The aggregate nature of human mesenchymal stromal cells in native bone marrow

Background aims. The clinical application of mesenchymal stromal cells (MSC) faces several obstacles, such as the lack of a standard method for direct isolation as well as a low frequency and concern about the safety of their in vitro expansion. Although the density-gradient separation technique is used as the first step in most methods of MSC isolation to enrich mononuclear cells, the efficiency of this method has not so far been examined. This study was designed to address this issue. Methods. Human bone marrow (BM) samples were laid over Ficoll-Paque, and after centrifugation the upper and lower fractions were cultured separately. Surface markers, differentiation potential and the number of emerged cells were determined. Results. The isolated cells from both the upper and lower fractions were characteristic of MSC. Although it is commonly believed that MSC are single suspending mononuclear cells and so are enriched in the upper fraction of Ficoll-Paque after density-gradient separation, our data showed that considerable numbers of these cells were accumulated in the lower fraction. Further data indicated that MSC were actually present as cell aggregates in BM and they could be enriched effectively by a simple filtration method. Conclusions. The aggregate nature of MSC in BM is in agreement with the concept that they are one of the main elements of the hematopoietic stem cell niche. In addition, the simple filtration method proposed here to isolate cell aggregates may provide opportunities for instant stem cell therapy without the need for extensive in vitro expansion.     

Proteomic techniques for characterisation of mesenchymal stem cell secretome

Mesenchymal stem cells (MSCs) are multipotent cells with a substantial potential in human regenerative medicine due to their ability to migrate to sites of injury, capability to suppress immune response and accessibility in large amount from patient's own bone marrow or fat tissue. It has been increasingly observed that the transplanted MSCs did not necessarily engraft and differentiate at the site of injury but might exert their therapeutic effects through secreted trophic signals. The MSCs secrete a variety of autocrine/paracrine factors, called secretome, that support regenerative processes in the damaged tissue, induce angiogenesis, protect cells from apoptotic cell death and modulate immune system. The cell culture medium conditioned by MSCs or osteogenic, chondrogenic as well as adipogenic precursors derived from MSCs has become a subject of intensive proteomic profiling in the search for and identification of released factors and microvesicles that might be applicable in regenerative medicine. Jointly with the methods for MSC isolation, expansion and differentiation, proteomic analysis of MSC secretome was enabled recently mainly due to the extensive development in protein separation techniques, mass spectrometry, immunological methods and bioinformatics. This review describes proteomic techniques currently applied or prospectively applicable in MSC secretomics, with a particular focus on preparation of the secretome sample, protein/peptide separation, mass spectrometry and protein quantification techniques, analysis of posttranslational modifications, immunological techniques, isolation and characterisation of secreted vesicles and exosomes, analysis of cytokine-encoding mRNAs and bioinformatics.     

A Systematic Comparison of Supervised Classifiers

Pattern recognition has been employed in a myriad of industrial, commercial and academic applications. Many techniques have been devised to tackle such a diversity of applications. Despite the long tradition of pattern recognition research, there is no technique that yields the best classification in all scenarios. Therefore, as many techniques as possible should be considered in high accuracy applications. Typical related works either focus on the performance of a given algorithm or compare various classification methods. In many occasions, however, researchers who are not experts in the field of machine learning have to deal with practical classification tasks without an in-depth knowledge about the underlying parameters. Actually, the adequate choice of classifiers and parameters in such practical circumstances constitutes a long-standing problem and is one of the subjects of the current paper. We carried out a performance study of nine well-known classifiers implemented in the Weka framework and compared the influence of the parameter configurations on the accuracy. The default configuration of parameters in Weka was found to provide near optimal performance for most cases, not including methods such as the support vector machine (SVM). In addition, the k-nearest neighbor method frequently allowed the best accuracy. In certain conditions, it was possible to improve the quality of SVM by more than 20% with respect to their default parameter configuration.     

