Current Stem Cell Research & Therapy - Volume 5, Issue 3, 2010

Since the first report of human embryonic stem cells (hESCs) in 1998, the field of stem cell research has witnessed tremendous progress in many areas of both basic and pre-clinical studies. Notable examples include the identification of factors in controlling pluripotency of the cells, optimization of methods for derivation and cell growth, demonstrations of differentiation into various cell types, improvement of differentiation efficiencies, the proof-of-principle preclinical studies and preparation for clinical trials, and the discoveries of inducible pluripotent stem cells (iPSCs). This issue of Current Stem Cell Research and Therapy is devoted to the progress and prospects of several of these areas in human pluripotent stem cell research. Lin and Xu concentrate on the major achievements in the optimization of hESC culture conditions. Several exogenous regulators including growth factors and small molecules have been found to play critical roles in maintaining pluripotency for hESCs. These findings have guided the development of many animal-free and/or defined culture systems including human feeders and defined xeno-free media. In addition, they describe the attempts to derive and expand clinical-grade hESCs and their derivatives in large-scale culture systems for transplantation. Blin et al. provide a summary of current understanding on the cardiogenic transcriptional network and known factors involved in early cardiogenesis in vivo. This knowledge has contributed to the development of advanced protocols for efficient cardiac differentiation from hESCs. The authors also review the progress on the development of iPSCs and their cardiogenic potential and discuss challenges in the translation of iPSC research into clinical applications. Asai et al. first address issues in current cardiotoxicity testing during drug development and then propose the use of hESC/iPSC-derived cardiomyocytes for this application. They introduce in details a novel assay QTempo: a system to examine QT prolongation using cardiomyocytes derived from pluripotent stem cells. This method allows high throughput screening of drugs and may improve the accuracy of predictions of clinical cardiotoxicity. Hannoun et al. focus on the differentiation of hepatic endoderm (HE). They review human liver biology and how this knowledge can be translated into the development of differentiation protocols to yield HE from hESCs. They also speculate a role for oxygen tension as a new regulatory mechanism in HE differentiation and highlight the importance of mitochondrial function in HE generation. Islam et al. present an example of how hESCs can be used in basic studies in human developmental biology. They illustrate the niche microenvironment for adult hematopoiesis and emphasize the potential of developing hematopoietic stem cells and niche components such as endothelial, osteoblast and osteoclast populations from hESCs for the study of their cellular and molecular interactions. The article by Dodla et al. provides an overview on the role of various factors including glial cells, glial and neuronal derived factors and cell adhesion molecules on the formation of functional synapses in neuronal cultures in vitro. They then discuss how these can be applied to derive functionally active hESC-derived neural networks, a particularly challenging goal yet to be achieved using current technology. The final article contributed by Levengood and Murphy focuses on biomaterials for the establishment of high-throughput stem cell culture screening system. They advocate this technology for the investigation of the effect of the complexity of the stem cell's microenvironment, such as interaction with cells, matrix and soluble factors, on cell growth and differentiation. hESCs and human iPSCs have unlimited proliferative capacity in the undifferentiated state while maintaining their pluripotency to develop into multiple cell types. Understanding the many aspects of stem cell biology will be critical to fully realize the potential of these cells. Progress described above and much more elsewhere will help pave the way to reaching the goals of utilizing these cells as model systems to study human developmental biology, physiologically relevant materials useful for drug development, and regenerative medicine to treat numerous diseases such as spinal cord injuries and other neurodegenerative diseases, cardiovascular disease, diabetes and liver disease.

The pressing demand to elucidate the biology of human embryonic stem (ES) cells and to realize their therapeutic potential has greatly promoted the progresses in the optimization of the culture systems used for this highly promising cell type. These progresses include the characterization of exogenous regulators of pluripotency and differentiation, the development of animal-free, defined, and scalable culture systems, and some pioneering efforts to establish good manufactory practice facilities to derive and expand clinical-grade human ES cells and their derivatives. All of these advancements appear to be also applicable to the derivation and culture of human induced pluripotent stem cells, an ES cell-like cell type derived from somatic cells via reprogramming. This review attempts to summarize these progresses and discuss some of the remaining challenges.

