Pluripotent Stem Cell Research Research Articles

The 3R principle: advancing clinical application of human pluripotent stem cells

The first derivation of human embryonic stem cells brought with it a clear understanding that animal models of human disease might be replaced by an unlimited supply of human cells for research, drug discovery, and drug development. With the advent of clinical trials using human pluripotent stem cell-based therapies, it is both timely and relevant to reflect on factors that will facilitate future translation of this technology. Human pluripotent cells are increasingly being used to investigate the molecular mechanisms that underpin normal and pathological human development. Their differentiated progeny are also being used to identify novel pharmaceuticals, to screen for toxic effects of known chemicals, and to investigate cell or tissue transplantation strategies. The intrinsic assumption of these research efforts is that the information gained from these studies will be more accurate, and therefore of greater relevance, than traditional investigations based on animal models of human disease and injury. This review will therefore evaluate how animals and animal-derived products are used for human pluripotent stem cell research, and will indicate how efforts to further reduce or remove animals and animal products from this research will increase the clinical translation of human pluripotent stem cell technologies through drug discovery, toxicology screening, and cell replacement therapies.

Cell transplantation therapy for diabetes mellitus: endocrine pancreas and adipocyte [Review

Experimental transplantation of endocrine tissues has led to significant advances in our understanding of endocrinology and metabolism. Endocrine cell transplantation therapy is expected to be applied to the treatment of metabolic endocriopathies. Restoration of functional pancreatic beta-cell mass or of functional adipose mass are reasonable treatment approaches for patients with diabetes or lipodystrophy, respectively. Human induced pluripotent stem (iPS) cell research is having a great impact on life sciences. Doctors Takahashi and Yamanaka discovered that the forced expression of a set of genes can convert mouse and human somatic cells into a pluripotent state [1, 2]. These iPS cells can differentiate into a variety of cell types. Therefore, iPS cells from patients may be a potential cell source for autologous cell replacement therapy. This review briefly summarizes the current knowledge about transplantation therapy for diabetes mellitus, the development of the endocrine pancreas and adipocytes, and endocrine-metabolic disease-specific iPS cells.

The future of the patient-specific Body-on-a-chip

As significant advancements in technology focused on Organ-on-a-chip continue, it is feasible to consider the future of Body-on-a-chip technology. With serious work being done to realize functioning artificial livers, kidneys, hearts, and lungs on chips, the next step is not only to interconnect these organs but also to consider the integration of stem cell technology to create interconnected patient-specific organs. Such a patient-specific Body-on-a-chip requires a sophisticated set of tools for micropattering cell cultures in 3D to create interconnected tissue-like organ structures. This review discusses advanced methods of the past two years in on-Chip organs, the complex 3D patterning of cultures and state-of-the-art scaffolding, and discusses some of the most relevant advancements in human-induced pluripotent stem cell (hiPSC) research applied to these organs and scaffolds for the future of a patient-specific Body-on-a-chip. We anticipate that such a technology would have a wide area of application, primarily benefiting drug development, chemical safety testing, and disease modeling.

Biophysical Characterization of Pluripotent Stem Cell Mass Accumulation Rate and Intracolony Motion

Despite the potential high impact of human pluripotent stem cell (hPSC) research in developmental biology, cancer biology, and regenerative medicine, surprisingly little is known about how hPSCs grow, divide, and respond to their environment. In this talk, we will introduce live cell interferometery (LCI) as a new, biophysical measurement approach for precisely quantifying hPSC colony mass distributions and growth rates (Figure 1A,B,D,F). LCI is a quantitative phase microscopy technique in which the phase shift of light as it passes through and interacts with matter inside a cell is measured. Our measurements with LCI show that retinoic acid-induced differentiation minimally slows the rate of mass accumulation, a surprising result considering the large metabolic and proliferative changes associated with the transition away from the pluripotent state. We also present methods to quantify the rate and coordination of intracolony motion from colony mass distribution measurements (Figure 1 C,G). Differentiated colonies exhibit a significantly slower rate of mass motion and significantly less coordination of motion, a previously unknown behavior that may provide new information on the health and differentiation potential of available hPSC lines.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Progress and Prospects: Induced Pluripotent Stem Cell Research for Neurological Diseases

Progress and Prospects: Induced Pluripotent Stem Cell Research for Neurological Diseases

