What is a stem cell?
Stem cells are undifferentiated cells, unique in both their proliferative capacity and ability to specialise into different cell types. The majority of cells that make up the human body have a specific function and lifespan and do not have the capacity to renew. In contrast, stem cells are able to divide and produce copies, replicating many times and serving as a biological repair system for the body, forming the basis of normal growth and development. In addition to their self-renewal properties, stem cells can differentiate to become more specific and specialized cell types. The classification of stem cells can be based upon their differentiating potential:
Stem cells can also be classified based on their origin and are present in both the embryo (embryonic stem cells) and adult (adult stem cells). Embryonic stem cells are derived from one of the earliest stages of development, thus they are pluripotent and can give rise to all human cell types. Adult stem cells are undifferentiated cells found within differentiated tissue, they are multipotent and can renew and become specialised to form any of the cell types of the tissue of origin. There are many different types of adult stem cell including:
All adult stem cells are considered multi- or uni-potent, however recent advances in stem cell research have enabled adult stem cells to be re-programmed to become embryonic cell-like, expressing genes that define and maintain the same properties as embryonic cells. These induced pluripotent stem cells have been shown in research settings to be capable of generating cells characteristic of all three germ lines and are currently being used in drug development, disease modelling, and are hopeful candidates in the field of transplantation medicine.
Stem cell research areas
Stem cell researchers are interested in understanding the unique properties of stem cells, the drivers behind differentiation and self-renewal, as well as practical and therapeutic applications of stem cells in medicine. The range of cloning and molecular interrogation products offered by Bioline covers a full suite of tools to assist efforts towards better understanding of stem cells and their unique properties. Plus, ISO13485 manufacturing standards support seamless transition into therapeutic applications.
Understanding stem cell development and regulators of potency
What are the properties within a stem cell that allow for numerous cycles of self-renewal? How does a stem cell remain undifferentiated? What are the factors that induce a pluripotent cell towards differentiation into the many hundreds of different cell types within the human body? These are just some of the questions researchers are interested in understanding to shed light on the unique properties of stem cells, to understand how an embryo develops, or how hematopoietic stem cells balance self-renewal and the provision of differentiated progenitors for the maintenance of the blood system throughout life. Identifying the molecular cues that regulate stem cell systems, as well as the microenvironments that contribute the various triggers and maintenance systems, are key to gaining greater understanding of the unique properties of stem cells.
Induced pluripotent stem cells
Pluripotent stem cells offer limitless possibilities for understanding human development and tissue formation, as well as the opportunity to model diseases and understand disease mechanisms, with ultimate aims toward cell therapy and regenerative medicine. For many years embryonic stem (ES) cells were the sole source of pluripotent cells - carrying the weight and complication of associated ethical issues, the availability and utility of ES cells was ultimately limited. It was the Nobel-prize winning breakthrough from Takahashi and Yamanaka in 2006 that led to the identification of four important reprogramming factors that enabled generation of induced pluripotent stem cells (iPSCs) from previously differentiated somatic cells. From a simple skin biopsy or blood sample iPSCs can now be generated, offering genetically matched pluripotent cells for virtually any patient – not surprisingly, there is excitement around the potential of this field for understanding disease mechanisms and development of new treatment options. There are still many hurdles and details to understand and overcome, including the molecular characterisation of iPSCs and whether they are indeed equivalent to ES cells – evidence indicates differences in efficacy of differentiation as well as DNA methylation patterns – plus identifying effective protocols to achieve appropriate and reproducible differentiation is also proving to be a challenge.
Cancer stem cells
Through investigations into cancer metastases, disease recurrence, and acquired resistance to therapy, researchers have uncovered an enormous level of intratumoral heterogeneity, most likely due to a combination of genetic mutations and interactions with the tumour microenvironment, as well as the presence of what is now known as cancer stem cells (CSCs). Similar to normal stem cells, CSCs have self-renewal properties, however the strict regulations surrounding differentiation appear to be deregulated, resulting in continuous expansion and production of aberrant progeny – CSCs form tumours in healthy animals when transplanted, where normal stem cells do not. CSCs have now been identified in a broad range of blood and solid tissue tumours, including leukaemia, breast and brain cancers. While CSCs appear to represent a very small percentage of the tumour cell population, their existence has been used to explain how cancer cells are able to proliferate and acquire all the necessary mutations for cancer development, as well as the recurrent nature of many tumours, as CSCs appear to survive many cancer therapeutic efforts. Understanding CSCs and how they drive cancer development, as well as the mechanisms for how cancer patients acquire CSCs, form a major part of cancer research today.
