Book Volume 1
Page: i-i (1)
Author: Glyn Stacey
Page: ii-iii (2)
Author: Kuldip S. Sidhu
Page: iv-ix (6)
Author: Kuldip S. Sidhu
Page: 3-15 (13)
Author: Kuldip S. Sidhu, Methichit Chayosumrit and Khun H. Lie
The human body is made up of about 220 different kinds of specialized cells such as nerve cells, muscle cells, fat cells and skin cells. All specialized cells originate from stem cells. Stem cells are not specialized and the process of their specialization is called differentiation. Depending on the source, stem cells can be classified into two broad categories i.e. embryonic stem cells that are derived from embryos and non-embryonic stem cells that are derived from adult and fetal tissues. Induced pluripotent stem cells, another types of pluripotent stem cells derived from any tissue by reprogramming and are the homologous source of stem cells. Stem cells are emerging as an important source of material for diseases in regenerative medicine. The non-embryonic stem cells like adult stem cells are in clinical use for many years and embryonic stem cells are now emerging as an alternative source for the same purpose with huge potentials in drug discovery and toxicological studies. The study of biology of stem cells is the hallmark of the recent emerging field of regenerative medicine and medical biotechnology.
Therapeutic Cloning: Derivation and Propagation of Embryonic Stem Cells by Somatic Cell Nuclear Transfer
Page: 16-29 (14)
Author: Zichuan Liu, Shuya Zhou, Shun Zhang, Baolong Xia and Qi Zhou
Embryonic stem cells (ESCs) can grow infinitely and give rise to all types of cells in human body, thus of tremendous therapeutic potentials for a variety of diseases, such as Parkinson’s disease, spinal cord injury, and diabetes. Moreover, the combination of gene modification and directed differentiation of ESCs provides perfect tool for disease modelling and drug discovery. However, tissue rejection following ESCs derivatives transplantation greatly hinders its application. Under such circumstances, the idea of “therapeutic cloning” was proposed, indicating the generation of ESCs from SCNT embryos for therapeutic purpose.
Mouse nuclear transfer embryonic stem cells (NT-ESCs) were first established in 2000, and then proved to be able to differentiate either in vivo or in vitro, and give rise to individual tissues through germ line transmission or tetraploid complementation. Fully reprogrammed NT-ESCs are indistinguishable from ESCs derived from fertilized eggs functionally and substantially. What is more, by deriving NT-ESCs from patient cells, the problem of immune rejection may be avoided. However, the derivation of human NT-ESCs goes with the destruction of clone embryos, leading to fierce ethical disputes. There has not been report of successful establishment of human NT-ES cells so far, and the limited resource of human eggs used for nuclear transfer cumbers the future application of NT-ESCs.
In this chapter, we will introduce therapeutic cloning in two aspects as SCNT and NT-ESCs, and the history, nowadays status and prospects of them will be reviewed.
Page: 30-40 (11)
Author: Romulo M. Brena
Human embryonic stem cells (hESCs) hold great therapeutic promise since they are capable of generating specialized cells that may be utilized to replace damaged tissues in patients suffering from various types of diseases. However, this application is dependent on a comprehensive understanding of the signalling mechanisms involved in ESC lineage restriction, an area in which further scientific proficiency is needed. Moreover, the development of clinical applications using hESCs rests on the formulation of appropriate culture conditions that would allow for the isolation and maintenance of homogeneous, genetically and epigenetically stable cell populations. The stability of these cells is critical to diminish potentially adverse effects following transplantation, such as host rejection, low survival of the grafted cells and maybe even tumor formation. In this chapter I will describe the current techniques for isolating, characterizing and maintaining hESCs in culture. I will address some of the challenges underlying these methods and, when possible, offer alternatives that may help overcome these challenges. Finally, I will discuss the current limitations that have made hESC-based therapy a discipline still in its infancy and we will provide my view of where the field of personalized stem cellbased medicine is likely to go in the future.