Flow field-flow fractionation: a pre-analytical method for proteomics

Proteome analysis requires a comprehensive approach including high-performance separation methods, mass spectrometric analysis, and bioinformatics. While recent advances in mass spectrometry (MS) have led to remarkable improvements in the ability to characterize complex mixtures of biomolecules in proteomics, a proper pre-MS separation step of proteins/peptides is still required. The need of high-performance separation and/or isolation/purification techniques of proteins is increasing, due to the importance of proteins expressed at extremely low levels in proteome samples. In this review, flow field-flow fractionation (F4) is introduced as a complementary pre-analytical separation method for protein separation/isolation, which can be effectively utilized for proteomic research. F4 is a set of elution-based techniques that are capable of separating macromolecules by differences in diffusion coefficient and, therefore, in hydrodynamic size. F4 provides protein separation without surface interaction of the analyte with packing or gel media. Separation is carried out in an open channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly-applied, secondary flow of the fluid. Therefore, biological analytes such as proteins can be kept under a bio-friendly environment without losing their original structural configuration. Moreover, proteins fractionated on a size/shape basis can be readily collected for further characterization or proteomic analysis by MS using, for instance, either on-line or off-line methods based on electrospray ionization (ESI) or matrix-assisted laser desorption-ionization (MALDI). This review focuses on the advantages of F4 compared to most-assessed separation/isolation techniques for proteomics, and on selected applications based on size-dependent proteome separation. New method developments based on the hyphenation of F4 with on-line or off-line MS, and with other separation methods such as capillary isoelectric focusing (CIEF) are also described.     

Total cell pooling in vitro: an effective isolation method for bone marrow-derived multipotent stromal cells

In vitro cellular proliferation and the ability to undergo multilineage differentiation make bone marrow-derived multipotent stromal cells (MSCs) potentially useful for clinical applications. Several methods have been described to isolate a homogenous bone marrow-derived MSCs population; however, none has been proven most effective, mainly due to their effects on proliferation and differentiation capability of the isolated cells. It is hypothesized that our newly established total cell pooling method may provide a better alternative as compared to the standard isolation method (density gradient centrifugation method). For the total cell pooling method, MSCs were isolated from rabbit bone marrow and were subsequently cultured in the growth medium without further separation as in the standard isolation method. The total cell pooling method was 65 min faster than the standard isolation method in completing cell isolation. Nevertheless, both methods did not differ significantly in the number of primary viable cells and population doubling time in the cultures (p?>?0.05). The isolated cells from both methods expressed CD29 and CD44 markers, but not CD45 markers. Furthermore, they displayed multilineage differentiation characteristics of chondroblasts, osteoblasts, and adipocytes. In conclusion, both methods provide similar efficiency in the isolation of rabbit bone marrow-derived MSCs; however, the total cell pooling method is technically simpler and more cost effective than the standard isolation method.     

肌源干细胞及其应用研究进展

研究证实,肌肉中存在2种类型的干细胞,即肌卫星细胞和多能干细胞,后者又可分为不同的细胞亚群。这些细胞群表现出自我更新能力和多潜能性,可分化为血细胞、成骨细胞、神经细胞等不同胚层的组织细胞。由于肌源干细胞具有治疗包括骨骼肌和心肌在内的肌肉疾病的可能性,并且潜在地可促进其他组织,如骨和软骨的再生和愈合,因此近几年来的相关研究取得很大进展。     

鸟类组织稳定同位素分析样品的预处理方法

稳定同位素分析样品在进行同位素质谱分析之前需要适当的处理,然而如何处理尚未形成统一的标准。本文结合鸟类组织稳定同位素分析样品的测试流程,介绍了鸟类学研究领域稳定同位素技术应用中涉及的分析样品解冻清洗、干燥、分离纯化、研磨和储存等前处理方法,并对不同组织常用的处理方法及分析样品测定中有待解决的问题展开了讨论。旨在抛砖引玉,为利用稳定同位素技术开展鸟类学方面的研究提供科学参考和技术支持。     

Separation technologies for stem cell bioprocessing

Stem cells have been the focus of an intense research due to their potential in Regenerative Medicine, drug discovery, toxicology studies, as well as for fundamental studies on developmental biology and human disease mechanisms. To fully accomplish this potential, the successful application of separation processes for the isolation and purification of stem cells and stem cell‐derived cells is a crucial issue. Although separation methods have been used over the past decades for the isolation and enrichment of hematopoietic stem/progenitor cells for transplantation in hemato‐oncological settings, recent achievements in the stem cell field have created new challenges including the need for novel scalable separation processes with a higher resolution and more cost‐effective. Important examples are the need for high‐resolution methods for the separation of heterogeneous populations of multipotent adult stem cells to study their differential biological features and clinical utility, as well as for the depletion of tumorigenic cells after pluripotent stem cell differentiation. Focusing on these challenges, this review presents a critical assessment of separation processes that have been used in the stem cell field, as well as their current and potential applications. The techniques are grouped according to the fundamental principles that govern cell separation, which are defined by the main physical, biophysical, and affinity properties of cells. A special emphasis is given to novel and promising approaches such as affinity‐based methods that take advantage of the use of new ligands (e.g., aptamers, lectins), as well as to novel biophysical‐based methods requiring no cell labeling and integrated with microscale technologies. Biotechnol. Bioeng. 2012; 109: 2699–2709. © 2012 Wiley Periodicals, Inc.     