Small Molecules in Stem Cell Research

Stem cells possess great promise as therapeutic tools for neurological disorders such as neurodegenerative diseases (Parkinson's disease and Huntington's disease), cerebrovascular diseases (stroke), neurotraumata (spinal cord injury) and demyelinating diseases (multiple sclerosis). This aspiration is based on the cells` ability to maintain a status of self-renewal and to differentiate into the various cell types of an organism. The use of the cells ranges from in vitro to in vivo studies in animal models, ending with clinical applications in humans. The self-renewal and commitment of stem/progenitor cells to differentiate and mature involves complex events leading to the generation of different phenotypes via distinctive developmental programs. Small molecules provide a tool with which to influence these regulatory changes in a controlled manner and to help understand the underlying mechanisms. Furthermore, substantial progress in generating induced pluripotent stem cells has been made using small molecules to replace reprogramming factors and enhance the reprogramming efficiency and kinetics, thus generating cells more compatible with the requirements for cell replacement therapies. In this review we will present the recent progress on the use of small molecules in embryonic and induced pluripotent stem cell research. In the final section we will give a short summary of the clinical approaches using these cells.

Microfluidic systems: A new toolbox for pluripotent stem cells

Conventional culture systems are often limited in their ability to regulate the growth and differentiation of pluripotent stem cells. Microfluidic systems can overcome some of these limitations by providing defined growth conditions with user-controlled spatiotemporal cues. Microfluidic systems allow researchers to modulate pluripotent stem cell renewal and differentiation through biochemical and mechanical stimulation, as well as through microscale patterning and organization of cells and extracellular materials. Essentially, microfluidic tools are reducing the gap between in vitro cell culture environments and the complex and dynamic features of the in vivo stem cell niche. These microfluidic culture systems can also be integrated with microanalytical tools to assess the health and molecular status of pluripotent stem cells. The ability to control biochemical and mechanical input to cells, as well as rapidly and efficiently analyze the biological output from cells, will further our understanding of stem cells and help translate them into clinical use. This review provides a comprehensive insignt into the implications of microfluidics on pluripotent stem cell research.

Communicating Risks and Benefits About Ethically Controversial Topics: the Case of Induced Pluripotent Stem (iPS) Cells

Many are supportive of approaches that incorporate lay citizens into policy making and risk management decisions. However, a great deal of learning must first take place about how citizen engagement for controversial topics is best accomplished. Online risk communication efforts are increasing in popularity but there is little empirical evidence accrued to demonstrate the effectiveness of such methods. The intention of our overall study is to create a powerful method for in-depth two-way communication with the public and expert communities about complex and sensitive issues at the heart of stem cell (SC) research. The fundamental objective is to raise awareness of SC science with lay citizens by fostering more holistic or "all things considered" ethical judgments. Our risk communication study demonstrates that lay citizens are both interested in, and capable of learning about, complex scientific issues provided the right tools are used to convey information and assess understanding. Our results show that it is worth the time and effort for SC researchers to continue posting podcasts and FAQ's about their work for non-expert communities to view. In addition, despite having increased our participants' risk perceptions about induced pluripotent stem (iPS) cell research, almost all were very supportive of this type of research in Canada by the end of the survey. In other words, participants understood that this research did in fact pose some risks and learned a great deal about both the risks and benefits of iPS cell research, and still thought this research was worthwhile to pursue.

Accelerating progress in induced pluripotent stem cell research for neurological diseases

In 2006, Yamanaka's group pioneered a method for reprogramming somatic cells by introducing definite transcription factors, which enabled the generation of induced pluripotent stem cells (iPSCs) with pluripotency comparable to that of embryonic stem cells. These iPSCs are attracting considerable attention for their potential in rejection-tolerance personalized replacement therapy. In recent years, patient-derived iPSCs have been used to recapitulate the phenotypes of neurological diseases and broaden our understanding of the pathogenesis of many neurological diseases, including those of late onset. It is now expected that iPSCs will serve as an unlimited source of disease-specific neural cells for use in disease modeling. This review outlines current progress in neurodegenerative disease research involving iPSCs and discusses the potential roles iPSCs may play in helping researchers elucidate the pathological processes of neurodegenerative diseases and in drug discovery and regenerative medicine.

The Impact of Cell Culture on Stem Cell Research

Induced pluripotent stem cell research has broadened possibilities for regenerative medicine and captured the world's attention in a way that science rarely does. However, clinical applications utilizing cultured stem cells have existed for >30 years and can assist benchers and bedsiders in identifying and expediting promising avenues for future therapies.