Page: 41-55 (15)
Author: Brock J. Conley, Mark Denham, Martin F. Pera and Mirella Dottori
The defining characteristic of a pluripotent stem cell is that is has the potential to self renew and the capacity for differentiation into derivatives of the three embryonic germ layers; endoderm, mesoderm and ectoderm, from which all somatic tissues in an organism can be traced. This full potentiality makes them particularly suitable for developing cell replacement therapies or establishing cellular model systems. Although ES cells may be ideal in terms of their pluripotency, from a therapeutic point of view, their great disadvantage is that they are not patient derived. More recently, advances in medical biology have shown that the genomic state of a somatic cell can be altered or ‘reprogrammed’ to become pluripotent. This is a significant leap forward for obtaining patient-specific pluripotent stem cells that can then be differentiated to the cell type of interest. Generation of reprogrammed somatic cells, known as induced pluripotent stem (iPS) cells, was initially performed by ectopic expression of four transcription factors, however, now this process is becoming more streamlined by improved methods. Through these advances, critical factors and mechanisms involved in driving pluripotency are being determined. This chapter provides a review on how pluripotency is defined and examined in vitro and in vivo. The various ways in which pluripotent stem cells are generated, particularly in relation to induced pluripotency, are also discussed. Finally an outline of disease-specific pluripotent stem cells is reviewed within the context of their capacity to generate the cell type of interest.
Page: 56-67 (12)
Author: Henry Chung and Kuldip S. Sidhu
Pluripotent stem cells are unique candidates for regenerative medicine owing to their differential and self renewal capabilities. With the rapid development of stem cell differentiation methodologies, a number of functional cell types have already been generated, though replacement of damaged or diseased cells/tissues/organs remains an exciting and challenging task. The most common pluripotent cell type are embryonic stem cells (ESCs), however in recent years, several pluripotent cell types have been emerged – nuclear transfer ESCs and induced pluripotent stem cells (iPSCs). Both of these cell types were generated for therapeutic cloning, hence facilitating the transition from lab bench to patients. However, before clinical implementation of pluripotent stem cell based therapies, it must firstly pass several tests that evaluate the long-term safety, with respect to genetic stability. Both nuclear transfer ESCs and iPSCs share many common features but due to their different origins, exhibit slight differences at the epigenomic and transcriptomic levels. This chapter attempts to elucidate some of the many differences that occur at each of the genomic, epigenomic and transcriptomic levels.
Page: 68-82 (15)
Author: Michael B. Morris
Embryonic stem cells (ESCs) serve as a model for development and as a potential source of cells for the treatment of a variety of human conditions. ESCs can undergo differentiation to each of the 3 germ layers and, upon successive elaboration, can generate all of the cell types of the developing embryo and adult. Multiple interconnected layers of regulatory networks/circuits control ESC self renewal, pluripotency, and differentiation. This complex biological system can only be partially understood and manipulated to advantage using reductive experimental approaches. Instead, complex systems analysis tools, which inherently take into consideration the nonlinear, non-intuitive, and highly interconnected nature of the system must be applied. In recent years, a combination of the development of high-throughput screening methods for measuring gross system properties, array technologies for measuring global system changes at various molecular levels, and mathematical algorithms and computer software for data handling and modelling has brought complex systems analysis to the fore. This Chapter highlights recent examples of complex systems applications to ESCs undergoing the earliest stages of differentiation to the germ layers via intermediate pluripotent populations.
Page: 83-94 (12)
Author: Loane Skene
This paper describes the law and the ethical and administrative guidelines that govern the development and use of human stem cells in Australia. It explains the history of the regulatory framework, particularly the process of periodic review of the relevant legislation in light of changes in the science and community views. It highlights a number of ethical issues that have been contentious and the regulatory responses to date.
Pluripotent Stem Cells & Neurodegenerative and Ischaemic Brain Diseases: Basic Science to Clinical Applications
Page: 95-112 (18)
Author: Kuldip S. Sidhu and Perminder S. Sachdev
Degenerative disorders of the central nervous system (CNS), such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD), represent a growing public health challenge worldwide. The ability to accurately diagnose and track neurodegenerative disorders is becoming increasingly important as we enter a new clinical era of disease-modifying therapies. Such treatments may be most effective in the earlier stages of the disease before neurodegeneration becomes too widespread, therefore, it may be important to identify subjects at early stages of the disease. Valid cellular models of these diseases are also needed to examine pathogenesis and test novel treatments at the preclinical stage. The recent advancement in stem cell technology, particularly in pluripotent stem cell biology, has opened up new vistas for neurodegenerative disease research. Both human embryonic stem cells and induced pluripotent stem cells possess the inherent ability to produce all cell types including neuronal tissues in large numbers. The greatest potential for induced pluripotent cells derived from affected individuals is likely to be their utility for modelling and understanding the mechanisms underlying neurodegenerative processes, and for searching for new treatments, including cell replacement therapies. However, much work remains to be done before pluripotent cells can be used for preclinical and clinical applications. In this chapter we discuss briefly, the clinical challenges in early diagnosis of these diseases, the progress so far in the use of stem cells for various neurodegenerative diseases at the levels of discovery, transition and translation, and the challenges of generating specific neural cell subtypes including the recent interest in the use of pluripotent stem cells to model these diseases in the dish. Progress in these areas will substantially accelerate effective application of pluripotent stem cells.