Therapeutic usage of stem cells
Stem cells have been referred to as an “Aladdin’s Lamp” with rich promise to cure many diseases, however the current reality is far from this ideal. Efforts continue towards a better understanding of stem cell characteristics in order to harness this potential in the therapeutic realm. Today, stem cells are being used to produce differentiated cell lines for drug testing, previously only possible on animal models. Knowledge of the signalling mechanisms that control specific differentiation is still limited, and much more work can be done in this area to improve reproducibility and increase the range of available differentiated cell types. Harvesting of hematopoietic stem cells (HSCs) from bone marrow and more recently from circulating blood, has been used to treat blood cancer patients and replenish blood components after chemotherapy rounds.
Perhaps the most exciting potential for stem cell therapy lies in the generation of new cells and tissues to replace diseased or ailing counterparts. Known as cell-based therapy, this treatment is being looked at to regenerate spinal cord damage, brain injury and heart disease. With the advent of induced pluripotent stem cells, this approach to regenerative medicine is looking even more promising. From cloning and vector reagents, PCR and real-time PCR kits or individual components, sample preparation, sequencing and even whole assay design services, Bioline is ready with a full suite of tools to support the growing spectrum of stem cell research and application advances.
"I am currently one of the QC scientists here at the London site. The most important aspect of my job is to ensure that, through rigorous laboratory testing, all reagents produced meet our own strict quality criteria prior their release. This is to maintain our standing as a provider of quality specialised bio-research reagents that simplify, accelerate and improve life science research.
I have been part of the team at Bioline for almost three years now and the time has flown by. I have seen many changes in this period, most of them have been quite an experience. However I have seen that the vast majority of these changes have directly improved our customer’s experiences; from the purchasing of our reagents to the development of new components used in the latest biomolecular techniques.
We have a number of big challenges ahead, but we as a team here in Bioline UK and internationally will overcome them, improve and continue to grow. To be greatest you must do great things!
Enough about me… I have work to do!"
Andrew Galeeba-Mugyenzi, QC Scientist
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The field of molecular diagnostics has seen much growth in the clinical setting, providing rapid and sensitive approaches for the detection and monitoring of a wide range of human ailments. There is very real potential for molecular diagnostics to revolutionise patient care, offering tools that go further than simple characterisation of disease, reaching into the domain of characterising the patient.
Bioline offers a range of products and services to support the research that contributes towards development of diagnostic products and testing services. With ISO13485 manufacturing standards, each reagent represents the level of quality required when developing tests for the clinical market. In addition, our custom assay development services can provide the edge required for optimal performance and expedient development of your next molecular diagnostic assay.
What is Molecular Diagnostics?
Diagnostic or clinical pathology plays an essential role in patient care – providing physicians with the specific information required to identify and treat the very broad range of ailments presented across primary doctor or hospital practice settings. Molecular diagnostic approaches utilise nucleic acid detection techniques to analyse target DNA or RNA from an affected individual. Molecular-based tests can cover a range of clinical conditions from inherited genetic disease, through the full range of cancers, infectious disease agents, drug-dose or treatment response scenarios (pharmacogenomics), and even personalised treatment and prognostic investigations based upon individual genetic make-up (personalised medicine). Results from molecular diagnostic tests are used in conjunction with the presented symptoms and clinical expertise of the serving physician to better understand disease aetiology, pathogenesis, diagnosis and prognosis.
Technology common within a research environment is not always readily adopted in a diagnostic setting. Diagnostic tests must demonstrate clinical utility, while at the same time adhere to strict quality requirements for reproducibility, along with appropriate sensitivity and specificity performance. Although molecular biology has been a field of study for over 50 years, the integration of molecular diagnostics into pathological fields has been variable. While clinical genetics has become almost entirely molecular-based, traditional morphological analysis, chemical analysis, and immunohistochemistry will always have a place in many areas.