Specimen Collection for Induced Pluripotent Stem Cell Research: Harmonizing the Approach to Informed Consent

Induced pluripotent stem cells (iPSCs) have elicited excitement in both the scientific and ethics communities for their potential to advance basic and translational research. They have been hailed as an alternative to derivation from embryos that provides a virtually unlimited source of pluripotent stem cells for research and therapeutic applications. However, research with iPSCs is ethically complex, uniquely encompassing the concerns associated with genomics, immortalized cell lines, transplantation, human reproduction, and biobanking. Prospective donation of tissue specimens for iPSC research thus requires an approach to informed consent that is constructed for this context. Even in the nascent stages of this field, approaches to informed consent have been variable in ways that threaten the simultaneous goals of protecting donors and safeguarding future research and translation, and investigators are seeking guidance. We address this need by providing concrete recommendations for informed consent that balance the perspectives of a variety of stakeholders. Our work combines analysis of consent form language collected from investigators worldwide with a conceptual balancing of normative ethical concerns, policy precedents, and scientific realities. Our framework asks people to consent prospectively to a broad umbrella of foreseeable research, including future therapeutic applications, with recontact possible in limited circumstances. We argue that the long-term goals of regenerative medicine, interest in sharing iPSC lines, and uncertain landscape of future research all would be served by a framework of ongoing communication with donors. Our approach balances the goals of iPSC and regenerative medicine researchers with the interests of individual research participants.

Expand and Regularize Federal Funding for Human Pluripotent Stem Cell Research

Human embryonic stem cell (hESC) research has sparked incredible scientific and public excitement as well as significant controversy. Because they are pluripotent, hESCs can in theory be differentiated into any type of cell found in the human body. Thus they evoke great enthusiasm about potential clinical applications. They are controversial because the method used to derive hESC lines destroys a 2-4 day old human embryo. Research and discoveries using human pluripotent stem cells are simultaneously cutting edge contributions to fundamental understanding and potentially invaluable sources of new treatments for some our most devastating diseases and injuries. Stem cell science represents an important case of “use-inspired basic research,” a class of scientific work that Donald Stokes (1997) compellingly argued could be used to reframe the increasingly fragile “contract” between science and society (Guston & Kenniston 1994). In this case, however, federal funding restrictions, legal challenges, and public controversy imposed on the field's development. Thus, hESC research also offers a “laboratory” for examining the effects high level science policy decisions have on the trajectory of an emerging scientific field. Today, nearly fifteen years after the discoveries that made human pluripotent stem cell science feasible, continued federal funding for this research is highly uncertain. We believe that federal funding for human pluripotent stem cell science should be expanded and stabilized through legislation. Explaining why requires that we begin with a simplified, schematic history of the field.

Induced pluripotent stem cell research: A revolutionary approach to face the challenges in drug screening

Discovery of induced pluripotent stem (iPS) cells in 2006 provided a new path for cell transplantation and drug screening. The iPS cells are stem cells derived from somatic cells that have been genetically reprogrammed into a pluripotent state. Similar to embryonic stem (ES) cells, iPS cells are capable of differentiating into three germ layers, eliminating some of the hurdles in ES cell technology. Further progress and advances in iPS cell technology, from viral to non-viral systems and from integrating to non-integrating approaches of foreign genes into the host genome, have enhanced the existing technology, making it more feasible for clinical applications. In particular, advances in iPS cell technology should enable autologous transplantation and more efficient drug discovery. Cell transplantation may lead to improved treatments for various diseases, including neurological, endocrine, and hepatic diseases. In studies on drug discovery, iPS cells generated from patient-derived somatic cells could be differentiated into specific cells expressing specific phenotypes, which could then be used as disease models. Thus, iPS cells can be helpful in understanding the mechanisms of disease progression and in cell-based efficient drug screening. Here, we summarize the history and progress of iPS cell technology, provide support for the growing interest in iPS cell applications with emphasis on practical uses in cell-based drug screening, and discuss some challenges faced in the use of this technology.

GENERATION OF RABBIT PLURIPOTENT STEM CELL LINES

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in the rabbit. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit full pluripotency in vivo. Moreover, similarly to several domestic species, markers used to characterize the putative ESCs are not fully adequate because studies in domestic species have revealed that they are not specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing rabbit embryo. The status of isolation and characterization of the putative pluripotency genes in rabbit will be discussed. Using rabbit specific pluripotency genes we might be able to reprogram somatic cells and generate induced pluripotent stem cells more efficiently thus overcome some of the challenges towards harnessing the potential of this technology. This study was financed by EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; RabPstem, PERG07-GA-2010-268422; PluriSys, HEALTH-2007-B-223485; AniStem, PIAP-GA-2011-286264), NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; Plurabbit, OMFB-00130-00131/2010 ANR-NKTH/09-GENM-010-01.