Human Embryonic or pluripotent stem cells hold many promises in regenerative medicine. They also provide the scientific community with powerful models of early human development including cardiogenesis under normal or pathological (congenital and genetic diseases) situations. Furthermore their cardiac derivatives turn out to be very useful to study human cardiac electrophysiology, pharmacology or cardiac toxicology. The current overview provides the basic knowledge on developmental biology of the heart which can be applied to stem cell research to study early cardiogenesis. We summarize both the cardiogenic transcriptional network and the role of morphogens involved in early cardiogenesis. We review protocols of cardiac differentiation of pluripotent stem cells so far available. We finally discuss the translation of basic stem cell research into clinical applications.

Human pluripotential stem cells including both embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) possess self-renewing potency and pluripotentency and can differentiate into virtually any somatic cell type. These features are a distinct advantage for the generation of specific types of human tissue cells in vitro for continuous use in drug development. Recently, an assay system for drug-induced QT interval prolongation using hESC/hiPSC-derived cardiomyocytes and microelectrode arrays (MEA) has been developed. Drug-induced QT interval prolongation (DIQTIP) can lead to sudden cardiac death and is a major safety concern for the drug industry. Regulatory authorities such as the US FDA and the European Medicines Agency require in-vitro testing of all drug candidates to identify potential risk of DIQTIP prior to clinical trials. To reduce the risk of DIQTIP, a routine assay system for in vitro electrophysiological properties using cell-based assays is effective and necessary in early phase of drug discovery. This review discusses developments over the last couple of years for a qualified drug testing method and provides some examples of how hESC/hiPSC-derived cardiomyocytes are beginning to find a practical use for drug discovery and development.

Primary human hepatocytes are a scarce resource with variable function which diminishes with time in culture. As a consequence their use in tissue modeling and therapy is restricted. Human embryonic stem cells (hESCs) could provide a stable source of human tissue due to their properties of self-renewal and their ability to give rise to all three germ layers. hESCs have the potential to provide an unlimited supply of hepatic endoderm (HE) which could offer efficient tools for drug discovery, disease modeling and therapeutic applications. There has been a major focus on developing protocols to derive functional HE from hESCs. This review focuses on human liver biology and the translation of observations of in vivo systems into developing differentiation protocols to yield hepatic endoderm. It also details the potential role of oxygen tension as a new regulatory mechanism in HE differentiation and points out the importance of mitochondrial function analysis in defining successful HE generation.

Intensive research of hematopoiesis using human embryonic stem cells (hESC) as a unique starting cell population has enabled differentiation and isolation of diverse hematopoietic cell lineages. However, there has been only limited success in derivation of hematopoietic stem cells (HSCs) capable of long-term, multi-lineage engraftment when transplanted into xenogeneic models. Better understanding of the HSC developmental niche, the home for hematopoietic stem and progenitor cells, will aid to advance strategies to derive and assay putative HSCs from hESCs. This review discusses recent status of hematopoietic development from the hESCs and highlights the possibility of developing HSC niche using hESC-derived niche components.

Availability of human embryonic stem cells (hESCs) and its neural derivatives has opened up wide possibilities of using these cells as tools for developmental studies, drug screening and cell therapies for treating neurodegenerative diseases. However, for hESCderived neurons to fulfill their potential they need to form functional synapses and spontaneously active neural networks. Until recently very few studies have reported hESC-derived neurons capable of forming such networks, suggesting lack of certain components in culture media to promote mature synaptogenesis. In this review we discuss the various factors that enhance functional synapse formation in primary and stem cell-derived neuronal cultures. These factors include astrocytes, astrocyte-derived factors, cell adhesion molecules and neurotrophins. We discuss the current literature on studies that have used these factors for functional differentiation of primary neural cultures, and discuss its implications for stem cell -derived neural cultures.