Pluripotent Stem Cells - A Novel Source of Haematopoietic Cells for Transplantation and Transfusion Medicine
Page: 113-142 (30)
Author: Catalina A. Palma, David Ma and Robert Lindeman
Haematopoietic stem cells (HSCs) are commonly used to reconstitute the bone marrow and blood of patients suffering from diseases such as leukaemias, lymphomas and non-malignant conditions like severe bone marrow aplasia and thalassemia major. The mature progeny of HSCs, especially erythrocytes and platelets, are used regularly for rapid transfusion into patients with acute conditions like life-threatening bleeding. For HSC transplantation (HSCT), cells must originate from an immunologically matched donor, and the chronic lack of supply of suitable cells drives the search for alternate HSC sources. Human embryonic stem cells (hESCs) hold the promise for development into every tissue type. Multiple laboratory based studies have demonstrated the careful, ontological approach to HSC differentiation from hESCs, leading to the differentiation of cells which are similar to bone marrow HSCs. Significant challenges remain in achieving HSC replicas, primarily improvement to both efficiency of HSC generation and sustained engraftment in vivo. The differentiation of mature blood cells from hESCs, in order to generate large numbers of “off the shelf” cells for transfusions, has been successful with the production of seemingly fully mature and functional cells, albeit in low numbers. Concerns about the need for immunosuppression after hESC transplantation, has meant that the development of patient-specific induced pluripotent stem cells, which have also been differentiated into blood cells, also has enormous potential for use in the clinic. Presently, these cell products have significant barriers to overcome before clinical translation, such as efficient in vivo function, large-scale manufacture of consistent cell products and short- and long-term safety issues.
Page: 143-167 (25)
Author: Khun H. Lie, Methichit Chayosumrit, Anand Hardikar and Kuldip S. Sidhu
Type 1 diabetes is caused by the loss of insulin-producing () cells in pancreas. A significant advance in cell therapy for diabetes has been the development of a protocol for islet transplantation from Dr. James Shapiro and colleagues at the University of Alberta in Edmonton, Canada (the Edmonton protocol). However, lack of suitable organ donors for transplantation is a critical factor that limits this therapy from majority of individuals suffering from diabetes. Research in the last decade has therefore been largely focused on generating insulin-producing cells that can be easily obtained (derived) and used in transplantation setting for replacement therapy in diabetes. Although insulinproducing cells have been obtained from various sources including human ES cells, bone marrowderived mesenchymal cells, umbilical cord-blood derived mesenchymal cells, transdifferentiation of liver / gallbladder cells, pancreatic duct cells, exocrine cells as well as islet-derived mesenchymal cells, the amount of insulin produced by most of these cell types is significantly less as compared to the amount of insulin produced by a normal adult pancreas. Present research is therefore focused on understanding signalling molecules and processes that would enhance differentiation of these cells. Although embryonic pluripotent/stem cells may be limited due to ethical issues, adult tissue-derived progenitor cells are believed to possess inherent traits that result in “commitment” to a particular phenotype, demonstrated by their relatively restricted differentiation capacity. In this chapter, we discuss the cell types that have been studied for replacement therapy in diabetes with specific reference to their possibility for use in a clinical setting.