Thiol redox proteomics seen with fluorescent eyes: the detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE

There is increasing evidence that several reversible oxidative post-translational modifications of protein cysteines participate in cell signalling. Specific proteomic techniques are required to identify these modifications and to study their regulation in different cell processes, that are collectively known as thiol redox proteomics. Recently, fluorescence derivatization methods have been developed that enable these post-translational modifications to be studied using proteomic workflows based on two-dimensional electrophoresis, which is a relatively accessible and affordable technique. As well as enabling a large number of samples to be processed, two-dimensional electrophoresis has the advantage that it does not rely on the intensive use of mass spectrometers. This methodology allows to "visualise" redox changes in a broad context and, although identification of the modified residues is not so straightforward, complementary derivatization can overcome this drawback. Here we review the different derivatization strategies that have been employed in these studies, comparing their advantages and potential limitations. We also review the applications and results obtained, with particular emphasis on those involving (patho)physiological stimuli, thereby showing the potential of these techniques to study the thiol redox proteome.     

Applications of field-flow fractionation in proteomics: presence and future

Field-flow fractionation (FFF) represents a group of elution separation methods where external force fields act perpendicularly on analytes in a carrier liquid flows with nonuniform velocity profiles. It is an elution separation method that enables to separate analytes in relatively short times and collect fractions for further characterization or for investigation of their properties. Other advantages of FFF are small consumption of samples and gentle experimental conditions. These make FFF uniquely qualified for separation and purification of biological samples. The most promising are applications of different variants of flow FFF utilizing a cross flow through membrane channel walls to separate proteins. The separation is based on differences in protein diffusion coefficients, which allows calculating the size of macromolecules. Other FFF techniques (e.g., electrical, isoelectric, and sedimentation FFF) were also used for separation of biomolecules. FFF appears to be not only promising rapid technique for protein separation but it offers some other advantages in sample preparation, especially, focusing (hyperlayer) FFF techniques that enable preparation of homogeneous fractions of cells. Selected applications of FFF to protein analysis are described and future trends in application of FFF to proteomics are discussed.     

A Difficult Time with the Permit Process

In the 1970s, new forms of public scrutiny were applied to the research methods of field biologists in the United States, particularly those studying endangered species and marine mammals. This paper shows how such scrutiny affected researchers’ choice of research methods through an analysis of a key moment in a decade-long controversy over the conservation of bowhead whales. In 1978, researchers at the Naval Arctic Research Laboratory received funding from the Bureau of Land Management to radio-tag bowhead whales. Although this promising but still largely untested technique might have answered one of the central scientific questions in the controversy, it ultimately went unused. Technical considerations played a role in the decision not to use the technique, but the most important factor was scientists’ concerns about potential backlash from Iñupiat whalers and animal protectionists. The same forces that had made marine mammalogists more influential than ever and that had put into their hands the resources necessary to develop more effective research techniques also placed serious constraints on where, when, and how they could do their research.     

Placental-derived stem cells: Culture,differentiation and challenges

Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice.     

Analysis of Brassinosteroids in Plants

Brassinosteroids (BRs) are the polyhydroxylated plant hormones sharing a common resemblance with animal steroids. They are active even at very low concentrations and are implicated for their pleiotropic involvement in diverse physiological processes and defense strategies during stress in plants. These compounds are well apparent in the plant kingdom with higher amounts in juvenile tissues. A total of 62 steroidal compounds have been identified so far. Keeping their significance in mind, researchers not only have worked extensively on their isolation, but also they were synthesizing their synthetic isomers. Different analytical techniques like HPLC, GC-MS, LC-MS/MS, UPLC-MS/MS, and bioassay-based methods have been used for their isolation, detection, and characterization from composite plant materials. Therefore, this review provides comprehensive information to the readers intending to isolate and characterize BRs, using either laborious techniques or modern-day more efficient methods.     

