Molecular genetic features of human mesenchymal stem cells after their osteogenic differentiation under the conditions of microgravity

Modified gravity is known to affect both the whole body and the molecular processes in individual cells. The purpose of the present study was to examine gene expression pattern in mesenchymal stromal cells (MSCs) of human bone marrow under the conditions of microgravity. Exposure of cell cultures on dRPM for 20 days resulted in significant changes in the expression of 144 genes. Thirty of these genes increased their expression, whereas the other 114 genes decreased their expression. Analysis of the genes revealed that most of them belonged to 11 principal groups according to their biological roles in the cell. Most genes with a changed expression were from the following groups: inflammatory responses and intercellular interactions, matrix and adhesion, metabolic processes, and signaling and regulation. We found that microgravity inhibited the expression of genes encoding the products, which play a key role in osteogenic differentiation including COL15A1, CXCL12, DPT, and WISP2 and are involved in intercellular interactions between MSCs and different types of bone marrow cells such as CXCL12 and SCG2. These molecular-genetic changes indicate the possible involvement of progenitor cells of osteogenic differon in the development of osteopeny under the conditions of microgravity.     

Antigen expression of cord blood derived stem cells under osteogenic stimulation in vitro

Tissue engineering strategies have become a promising option for treating musculoskeletal defects in future. Cord blood includes mesenchymal stem cells which are able to differentiate into several cell lines under lineage specific stimulation including osteoblasts, chondroblasts and adipoblasts. In this study the antigen pattern of cord blood stem cells cultivated onto a porous porcine collagen I/III scaffold is investigated. The cultures were stimulated with osteogenic mixture (dexamethasone, ascorbic acid, glycerolphosphatate [DAG]) over 21days in vitro. The following antigens and markers served for immuncytochemical evaluation: bone sialoprotein, osteocalcin, osteonectin, osteopontin, cartilage proteoglycan, chondrogenic oligomeric matrix protein, collagen I/II/III/X, CD13, CD 31, CD34, CD44, CD45, CD105, fibroblast growth factor receptor 2, vascular endothelial growth factor, vimentin and von-Kossa and HE stainings. We showed that a collagen I/III scaffold is an appropriate cellular carrier for cord blood progenitor cells and allows for an osteoblastic differentiation. Moreover there were differences in antigen pattern, dependent on the location of the adherent cells. CD105 and VEGF were only expressed at the bottom of the biomaterial. Future investigations should show the role of cytomechanical forces in the differentiation of cord blood derived progenitor cells and also if a cell-loaded collagen I/III scaffold is appropriate to stimulate bone regeneration in vivo.     

In vitro osteogenic differentiation of human ES cells

Since their isolation in 1998, human embryonic stem (hES) cells have been shown to be capable of adopting various cell fates in vitro. Here, we present in vitro data demonstrating the directed commitment of human embryonic stem cells to the osteogenic lineage. Human ES cells are shown to respond to factors that promote osteogenesis, leading to activation of the osteogenic markers osteocalcin, parathyroid hormone receptor, bone sialoprotein, osteopontin, cbfa1, and collagen 1. Moreover, the mineralized nodules obtained are composed of hydroxyapatite, further establishing the similarity of osteoblasts in culture to bone. These results show that osteoblasts can be derived from human ES cultures in vitro and provide the basis for comparison of adult and embryonic-derived osteogenesis, and for an investigation of potential applications for hES cells in orthopaedic tissue repair.     

c-fos expression precedes osteogenic differentiation of cartilage cells in vitro

We have investigated the temporal pattern of expression of c-fos in cartilage cells in mouse mandibular condyles. During in vitro cultivation, the progenitor cells in this organ differentiate to osteoblasts, and hypertrophic chondrocytes start to show features indicative of osteogenic differentiation. Prior to these processes we observed two distinct patterns of c-fos expression. High, transient c-fos expression was found in the entire tissue within 30 min of culture. This type of c-fos expression appeared to result from mechanical forces applied during dissection. The second type of c-fos expression appeared in individual cells in the zone of hypertrophic chondrocytes. A varying number of formerly quiescent chondrocytes expressed high levels of c-fos mRNA after between 30 min and 10 d in culture, with a peak in the number of cells between days 1 and 3. c-fos expression in these cartilage cells was followed by DNA replication and expression of genes typifying osteoblastic differentiation. After 7 d in culture, groups of cells with the typical ultrastructural features of osteoblasts, and surrounded by an osteoid-like matrix, were observed in single chondrocyte-type lacunae, suggesting division of chondrocytes and differentiation to osteoblasts. The data suggest that c-fos may play a crucial role in the perturbation of determined pathways of skeletoblast differentiation and in the regulation of endochondral bone formation.     