Molecular diagnostics covers a range of techniques from fluorescent in-situ hybridisation (FISH), DNA-chip technology, mass spectrometry, as well as nucleic acid amplification tests (NAATs). The revolution in molecular diagnostics came with the adoption of the Polymerase Chain Reaction (PCR) and the completion of the Human Genome Project. Both scientific milestones have served to expand the usefulness and range of applicability for molecular diagnostic approaches. Data from the Human Genome Project have opened up many possible targets for detection, prevention and/or treatment of disease. PCR is now the most commonly used molecular diagnostic tool, offering a very sensitive and rapid approach for the detection, identification, and quantification of specific DNA or RNA targets. More recently Real-Time PCR, utilising fluorescent dye detection, has streamlined the use of NAATs - improving quantification applications, turn-around times, and significantly reducing the risk of carry-over contamination.
Molecular Diagnostics – Current Applications
From individual buffers and enzymes, sample preparation reagents and Real-Time PCR Kits, right through to fully designed PCR assays, Bioline molecular reagents and services are quality manufactured for your nucleic-acid based testing (NAAT) needs. Molecular diagnostic approaches routinely use NAATs across a range of applications, some of which are summarised here.
Although traditional culture methods are still invaluable for pathogen identification and investigation of specific treatment sensitivities (such as antibiotic resistance screening), molecular diagnostics, in particular PCR and Real-Time PCR NAATs, are routinely used for a range of infectious disease testing. The very sensitive and rapid approach of molecular tests support clinical decisions for diagnosis and treatment of a range of bacterial and viral infections, allowing for very specific discrimination of individual target strains, as well as accurate quantification for monitoring organism load across a treatment regime. Molecular diagnostics are also routinely used for screening purposes - from sexually transmitted diseases right through to screening blood products for potential pathogens.
Mutations in a DNA sequence can change the resulting translated protein with the potential for ongoing effects on the subsequent phenotype or active characteristics of an organism. The most common genetic variants are in the form of single nucleotide polymorphisms (SNPs), and these can confer a broad range of characteristics such as antibiotic resistance in bacteria, or a change in likely response to certain drug treatments, thus there is a range of SNP molecular diagnostic tests in routine practice today. SNPs can be detected using a range of methods including sequence-specific PCR, Dual-labelled hybridisation probe discrimination, and post-PCR analyses, such as amplicon melting or restriction fragment polymorphism approaches. As deep sequencing technologies improve and more genome data is gathered across a range of organisms, the utility and application of SNP testing will only increase.
Biomarkers are objectively measured characteristics, used as indicators to monitor biological or pathological processes, as well as pharmacological responses to treatment or therapy. Biomarkers can cover a range of substances and molecules - such as whole cells, enzymes or hormones. Specific genes or gene products are often targeted for biomarker applications, particularly in the field of drug development, allowing for stratification of a population based on genotype or presence of RNA marker. Although molecular biomarkers show real promise in research and development settings, their routine use in a clinical setting are often hampered by the logistical challenges of standardised measurement processes, along with requirements for robust validation of analytical procedures, and heavy data requirements for clinical validity.
Emerging trends in personalised medicine
The recent boom in deep sequencing technologies and computational biology has expanded biological study into the era of “omics” – such as genomics and proteomics – with simultaneous analysis of hundreds or thousands of genes or proteins. The most direct and current application of this high-volume, high-throughput approach is in the area of array-based technologies, where hundreds of targets can be tested in parallel to gain a large-scale gene expression profile. A clinical setting example of this approach is the array test for detection of mutations or polymorphisms in the genes of the cytochrome P450 system, responsible for metabolism of a range of medications. However, in general, the transition of this rise in genetic information has been slow to cross into the molecular diagnostic laboratory. The challenge lies in understanding the vast amount of data and translating that into clinically useful information. True personalised medicine is the ultimate goal – identifying the individual differences that lead to disease susceptibility, treatment response differences, ultimate disease progression and therapeutic outcomes, and research is continuing toward that end.
