The science and engineering of stem cell-derived organoids-examples from hepatic,biliary, and pancreatic tissues

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.     

Clinical diagnostics and therapy monitoring in the congenital disorders of glycosylation

Abnormal protein glycosylation is observed in many common disorders like cancer, inflammation, Alzheimer’s disease and diabetes. However, the actual use of this information in clinical diagnostics is still very limited. Information is usually derived from analysis of total serum N-glycan profiling methods, whereas the current use of glycoprotein biomarkers in the clinical setting is commonly based on protein levels. It can be envisioned that combining protein levels and their glycan isoforms would increase specificity for early diagnosis and therapy monitoring. To establish diagnostic assays, based on the mass spectrometric analysis of protein-specific glycosylation abnormalities, still many technical improvements have to be made. In addition, clinical validation is equally important as well as an understanding of the genetic and environmental factors that determine the protein-specific glycosylation abnormalities. Important lessons can be learned from the group of monogenic disorders in the glycosylation pathway, the Congenital Disorders of Glycosylation (CDG). Now that more and more genetic defects are being unraveled, we start to learn how genetic factors influence glycomics profiles of individual and total serum proteins. Although only in its initial stages, such studies suggest the importance to establish diagnostic assays for protein-specific glycosylation profiling, and the need to look beyond the single glycoprotein diagnostic test. Here, we review progress in and lessons from genetic disease, and review the increasing opportunities of mass spectrometry to analyze protein glycosylation in the clinical diagnostic setting. Furthermore, we will discuss the possibilities to expand current CDG diagnostics and how this can be used to approach glycoprotein biomarkers for more common diseases.     

Circulating tumor cell isolation,culture, and downstream molecular analysis

Circulating tumor cells (CTCs) are a major contributor of cancer metastases and hold a promising prognostic significance in cancer detection. Performing functional and molecular characterization of CTCs provides an in-depth knowledge about this lethal disease. Researchers are making efforts to design devices and develop assays for enumeration of CTCs with a high capture and detection efficiency from whole blood of cancer patients. The existing and on-going research on CTC isolation methods has revealed cell characteristics which are helpful in cancer monitoring and designing of targeted cancer treatments. In this review paper, a brief summary of existing CTC isolation methods is presented. We also discuss methods of detaching CTC from functionalized surfaces (functional assays/devices) and their further use for ex-vivo culturing that aid in studies regarding molecular properties that encourage metastatic seeding. In the clinical applications section, we discuss a number of cases that CTCs can play a key role for monitoring metastases, drug treatment response, and heterogeneity profiling regarding biomarkers and gene expression studies that bring treatment design further towards personalized medicine.     

New Insight into Neurodegeneration: the Role of Proteomics

Recent advances within the field of proteomics, including both upstream and downstream protocols, have fuelled a transition from simple protein identification to functional analysis. A battery of proteomics approaches is now being employed for the analysis of protein expression levels, the monitoring of cellular activities and for gaining an increased understanding into biochemical pathways. Combined, these approaches are changing the way we study disease by allowing accurate and targeted, large scale protein analysis, which will provide invaluable insight into disease pathogenesis. Neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), prion disease, and other diseases that affect the neuromuscular system, are a leading cause of disability in the aging population. There are no effective intervention strategies for these disorders and diagnosis is challenging as it relies primarily on clinical symptomatic features, which often overlap at early stages of disease. There is, therefore, an urgent need to develop reliable biomarkers to improve early and specific diagnosis, to track disease progression, to measure molecular responses towards treatment regimes and ultimately devise new therapeutic strategies. To accomplish this, a better understanding of disease mechanisms is needed. In this review we summarize recent advances in the field of proteomics applicable to neurodegenerative disorders, and how these advances are fueling our understanding, diagnosis, and treatment of these complex disorders.     

Quantitative Fusarium spp. and Microdochium spp. PCR assays to evaluate seed treatments for the control of Fusarium seedling blight of wheat