Comprehensive gene expression analyses in pluripotent stem cells of a planarian, Dugesia japonica

The neoblasts are the only somatic stem cells in planarians possessing pluripotency, and can give rise to all types of cells, including germline cells. Recently, accumulated knowledge about the transcriptome and expression dynamics of various pluripotent somatic stem cells has provided important opportunities to understand not only fundamental mechanisms of pluripotency, but also stemness across species at the molecular level. The neoblasts can easily be eliminated by radiation. Also, by using fluorescence activated cell sorting (FACS), we can purify and collect many neoblasts, enabling identification of neoblast-related genes by comparison of the gene expression level among intact and X-ray-irradiated animals, and purified neoblasts. In order to find such genes, here we employed the high coverage expression profiling (HiCEP) method, which enables us to observe and compare genome-wide gene expression levels between different samples without advance sequence information, in the planarian D. japonica as a model organism of pluripotent stem cell research. We compared expression levels of ~17,000 peaks corresponding to independent genes among different samples, and obtained 102 peaks as candidates. Expression analysis of genes identified from those peaks by in situ hybridization revealed that at least 42 genes were expressed in the neoblasts and in neoblast-related cells that had a different distribution pattern in the body than neoblasts. Also, single-cell PCR analysis of those genes revealed heterogeneous expression of some genes in the neoblast population. Thus, using multidimensional gene expression analyses, we were able to obtain a valuable data set of neoblast-related genes and their expression patterns.

Unfinished Business: Ongoing Ethical Exceptionalism in the Oversight of Human Pluripotent Stem Cell Research in Canada

In this article, we critically examine the arguments for and against the exceptional status given human pluripotent stem cell research in Canada (through the latest [December 2010] revision of the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans), and conclude that this exceptionalism is unwarranted and ethically unsound. In our view, the three federal research granting agencies should honor their longstanding commitment that researchers, research sponsors, and Research Ethics Boards in Canada have access to “a single reference document for all research involving humans conducted under the auspices of institutions eligible for Agency funding.” As well, responsibility for the development, interpretation, and implementation of Canada's research ethics guidelines should be under the authority of a single oversight body that is independent of the federal research granting Agencies.

Concise Review: Embryonic Stem Cells Versus Induced Pluripotent Stem Cells: The Game Is On

Extraordinary advances in pluripotent stem cell research have initiated an era of hope for regenerative strategies to treat human disease. Besides embryonic stem cells, the discovery of induced pluripotent stem cells widened the possibility of patient-specific cell therapy, drug discovery, and disease modeling. Although similar, it has become clear that these two pluripotent cell types display significant differences. In this review, we explore current knowledge of the molecular and functional similarities and differences between these two cell types to emphasize the necessity for thorough characterization of their properties as well as their differentiation capabilities in the pluripotent state. Such comparative studies will be crucial for determining the more suitable cell type for future stem cell-based therapies for human degenerative diseases.

Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function

Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell-derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25 cm/s, and 3) significant contractile forces of up to 2 mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease.

Developing Defined Culture Systems for Human Pluripotent Stem Cells

Human pluripotent stem cells hold promising potential in many therapeutics applications including regenerative medicine and drug discovery. Over the past three decades, embryonic stem cell research has illustrated that embryonic stem cells possess two important and distinct properties: the ability to continuously self-renew and the ability to differentiate into all specialized cell types. In this article, we will discuss the continuing evolution of human pluripotent stem cell culture by examining requirements needed for the maintenance of self-renewal in vitro. We will also elaborate on the future direction of the field toward generating a robust and completely defined culture system, which has brought forth collaborations amongst biologists and engineers. As human pluripotent stem cell research progresses towards identifying solutions for debilitating diseases, it will be critical to establish a defined, reproducible and scalable culture system to meet the requirements of these clinical applications.

Conference Scene: Translating Pluripotent Stem Cells in Regenerative Medicine: a Platform for Applications and Issues to Consider

Euroscicon hosted a discussion workshop on the rapidly moving area of pluripotent stem cell research on the 6 May 2011. The event brought together a panel of seven experts as well as three invited speakers to demonstrate and discuss important areas of their work. The workshop was split into two main sessions: the morning session allowed each panel expert to give a short talk on their area of specialty. In the afternoon sessions the delegates were divided into small groups for informal discussions with each expert panelist. There was also an exhibition session organized by various companies presenting their products.