Molecular Diagnostics – Maintaining the Gold Standard
In addition to demonstrating the clinical utility and scientific validity of a test, in order for it to be applied in a clinical or diagnostic setting, a number of performance characteristics need to be established, including precision, accuracy, analytical sensitivity and specificity, along with a range of quality determinants covering manufacturing processes, reproducibility, and laboratory staff training requirements. These processes are monitored by local governing bodies, and molecular diagnostic laboratories are required to undergo routine testing and certification updates to maintain their status for reporting clinical results. For these reasons alone, introduction of new technologies or tests is a slow process in molecular diagnostics, and examples of erroneous results in the form of false positive, false negative, or those of no clear relevance to disease state, continue to be reported across the field. Although PCR is now widely adopted in molecular diagnostics, this technology holds its own inherent caveats, including finding appropriate reference sequences, or endogenous control genes for accurate detection or gene expression analysis. Finding a trusted source for core reagents that offer reliability, reproducibility and performance standards that support the demands of clinical specificity and sensitivity is always key to a successful application in a molecular diagnostic setting, and with ISO13485 manufacturing standards and a commitment to quality assay design services, Bioline is a well-placed choice to support your laboratory quality standards.
Cancer cells represent the manifestation of a breakdown in any number of normal cell states involving a wide range of cellular processes and cell types, thus the complexity of this disease cannot be understated. Mutations or changes anywhere from somatic DNA, through to active stem cells, as well as epigenetic changes, and environmental factors can all contribute to the resulting disease, and cancer research areas are just as diverse as these contributing factors. The range of reagents, kits and assays from Bioline support all aspects of cancer research, including oncogenes and genetic markers, epigenetic changes, as well as biomarkers and personalised medicine approaches.
Cancer - a mixed bag of disease states
Our bodies are composed of a broad range of cell types that grow, divide, and function in a controlled manner - interacting with other cells to form organised matrices, differentiating into specific types of cells where required, and breaking down when damaged or no longer needed. The processes that control normal cell activity are complex and multi-level, and mutations in any number of them can lead to cancer – the continual, unregulated proliferation of cells that grow, reproduce and eventually migrate throughout the body.
There are over 200 different types of known cancers, classified based on the type of cell from which they arise, falling into three main groups:
Further classification is based on the type of cell involved and the tissue of origin – for example erythroid leukemias are precursors of erythrocytes. The four most common cancers are breast, prostate, lung and colon, accounting for more than 50% of all cancer cases.
Cancer – mutations within basic cellular processes
The genes within a genome hold the blueprint for creation of the protein molecules that perform many of the important functions of a healthy cell. Mutations within this blueprint have the potential to affect protein production and resulting cellular function. Bioline offers an excellent range of end-point and Real Time PCR reagents and kits to support research into the genomics of cancer and cancer-related mutations. Usually a number of mutations are required before a cell exhibits cancerous properties, typically involving abnormalities in the mechanisms that control cell proliferation, differentiation and survival.
A primary hallmark of cancerous cells is their ability to sustain chronic cell proliferation – growing to high cell densities and larger cell sizes, with limited spatial regulation. Normal cells exhibit density-dependent growth and proliferation, controlled by complex pathways of growth-promotion and inhibitory signalling. There are a number of ways through which cancer cells can bypass normal proliferative mechanisms including producing their own growth-factor ligands, stimulating neighbour cells to produce various growth factors, or by altering cell surfaces to become hyper-responsive to growth factors. Research in this area looks at a number of different aspects of cell-growth mechanisms, such as mutations that lead to disruptions in the negative feedback mechanisms that reduce proliferative signalling, or those that aid in the evasion of growth suppressors, as well as somatic mutations that activate downstream pathways involved in cell proliferation.
All normal cells are subject to a process called apoptosis, the programmed cell death process that occurs in response to the absence of growth factors or other environmental stimulations, or as a result of DNA damage. The ability to evade apoptotic processes is another hallmark of cancerous cells, increasing their lifespans and significantly contributing to tumour growth. Upstream regulators and downstream effector components contribute to the apoptotic machinery of the cell environment and consist of both extra- and intra-cellular signalling pathways. There are a number of ways through which tumour cells circumvent or limit apoptosis, such as increasing expression of antiapoptotic regulators, downregulating proaptotic factors, or through the total loss of tumour suppressor genes.