Page: 168-179 (12)
Author: Indumathi Mariappan, Subhash Gaddipati, Taraprasad Das, Geeta K. Vemuganti and Virender S. Sangwan
The visual function of an eye gets affected due to defects involving some of its major parts, such as the cornea, lens and retina. Simple refractive error corrections and advanced surgical procedures like cataract surgeries, corneal transplantations and others have revolutionized the treatment of ocular diseases in the past. However, the past decade has witnessed the promise of adult limbal stem cell transplantation (LSCT) for the reconstruction of ocular surface that has been damaged by chemical and thermal burns. LSCT has now become a widely accepted treatment modality in several clinics around the world. While this approach works well for the corneal epithelium, reconstruction of other damaged ocular tissues pose a greater challenge. The current interest is therefore to look for various sources of stem cells that can generate corneal endothelial cells for corneal endothelial dystrophies, glandular cells of the lacrimal epithelium for various dry eye conditions, retinal neurons and the retinal pigmented epithelium (RPE) for treating glaucoma-induced vision loss and other age-related and hereditary retinal dystrophies. Apart from some of the adult ocular stem cell sources, the embryonic stem cells (ESCs) and the recently introduced induced pluripotent stem cells (iPSCs) have come to the forefront and have kindled a lot of hope for ocular regenerative medicine in the future.
Page: 180-190 (11)
Author: Jinlian Hua, Shun Zhang, L. Wang, H. Cao, H. Zhu, J. Sun and Kuldip S.Sidhu
Micro RNAs (miRNAs), recently discovered specific small molecules, have emerged as crucial regulators of diverse developmental processes, such as cell maintenance, proliferation, differentiation, apoptosis, organ formation, tumor formation and so on. They exert widespread functions as regulatory molecules mainly in post-transcriptional levels (also in translation levels). Germ cells, as a kind of unique cells, carry the responsibility for passing genetic information to the next generation. miRNAs play a crucial role in germline development including the formation of primordial germ cells (PGCs), germline stem cells (GSCs) and gametes. We have reviewed here mainly the effects of miRNAs on germ cell development and its related progress.
Page: 191-204 (14)
Author: Martina Klarić, Roberto E.-Waser, Kinga Vojnits and Susanne B.-Hoffmann
Chemicals produced by various industrial sectors such as drugs, cosmetics, agrochemicals and industrial chemicals are obliged to undergo a rigorous safety assessment before their entry into the market. Today the standard information requirements providing the necessary details on the toxicity of the individual compounds are mainly derived from tests performed in laboratory animals. The principal drawbacks of such in vivo tests are their length, cost, the remaining uncertainty for humans and the associated ethical concerns. Therefore there is a strong desire of toxicologists to compile the increasing scientific knowledge as well as emerging technologies and methodologies in a framework that drives safety assessments towards a mechanistic understanding of chemically induced toxicity.
An integer part of such modern toxicology are human based in vitro tests that are designed to allow a high throughput of substances and to specifically elucidate relevant toxicological mechanisms. The most promising source of human cells are stem cells since these cells can be propagated in vitro indefinitely, provide all toxicologically relevant target cells and they do not exhibit unwanted characteristics of cell lines with a carcinogenic origin.
However, the establishment of in vitro toxicity tests based on human pluripotent stem cells is challenging since differentiation protocols are often unstable, the cell cultures are not pure and the differentiation is not leading to fully matured cells that are toxicologically relevant. A first step in test development to tackle these challenges is the establishment of high quality standards which will support the reproducibility of test results within a laboratory set up but also between laboratories.
In the last years, many progress have been made to develop test systems particularly in areas of toxicity where human cellular models are not available but strong interspecies variations exists such as developmental toxicity, cardiotoxicity and hepatotoxicity. However, no pluripotent stem cell- based toxicity test has been formally validated yet.
Page: 205-219 (15)
Author: Greg T. Sutherland and Kuldip S. Sidhu
The prevalence of late-onset neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease is increasing alarmingly worldwide but their causes remain largely unknown. In order to study the pathogenesis of these diseases researchers use a variety of models from cell culture through to full organisms. As the complexity of the model system increases so does the potential variables that can confound experimental results and interpretation. Transgenic animal models are the workhorses of disease research but they have been purposefully intra-bred to remove their genetic variability, a major factor in human neurodegenerative disease. Prior to stem cell advances, researchers could obtain differentiated cell types such as fibroblasts from patients and these had reasonable longevity in the laboratory. However it was questionable how much fibroblasts could tell us about the relative susceptibilities of cells in the human brain. Embryonic stem cell lines provided the flexibility to produce all the different cell types of the body including neurons but they were derived from a limited number of individuals. It was the discovery of induced pluripotency by Yamanaka and colleagues that allowed neurons to be produced from a number of patients and unaffected controls. Now individual genetic variability could be incorporated into our experimental paradigms. Looking forward, induced pluripotent stem cells (iPSC) can be perturbed with putative toxins to re-create the natural history of a disease. This in vitro phenotype can then be used for the high throughput screening of therapeutic agents. The latter is akin to a “cure in a dish”.