Cell interactions in microgravity: cytotoxic effects of natural killer cells in vitro

To study cell-to-cell interactions in microgravity we examined the functional activity of natural killer cells on board of the ISS. NK cells are the effector cells with direct cytotoxic activity to oncogenic, virus-infected cells and cells with modified differentiation. Ground-based experiments have shown that the examination of target cell lysis after incubation with NK cells is a simple and informative model for studying the influence of microgravity. NK cytotoxicity was measured as the value of non-degradeted labeled myeloblasts (K-562) after 24 hrs exposure with human lymphocytes in suspension. A special device was developed for space flight experiments. Human cultured lymphocytes and labeled K-562 cells were loaded into special syringes and delivered to the Russian segment of the ISS. Cosmonauts prepared co-cultured suspensions during the first day of microgravity, exposed them at 37 degrees C for 24 hrs and then separated H3-labeled cells on special filters. The results of ISS-8 mission showed that human NK cells in vitro remain lysis activity toward target cells in microgravity. The basal level of NK cytotoxicity was low and we did not found significant differences between "control" and "flight" values. Interferon production during the interaction between immune and target cells (ratio 10:1) in microgravity did not differ compared with ground-based control experiments. Ground exposure of the same lymphocyte samples with K-562 cells to 24 hrs clinorotation also did not lead to significant differences. These experiments paved the way for understanding the cell interaction mechanisms in space flight and the obtained results suggest that microgravity does not disrupt the interaction of NK cells with tumor cells.     

Interactions of cells in zones of bone resorption under microgravity and hypokinesia

With the use of the methods of electron microscopy and autoradiography employing 3H-glycine the study was made of some morpho-functional cells-cells interactions (osteoblasts, osteocytes, macrophages, fibroblasts) in zones of adaptive remodeling of bone structures of the metaepiphyseal femoral bones of white rats which were during 28 days under experimental hypokinesia conditions, as well as of rats, flown on SLS-2 during 2 weeks. It is established that in zones of an increase of mineral matrix resorption some osteoblasts and osteocytes undergo destruction; a part of osteoblasts remains intact. The osteoclasts don't take part in destruction of osteoblasts and osteocytes. The utilization of the osteogenic cells detritus is accomplished by macrophages, coming to these zones. The resorption loci are filled not with the differentiating osteoblastic cells, as it is the case in the norm, but with fibroblasts and the bundles of collagen fibrils (fibrotic tissue) which do not undergo mineralization. Such changes are considered as one of the mechanisms of bone tissue response to a reduction of the supporting load.     

Cytogenetic analysis of human bone marrow-derived mesenchymal stem cells passaged in vitro

This study concerns the cytogenetic stability of in vitro human bone marrow-derived mesenchymal stem cells (MSCs) in primary culture and after passaging. Bone marrow samples were collected from seven brain malfunction patients involved in autologous MSC transplantation trials. Chromosome preparations from primary MSC cultures and after 3 passages were analyzed by conventional staining and G-banding techniques. All MSCs showed normal diploid karyotypes, 46 XY or 46 XX, without aneuploidy or polyploidy; chromosome structural abnormalities were not detected. The results indicate that the in vitro cultured MSCs retained normal cytogenetics before being transplanted back into the patients.     

Donepezil hydrochloride as a novel inducer for osteogenic differentiation of mesenchymal stem cells on PLLA scaffolds in vitro

Over the past decades, bone defects caused by illness or trauma have been the most common traumatic injuries in humans and treatment of orthopedic infections has always been a serious challenge to experts in the world. In this project, poly L-lactic acid (PLLA) nanofibrous scaffolds were synthesized as a nontoxic, eco-friendly, and cost-effective scaffold by the electrospinning technique. Then, the impact of PLLA on the cell proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assayed in the presence and absence of donepezil hydrochloride (DH) which was prescribed in patients with Alzheimer's disease. Also, hMSCs were seeded on PLLA scaffold in the presence (PLLA-DH) and absence of 1 μg mL^-1 of DH under osteogenic induction media. Osteogenic differentiation of hMSCs was assessed by specific bone-related tests including alkaline phosphatase (ALP) activity, Alizarin red and von Kossa staining, calcium content assay. Also, Osteocalcin and osteopontin were evaluated as osteogenic proteins as well as ALP, osteonectin, osteocalcin, collagen type I (Col-I) and Runx2 as osteogenic genes via immunocytochemistry (ICC) and Real-time PCR analyses. The obtained data showed the higher ALP enzyme activity and biomineralization, more intensity during von Kossa staining as well as the increase in the expression rate of osteogenic related gene and protein markers in differentiated hMSCs on PLLA-DH. In conclusion, the present study revealed that the combination of PLLA scaffold with DH provides a scope to develop a suitable matrix in bone tissue engineering applications.     