Tissue Invasion and Metastasis
Cell-cell interactions and the phenomenon of contact inhibition control the orderly way in which normal cells grow, migrate and adhere to each other to form a healthy cell matrix. In contrast, cancerous cells continue migration regardless of cell contact, growing in multi-layered and disorderly patterns. In addition, many malignant cells secrete proteases to digest the components of the extracellular matrix and enable invasion of adjacent tissue. Another hallmark of cancer cells is the ability to promote the formation of new blood vessels – angiogenesis – required to supply much needed oxygen and nutrients to the proliferating tumour. The activation of this
angiogenic switch can be related to factors that either induce or oppose angiogenesis, and most likely a number of countervailing factors. The ability to metastasise, that is to migrate or spread to another part of the body not directly connected with the original tumour, involves a complex invasion-metastasis cascade that is still being understood, and includes mechanisms that both allow for physical dissemination from the primary tumour, as well as adaptation to the foreign tissue environment at the secondary destination.
Cancer research – genetic predisposition through to personalized medicine
Bioline offers reagents that support all areas of cancer research, from individual reagents and kits for PCR and Real Time PCR, through to fully optimised custom designed assays. Real Time PCR Assay panels of a full range of miRNA targets associated with cancer-related processes are also available to assist researchers in identifying appropriate targets for further study, and ISO13485 manufactured reagents provide the required quality for developing assays for personalised medicine approaches.
Oncogene/tumour suppressor research
Oncogenes can be defined as any gene or gene cluster, typically involved in an important cellular function such as differentiation, proliferation or apoptosis, which can turn a normal cell cancerous under specific circumstances. Since the completion of the Human Genome Project and the continued progression of deep sequencing approaches for a range of tumours and cancer types, there has been much hope that identification of key oncogenes would become evident, and once identified, ways in which to counter their activation could be devised. What has instead come to light is an unpredicted level of diversity both within and between cancers that have been sequenced thus far. Mutations in several hundred different genes have been identified as drivers of cancer, and while data is still being gathered, no clear candidates for subsequent treatment development have emerged thus far, so research efforts continue.
Epigenetic cancer research
The presence of a gene, or indeed a gene mutation, does not necessarily result in subsequent translation of an affected protein. Physiological or phenotypic variations can be observed in genetically identical cells that contain, for example, differences in DNA methylation or histone modifications, properties that alter the transcriptional potential of a cell. Epigenetic changes can occur as a result of environmental influences, they are heritable as well as reversible, and epigenetic changes contribute to carcinogenesis, thus studies in cancer epigenetics are thriving and this area holds promise for the development of cancer treatment options.
Tumour microenvironment research
The tumour, or cancer microenvironment is the cellular environment within which a tumour exists, comprised of immune cells, fibroblasts, blood vessel cells, as well as the proteins produced by these non-cancerous cells that support the growth of cancer cells. It is difficult to dissociate the microenvironment from traditionally defined cancer cells, however data suggests that dysfunction within the microenvironment is linked to carcinogenesis, thus understanding the pathophysiology of the microenvironment is a path towards development of chemopreventive agents. Research in this area attempts to understand the dynamic and reciprocal interactions between tumour cells and the cells that orchestrate their growth, metastatic properties or drug resistance progression.
Cancer biomarkers and personalised medicine
Biomarkers are biological molecules, such as proteins or nucleic acids, which are found in body fluids or tissues and can be used to assess the state of a biological process, or differentiate normal from disease state. A wide range of biomarkers for cancer have been identified across the full spectrum of cellular processes involved in the disease, and these biomarkers can be used to estimate risk towards developing disease, screen for active disease, determine disease prognoses, and both predict response to therapy and monitor therapeutic responses. There are many researchers committed to unravelling the immense complexity of biological and cellular information that has been collected thus far across the 200-odd diseases that are collectively known as cancer. Insights into this diversity and individual correlations within disease state form the foundation of biomarker development and subsequent personalised medicine. Today almost half of the cancer medicines and treatments in development are linked to small-molecules or novel biologic agents that have been identified as biomarkers for disease, and this figure is only likely to increase as research continues in this area.