AIMS: To develop sensitive quantitative PCR assays for the two groups of pathogens responsible for Fusarium seedling blight in wheat: Fusarium group (Fusarium culmorum and Fusarium graminearum) and Microdochium group (Microdochium nivale and Microdochium majus); and to use the assays to assess performance of fungicide seed treatments against each group. METHODS AND RESULTS: Primers conserved between the species within each group were used to develop competitive PCR assays and used to quantify DNA of each group in wheat seed produced from inoculated field plots. Seed was used in seed treatment efficacy field experiments and the amount of DNA of each group was determined in emerged seedlings. The performance of treatments towards each group of pathogens was evaluated by comparison of the reduction in DNA in seedlings emerged from treated seed compared with untreated seed. CONCLUSIONS: DNA from the two groups of pathogens causing Fusarium seedling blight of wheat can be quantified separately using the competitive PCR assays. These assays show improved sensitivity compared with those previously reported for the individual species and allowed the quantification of pathogen DNA in seed and seedlings. Significant reductions in pathogen DNA were evident for each seed treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantification of DNA for each group allows the evaluation of seed treatment performance towards the two components of Fusarium seedling blight disease complex. The approach taken and the assays developed in this study will be of use for the study of other Fusarium disease complexes and their control. Based on the results reported here on the seedling stage of crop development, further studies that examine the control of seed-borne pathogens through fungicide seed treatments throughout the growing season are warranted.     

A Guide to Harmonisation and Standardisation of Measurands Determined by Liquid Chromatography – Tandem Mass Spectrometry in Routine Clinical Biochemistry

Globally, harmonisation in laboratory medicine is a significant project. The relatively new implementation of liquid chromatography coupled with tandem mass spectrometry (LC-MSMS) techniques as routine assays in diagnostic laboratories provides the unique opportunity to harmonise, and in many cases standardise, methods from an early stage. This guide aims to provide a practical overview of the steps required to achieve agreement between LC-MSMS analytical procedures for routine clinical biochemistry diagnostic assays, with particular focus on the harmonisation and standardisation of methods currently implemented.To achieve harmonisation, and where practical standardisation, the approach is more efficient if divided into sequential stages. The suggested division entails: (i) planning and preliminary work; (ii) initial assessment of performance; (iii) standardisation and harmonisation initiative; (iv) establishing common reference intervals and critical limits; (v) developing best practice guidelines; and (vi) performing an ongoing review.The profession has a unique and significant opportunity to bring clinical mass spectrometry-based assays into agreement. Harmonisation of assays should ultimately provide the same result and interpretation for a given patient’s sample, irrespective of the laboratory that produced the result. To achieve this goal, we need to agree on the best practice LC-MSMS methods for use in routine clinical measurement.     

Social dimensions of preimplantation genetic diagnosis: a literature review

The study provides a systematic overview of research on the social implications of preimplantation genetic diagnosis (PGD). The analysis focuses on empirical studies that provide insights into its heterogeneous fields of application and the concrete experiences of its users. The literature review shows that three areas of concern and controversial topics are particularly relevant for a social-scientific evaluation of PGD. Firstly, we present attitudes towards and assessments of PGD based on an ongoing expansion and transformation of its fields of application. This process includes not only more and more disorders and disease risks but also applications that are not disease-related. Secondly, there is evidence of significant gender asymmetries and financial concerns regarding the use of and access to PGD. Thirdly, the empirical studies point to a shift in normative expectations towards the idea of “genetic reproductive responsibility” and possible discriminatory consequences for individuals and families with diseases and disabilities.     

Using Current Data to Define New Approach in Age Related Macular Degeneration: Need to Accelerate Translational Research

Age related macular degeneration (AMD) is one of the major retinal degenerative disease of ageing whose complex genetic basis remains undeciphered. The involvement of various other factors like mitochondrial genes, cytoskeletal proteins and the role of epigenetics has been described in this review. Several population based AMD genetic studies have been carried out worldwide. Despite the increased publication of reports, clinical translation still eludes this davastating disease. We suggest models to address roadblocks in clinical translation hoping that these would be beneficial to drive AMD research towards innovative biomarkers and therapeutics Therefore, addressing the need large autopsy studies and combining it with efficient use of bioinformatic tools, statistical modeling and probing SNP-biomarker association are key to time bound resolution of this disease.     

Overview of Strain Characterization in Relation to Serological and Molecular Detection of Citrus tristeza <i>Closterovirus</i>

Tristeza is a devastating viral disease in all the citrus growing countries throughout the world and has killed millions of citrus trees in severely affected orchards. The citrus species grafted on sour orange rootstock are affected by this disease. Predominantly, the sweet orange, grapefruit and lime trees grafted on sour orange exhibit severe symptoms like quick decline, vein clearing, pin holing, bark scaling and degeneration leading to variable symptoms. Symptomatic expression of Citrus tristeza virus (CTV) in different hosts has been attributed to virus isolates which are from severe to mild. Different serological and molecular assays have been deployed to differentiate the strains of CTV. Citrus tristeza virus is diversified towards its strains on the basis of biological, serological and molecular characterization. Phenotypic expression is due to genetic alteration and different molecular basis have now been adopted for strain differentiation. This review will give a brief idea about the different CTV isolates, their characterization based on nucleic acid and serological assays. Different methods along with salient features for strain characterization has also been reviewed. This review will also open the new aspects towards formulation of management strategies through different detection techniques.     