Page: 220-226 (7)
Author: Jürgen Götz and Sven Büttner
With an ageing population, Alzheimer’s disease (AD) represents a serious social and economic threat to most societies. While AD’s key histopathological features, the amyloid- plaques and the tau tangles, have been described more than a century and the first disease-causing mutations in familial cases of AD already two decades ago, there is still no cure for this debilitating disease. Current treatment is limited to various acetylcholine esterase (AChE) inhibitors and the NMDA receptor antagonist, memantine, yet neither of these strategies halts the degenerative process that characterizes AD . More recently, strategies have been developed that target both amyloid- and tau, but none of these has reached the patient . Here we review what we have learned from animal models about the pathogenesis of AD and treatment options. We further review current attempts to use stem cells to restore the function of degenerating neurons or to replace them.
Page: 227-233 (7)
Author: Kuldip S. Sidhu
With the emergence of induced pluripotent stem cell (iPSC) technology, the somatic cells can be reprogrammed to pluripotent state, and thus the new paradigm to generate patient-specific pluripotent stem cells for disease pathogenesis and cell therapy. These iPSCs produced from a variety of somatic cell sources are found to be very similar if not identical to embryonic stem cells. Currently the most efficient way to produce such cells is by viral transduction with a combination of transcriptional factors and as such that renders these cells unfit for therapeutic purposes. However, recently there is further development in methods for generating iPSCs with minimal or no genetic modifications via excisable lentiviral and transposon vectors or through repeated application of transient plasmid, episomal, and adenovirus vectors and very recently the use of small molecules, synthetic mRNA and microRNAs. However, as it is becoming evident now that the cell type of origin influences the molecular and functional properties of derived iPSC. The indications that reprogramming may erase the cell memory also raises the question if the disease phenotype may not be correctly represented or also erased in iPSC unless coaxed by further perturbation in vitro culture conditions. Similarly other tissue-derived stem cells and hESC-derived lineages offer an unprecedented opportunity in biomedical research, cell therapy and regenerative medicine. However, to harness the full potential of these technologies, a number of issues need to be resolved pertaining to their safety, stability, culture variability, and better ways to direct a specific reprogramming process including lineage specifications. Both hESC and iPSC fields offer phenomenal opportunities to study the basic molecular mechanistic of human development, their use in disease modelling, regenerative medicine and drug discovery. A steady progress in transitional and translational research particularly in various disease animal models and very recently some human trials with these cells are very encouraging. This chapter briefly summarises (not review) the recent developments in stem cell therapy, pitfalls and development.
Page: 234-237 (4)
Author: Kuldip S. Sidhu
Pluripotent stem cells have garnered tremendous interest in recent years, which is primarily driven by the hope of finding a cure for several debilitating human diseases. Cell transplantation (regeneratve medicine) offers considerable therapeutic potential. The procedure employs pluripotent stem cells as these have the inherent ability to reproduce indefinitely and have the ability to produce over 200 different types of cells constituting the human body. The isolation of human embryonic stem cells (hESCs) from embryos and their successful culture in a petri dish in 1998 has been considered as a major breakthrough that is set to shape stem cell research in the 21st century. This has been followed by another remarkable breakthrough in 2006 when scientists demonstrated for the first time that such pluripotent stem cells could be produced from adult somatic tissues without having to use human embryos. These pluripotent stem cells are called the induced pluripotent stem (iPS) cells. Both hESCs and iPS cells - highly versatile cells – could pave the way for alleviating patients suffering from diabetes, Parkinson’s disease, Alzheimer’s disease, and many more. This eBook brings together the information from the last decade on stem cells, compiled by reputed research experts. Readers will learn all aspects of pluripotent stem cells from basic biology to their use in understanding disease process, toxicology, drug discovery and in developing therapeutic strategies. Research on these cells, including transitional and translational aspects, is explained with the aid of extensive figures, colour photographs, and tables. This eBook is a valuable resource for undergraduates, postgraduates, scientists, embryologists, tissue engineers, doctors and biomedical scientists interested in stem cell research and medicine.
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