BMP7 can promote osteogenic differentiation of human periosteal cells in vitro

To study and evaluate BMP7s functions in osteogenic differentiation of human periosteal cells in vitro. Human periosteal cells from adult tibia were collected and cultured as experimental samples. BMP7 was used to induce periosteal cells in the experiment group with common osteogenic medium. The proliferative activity of periosteal cells was detected by CCK-8. The potentials of osteogenic differentiation were demonstrated as follows: (1) realtime-PCR and ELISA to confirm the expression of the OC, ALP and OPN, (2) Colorimetry, ALP staining and Von Kossa staining were performed to identify ALP activity, ALP expression and calcium nodules, respectively. Based on the significant different expression of OC, ALP and OPN, BMP7 ability of osteogenic differentiation can be identified. ALP activity detection, calcium nodules staining and toluidine staining also provide the power evidence to support BMP7 can promote osteogenic differentiation of human periosteal cells in vitro. To human periosteal cells, BMP7 is a good inducer for osteogenic differentiation. Therefore, it's maybe a potential tool for clinical application.     

Redistribution of bodily fluids under conditions of microgravity and in microgravity models

This review is focused on the redistribution of blood and other bodily fluids along the body axis in the cranial direction under conditions of microgravity or during simulation of the physiological effects of microgravity. This redistribution of bodily fluids in the direction of the thorax or head results in respective physiological responses and induces a whole cascade of secondary adaptation mechanisms. Changes in central venous pressure, heart cavity volume, kidney functioning, and hormonal volume regulation lead to adaptive modifications in bodily fluid sectors. Modification of the hemodynamic in the splanchnic vascular system influences the organs of the abdominal cavity. Pharmacological correction accelerates the adaptation of the human body to unusual living conditions.     

Periodically discontinuous induction of bone marrow stem cells toward osteogenic differentiation in vitro

This paper reveals that a discontinuous in vitro induction, namely, the periodic presence and absence of foreign induction factors, might be, under a certain condition, more effective to stimulate stem cells' differentiation than a continuous induction. Bone marrow stem cells (BMSCs) derived from Sprague Dawley rats were employed to examine the effects of discontinuous additions of osteogenic supplements with a series of alternate frequency in contrast to those with continuous induction or no induction. The results demonstrated that a suitable discontinuous induction was more able to achieve osteogenesis than not only no induction but also the associated continuous induction. Additionally, the osteogenic supplements were confirmed to enhance cell differentiation but suppress cell proliferation. So, the combination of differentiation extent per cell and cell number accounts for the "unexpected" good osteogenic effect of the discontinuous induction. The induction effect was found to be dependent upon alternate frequency, and the optimum alternate period in our experimental systems was determined to be around 4 days. Since it is very common to change culture medium every 2-4 days, such a strategy of discontinuous induction does not bring any extra manual work but reduces the consumption of foreign induction factors and significantly enhances the global differentiation efficacy. Our work thus affords a convenient and practical approach to achieve differentiation of BMSCs, which might be useful for potential large-scale culture and differentiation of stem cells. Meanwhile, the existence of optimum frequency implies some unknown inherent rhythms of cell proliferation and differentiation.     

Experimental study of rat beta islet cells cultured under simulated microgravity conditions

To observe the effects of simulated microgravity on beta islet cell culture, we have compared the survival rates and the insulin levels of the isolated rat islet cells cultured at micro- and normal gravity conditions. The survival rates of the cells cultured were determined by acridine orange-propidium iodide double-staining on day 3, 7 and 14. The morphology of the cells was observed by electron microscopy. Insulin levels were measured by radio immuno assays. Our results show that the cell number cultured under the microgravity condition is significantly higher than that under the routine condition (P<0.01). Some tubular structure shown by transmission electron microscopy, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7. There were also abundant secretion particles and mitochondria in the cytoplasm of the cells. Scanning electron microscopy showed that there were holes formed between each islet, possibly connecting with the nutrient trans     

Combining predictors for classification using the area under the receiver operating characteristic curve

No single biomarker for cancer is considered adequately sensitive and specific for cancer screening. It is expected that the results of multiple markers will need to be combined in order to yield adequately accurate classification. Typically, the objective function that is optimized for combining markers is the likelihood function. In this article, we consider an alternative objective function-the area under the empirical receiver operating characteristic curve (AUC). We note that it yields consistent estimates of parameters in a generalized linear model for the risk score but does not require specifying the link function. Like logistic regression, it yields consistent estimation with case-control or cohort data. Simulation studies suggest that AUC-based classification scores have performance comparable with logistic likelihood-based scores when the logistic regression model holds. Analysis of data from a proteomics biomarker study shows that performance can be far superior to logistic regression derived scores when the logistic regression model does not hold. Model fitting by maximizing the AUC rather than the likelihood should be considered when the goal is to derive a marker combination score for classification or prediction.