Recommendations for safety testing with the in vivo comet assay

While the in vivo comet assay increases its role in regulatory safety testing, deliberations about the interpretation of comet data continue. Concerns can arise regarding comet assay publications with limited data from non-blind testing of positive control compounds and using protocols (e.g. dose concentrations, sample times, and tissues) known to give an expected effect. There may be a tendency towards bias when the validation or interpretation of comet assay data is based on results generated by widely accepted but non-validated assays. The greatest advantages of the comet assay are its sensitivity and its ability to detect genotoxicity in tissues and at sample times that could not previously be evaluated. Guidelines for its use and interpretation in safety testing should take these factors into account. Guidelines should be derived from objective review of data generated by blind testing of unknown compounds dosed at non-toxic concentrations and evaluated in a true safety-testing environment, where the experimental design and conclusions must be defensible. However, positive in vivo comet findings with such compounds are rarely submitted to regulatory agencies and this data is typically unavailable for publication due to its proprietary nature. To enhance the development of guidelines for safety testing with the comet assay, and with the permission of several sponsors, this paper presents and discusses relevant data from multiple GLP comet studies conducted blind, with unknown pharmaceuticals and consumer products. Based on these data and the lessons we have learned through the course of conducting these studies, I suggest significant adjustments to the current conventions, and I provide recommendations for interpreting in vivo comet assay results in situations where risk must be evaluated in the absence of carcinogenicity or clinical data.     

Review of biophysical factors affecting osteogenic differentiation of human adult adipose-derived stem cells

Developing bone is subject to the control of a broad variety of influences in vivo. For bone repair applications, in vitro osteogenic assays are routinely used to test the responses of bone-forming cells to drugs, hormones, and biomaterials. Results of these assays are used to predict the behavior of bone-forming cells in vivo. Stem cell research has shown promise for enhancing bone repair. In vitro osteogenic assays to test the bone-forming response of stem cells typically use chemical solutions. Stem cell in vitro osteogenic assays often neglect important biophysical cues, such as the forces associated with regular weight-bearing exercise, which promote bone formation. Incorporating more biophysical cues that promote bone formation would improve in vitro osteogenic assays for stem cells. Improved in vitro osteogenic stimulation opens opportunities for “pre-conditioning” cells to differentiate towards the desired lineage. In this review, we explore the role of select biophysical factors—growth surfaces, tensile strain, fluid flow and electromagnetic stimulation—in promoting osteogenic differentiation of stem cells from human adipose. Emphasis is placed on the potential for physical microenvironment manipulation to translate tissue engineering and stem cell research into widespread clinical usage.     

The use of human tissue in drug discovery

Experiments conducted on human tissue samples are a key component of modern drug discovery programs and complement the use of animal tissue based assays in this process. Such studies can (i) enhance our understanding of disease pathophysiology, (ii) increase (or decrease) confidence that modulating the function of particular molecular targets will have therapeutic benefit (iii) allow comparison of the activities of different agents on particular mechanisms/processes and (iv) provide information on the potential safety risks associated with targets. All of this information is critical in identifying the targets that are most likely to deliver efficacious and safe medicines to address unmet clinical needs. With the introduction of new technologies, human tissue samples are also increasingly being incorporated into drug project screening cascades, including their use in high throughput assays. Improved access to human tissue would undoubtedly further extend the utility of this valuable resource in the drug discovery process.     

Invitrogen & HUPO enter partnership

Neuroblastoma (NB) is one of the most common solid tumors of childhood and displays a remarkable diversity in both biologic characteristics and clinical outcomes. Availability of high-throughput ‘omics technologies and their subsequent application towards oncology has provided insight into the complex pathways of tumor formation and progression. Investigation of NB ‘omics profiles may better define tumor behavior and provide targeted therapy with the goal of improving outcomes in patients with high-risk disease. Utilization of these technologies in NB has already led to advances in classification and risk stratification. The gradual emergence of NB-directed proteomics adds a layer of intricacy to the analysis of biologic organization but may ultimately provide a better comprehension of this complex disease. In this review, we cite specific examples of how NB-directed proteomics has provided information regarding novel biomarkers and possible therapeutic targets. We finish by examining the impact of high-throughput ‘omics in the field of NB and speculate on how these emerging technologies may further be incorporated into the discipline.     