A cell's microenvironment plays a primary role in defining cell fate during tissue development, physiological function, and pathological dysfunction. Understanding the key components and interactions within these microenvironments is critical for effective use of stem cells for disease modeling and therapeutic applications. Yet cell microenvironments are difficult to study, as there are tens or hundreds of parameters that can influence cell behavior simultaneously. Additionally, parameters such as cell-cell interactions, cell-ECM interactions, cell shape, soluble signals, and mechanical forces vary dynamically in 3-dimensional space and time. The number of relevant experimental conditions in these cell-based biological systems quickly becomes intractable using standard experimental platforms and techniques. A new set of strategies involving high-throughput experimental formats and 3-dimensional culture is required to achieve significant progress in understanding and exploiting stem cell biology. This mini-review describes bioengineering approaches that are enabling for high-throughput stem cell culture, screening and analysis.

During the past several years, mesenchymal stem cells (MSCs) derived from adult tissue have rapidly moved from in vitro and animal studies into clinical trials as a therapeutic modality for a diverse group of clinical applications, including head and neck reconstruction. For many diseases, cell therapy could affect the underlying pathophysiologic processes through multiple pathways providing an advantage over current treatment modalities. There is an emerging body of evidence that MSCs have unique immunomodulatory properties in addition to the ability to differentiate into multiple tissue lineages which make them even more attractive for regenerative medicine. A variety of pre-clinical and clinical studies have shown that MSCs may have a useful role in tissue repair as well as engineering strategies in head and neck reconstructive surgery. Clinically, this has ranged from injection laryngoplasty to the implantation of a tracheal construct seeded with MSC-derived chondrocytes. Recent advances in stem cell immunobiology can offer insight in the multiple mechanisms through which MSCs could affect underlying pathophysiologic processes ranging from vocal fold scarring to composite tissue defects. Thorough evaluation of the current literature is necessary in understanding how MSCs could potentially revolutionize our approach to head and neck defects. The purpose of this review is to highlight the advances in MSC-based therapies in head and neck surgery, specifically laryngotracheal reconstruction. The clinical role of tissue-derived MSCs, though not well understood, holds promise for many therapeutic applications in regenerative medicine and reconstruction.

Neural stem cells (NSCs) have been investigated in preclinical models as delivery vehicles for therapeutic genes for treatment of tumors in the central nervous system and other organs. Melanoma at early stages is effectively treated with surgery and radiotherapy, however metastatic disease is almost universally fatal, thus novel therapeutic approaches are needed. We studied the use of HB1.F3.CD NSCs, a well-characterized clonal cell line derived from human fetal telencephalon, for their potential to secrete a prodrug-activating enzyme. HB1.F3.CD cells were transduced by adenovirus encoding rabbit carboxylesterase (rCE), which converts CPT-11 (irinotecan) into SN-38, a potent topoisomerase 1 inhibitor and anti-cancer agent. In vitro cell migration assays revealed robust migration of NSCs to conditioned media from human melanoma cells. Cytokine profiles showed that IL-6, IL-8, MCP-1 and TIMP-2, known chemoattractants for stem cells, were highly expressed by melanoma cells. Exposure of melanoma cells to conditioned media from the HB1.F3.CD.rCE cells in the presence of CPT-11 increased the tumor cell-killing effect by approximately 100-fold when compared to CPT-11 alone. Our data demonstrate the rationale for a NSC-based enzyme/prodrug therapeutic approach to target metastatic melanoma. Future experiments will evaluate the therapeutic efficacy of NSC-mediated melanoma therapy in animal models, which will provide the basis for targeted therapy in patients with advanced melanoma.

Acute leukemias are the result of aberrant hematopoietic processes initiated by rare leukemia stem cells that have maintained or acquired the capacity of indefinite proliferation through accumulated mutations and/or epigenetic changes. The most likely view of hematopoietic cell lineage organization is that of complex reactive or adaptative systems. The properties of leukemia stem cells indicate that current chemotherapy will not be effective. Furthermore, recent advances indicate that stem cell microenvironment directly affects cell fate decisions. Because they are quiescent, leukemia stem cells do not respond to cell cycle-specific cytotoxic agents used in treating leukemia, which contribute to treatment failure. New strategies are required that specifically target the malignant stem cell population.