cells are very resistant to adverse effects and stress induced by altered gravity. Tissue engineering of chondrocytes is a new strategy for cartilage regeneration. Using a three-dimensional Random Positioning Machine and a 2D rotating clinostat, devices designed to simulate microgravity on Earth, we investigated the early effects of microgravity exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis; and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-1 was enhanced for 24 h. Based on the results achieved, we suggest that chondrocytes exposed to simulated microgravity seem to change their extracellular matrix production behavior while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.T is a pressing global medical need for the development of tissue-engineered alternatives to supplement the scarce supply of human donor corneal tissue for transplantation and vision restorative therapy. There are currently over 10 million people blind due to corneal scarring or disease, with 1-2 million new cases of corneal blindness arising annually. To meet this enormous global demand, our research has focused on developing transparent, tissue-engineered collagen-based hydrogels for implantation in the cornea. In preclinical studies, designs have evolved with respect to collagen composition and use of various synthetic cross-linking agents. Utilizing advanced in vivo imaging techniques such as optical coherence tomography and in vivo confocal microscopy, we have developed methods to visualize and longitudinally quantify bio-integration, wound healing, and cellular and neural compatibility of the biomaterial implants in rabbit and pig models, in real time. The implantable biomaterials and imaging techniques have also been extended into the clinic, where 10 patients received the first tissueengineered corneal substitutes, and were followed and documented extensively for over six years postoperatively. In more recent work, our clinical experiences have resulted in refined designs of biomaterials and surgical implantation techniques, with a focus on robustness of materials in the operating room and optimization of the degradation profile of biomaterials in vivo, to facilitate host cell invasion and regeneration of tissue. Considerations of cost, raw material availability, and safety for human use are also becoming increasingly important as future efforts are aiming towards large-scale production of tissueengineered corneas to meet an increasing global demand.