From the laboratory bench to the patient's bedside: an update on clinical trials with mesenchymal stem cells

Mesenchymal Stem Cells (MSCs) are non-hematopoietic multi-potent stem-like cells that are capable of differentiating into both mesenchymal and non-mesenchymal lineages. In fact, in addition to bone, cartilage, fat, and myoblasts, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes in vitro and in vivo. MSCs are of interest because they are isolated from a small aspirate of bone marrow and can be easily expanded in vitro. As such, these cells are currently being tested for their potential use in cell and gene therapy for a number of human diseases. Nevertheless, there are still some open questions about origin, multipotentiality, and anatomical localization of MSCs. In this review, we discuss clinical trials based on the use of MSCs in cardiovascular diseases, such as treatment of acute myocardial infarction, endstage ischemic heart disease, or prevention of vascular restenosis through stem cell-mediated injury repair. We analyze data from clinical trials for treatment of osteogenesis imperfecta (OI), which is a genetic disease characterized by production of defective type I collagen. We describe progress for neurological disease treatment with MSC transplants. We discuss data on amyotrophic lateral sclerosis (ALS) and on lysosomal storage diseases (Hurler syndrome and metachromatic leukodystrophy). A section of review is dedicated to ongoing clinical trials, involving MSCs in treatment of steroid refractory Graft Versus Host Disease (GVHD); periodontitis, which is a chronic disease affecting periodontium and causing destruction of attachment apparatus, heart failure, and bone fractures. Finally, we will provide information about biotech companies developing MSC therapy.     

Nonradiographic axial spondyloarthritis: clinical and therapeutic relevance

Current classification criteria for axial spondyloarthritis (axSpA) provide for the inclusion of patients with a wide range of presentations and manifestations. While not considered a formal subclassification, patients are often divided into radiographic or nonradiographic axSpA based on the presence or absence of radiographic sacroiliitis. This review will focus on nonradiographic axSpA and will discuss clinical manifestations of disease that distinguish, or in many cases do not distinguish, this entity from other individuals with axSpA. This review will also cover treatment paradigms for nonradiographic axSpA, particularly the use of biologic therapies, where current data suggest that nonradiographic disease should be managed largely the same as radiographic disease, or classical ankylosing spondylitis.     

Regulatory perspective on in vitro potency assays for human T cells used in anti-tumor immunotherapy

The adaptive immune system is known to play an important role in anti-neoplastic responses via induction of several effector pathways, resulting in tumor cell death. Because of their ability to specifically recognize and kill tumor cells, the potential use of autologous tumor-derived and genetically engineered T cells as adoptive immunotherapy for cancer is currently being explored. Because of the variety of potential T cell-based medicinal products at the level of starting material and manufacturing process, product-specific functionality assays are needed to ensure quality for individual products. In this review, we provide an overview of in vitro potency assays suggested for characterization and release of different T cell-based anti-tumor products. We discuss functional assays, as presented in scientific advices and literature, highlighting specific advantages and limitations of the various assays. Because the anticipated in vivo mechanism of action for anti-tumor T cells involves tumor recognition and cell death, in vitro potency assays based on the cytotoxic potential of antigen-specific T cells are most evident. However, assays based on other T cell properties may be appropriate as surrogates for cytotoxicity. For all proposed assays, biological relevance of the tests and correlation of the read-outs with in vivo functionality need to be substantiated with sufficient product-specific (non-)clinical data. Moreover, further unraveling the complex interaction of immune cells with and within the tumor environment is expected to lead to further improvement of the T cell-based products. Consequently, increased knowledge will allow further optimized guidance for potency assay development.     

From morphologic to molecular: established and emerging molecular diagnostics for breast carcinoma

Diagnostics in the field of breast carcinoma are constantly evolving. The recent wave of molecular methodologies, both microscope and non-microscope based, have opened new ways to gain insight into this disease process and have moved clinical diagnostics closer to a 'personalized medicine' approach. In this review we highlight some of the advancements that laboratory medicine technology is making toward guiding the diagnosis, prognosis, and therapy selection for patients affected by breast carcinoma. The content of the article is largely structured by methodology, with a distinct emphasis on both microscope based and non-microscope based diagnostic formats. Where possible, we have attempted to emphasize the potential benefits as well as limitations to each of these technologies. Successful molecular diagnostics, applied in concert within the morphologic context of a patient's tumor, are what will lay the foundation for personalized therapy and allow a more sophisticated approach to clinical trial stratification. The future of breast cancer diagnostics looks challenging, but it is also a field of great opportunity. Never before have there been such a plethora of new tools available for disease investigation or candidate therapy selection.