Rapid chiral separation methods development by cyclodextrin-mediated capillary electrophoresis for acidic and basic compounds

Chiral separation methods development using conventional techniques such as GC or HPLC requires a lot of experience, effort, and expense, due to the wide diversity of the optically active solutes and their possible chiral selectors. Capillary electrophoresis has received increased attention as an alternative technique for chiral separation due to its inherent high efficiencies and ease of methods development. However, due to the wide variety of chiral selectors available in CE, the benefits of this technique might be diminished without an appropriate methods development scheme. In this paper detailed examples are shown for fast, efficient, and predictable chiral capillary electrophoresis separation methods development based on a new and systematic theory. Optimized separations and their parameters are presented for several enantiomeric acids and bases. All the three possible cases, such as the use of low and high pH, as well as pH = pK buffer systems are thoroughly discussed. © 1995 Wiley-Liss, Inc.     

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.     

Tag–probe labeling methods for live-cell imaging of membrane proteins

Instead of using reconstituted proteoliposomes, in situ investigations of membrane proteins in living cell membranes are important because the heterogeneous and dynamic nature of biomembranes significantly affects their behavior. Protein-specific labeling is a key technique for the detection of a target protein by fluorescence measurements, particularly fluorescence microscopy. However, conventional genetic fusion with fluorescent proteins has several shortcomings. Post-translational labeling methods using a genetically encodable tag and synthetic probes targeting to the tag can overcome these limitations. This review summarizes emerging tag–probe techniques for labeling specific membrane proteins and their applications, including endocytotic internalization, partitioning to specific membrane domains, interprotein interactions, and conformational changes.     

New techniques for isolation of single prokaryotic cells

Since the 1960s, several new attempts have been made to improve the management of single prokaryotic cells using micromanipulator techniques. In order to facilitate the isolation of pure cultures we have recently developed an improved micromanipulation method for routine work. With the aid of this method single prokaryotic cells can be picked out of a mixed community under direct visual control. The isolated aerobic or anaerobic cells can be grown in pure culture or can be subjected to single cell PCR. Other powerful and completely new approaches are the applications of laser micromanipulation systems, such as optical tweezers or laser microdissection techniques. Of the latter two methods only optical tweezers have been successfully applied to cloning prokaryotic cells.     

Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by “horizontal” exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional EV features are usually studied in vitro. Several methods of EV isolation from cell culture medium are currently used; however, selection of a particular method may have a significant impact on obtained results. The choice of the optimal method is usually determined by the amount of starting biomaterial and the aims of the research. We have performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with 30% sucrose/D₂O “cushion,” precipitation with plant proteins and latex-based immunoaffinity capturing. EV isolated from several human glial cell lines by different approaches were compared in terms of the following parameters: size, concentration, EV morphology, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, total RNA, and several glioma-associated miRNAs. The applied methods included nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. Based on the obtained results, we have developed practical recommendations that may help researchers to make the best choice of the EV isolation method.     

Statistical methods suitable for the analysis of plant tissue culture data

Statistical analyses are an essential part of biological research. Statistical methods are available to biological researchers that range from very simple to extremely complex. Therefore, caution should be used when selecting a statistical method. When possible it is best to avoid complicated statistical procedures that are difficult to interpret and may hinder the researcher's ability to make treatment comparisons. Instead a method should be chosen that compliments a logical and practical treatment design. Statistics should be used as a tool to compare treatments of interest and should not dictate the treatments. Experimental designs should take into account the eventual analysis, otherwise one could conceive of a design that could not be analyzed or, when analyzed, would not answer the desired questions. Therefore, time should be spent before conducting an experiment to plan an experimental design and analysis that best compliments the treatment scheme and questions to be answered. The purpose of this paper is to present examples of experimental designs, means separation procedures, data transformations and presentation methods suitable for plant cell and tissue culture data.Abbreviations ANOVA analysis of variance - BA benzyladenine - CV coefficient of variation - DF degrees of freedom - IAA indole-3-acetic acid - IBA indole-3-butyric acid - LOF lack-of-fit - MSE mean square error - P-ITB phenyl indole-3-thiolobutyrate - S standard deviation - SE standard error of the mean - TDZ thidiazuron     

How to analyze binding,enzyme and uptake data: The simplest case,a single phase

The results of many biochemical, pharmacological and physiological experiments fit a rectangular hyperbola. Several methods for analysis of this curve have been developed but these techniques have not been readily available to researchers who are not interested in becoming mathematical statisticians. We have reviewed these analytical methods and concluded that, the Eadie-Hofstee technique is the best choice. We explain how to use this method and include sample calculations and calculator programs. We are especially interested in showing scientists who are not particularly mathematically inclined how to use these methods effectively and thus save a great deal of time and expense.