Since the first successful isolation and cultivation of human embryonic stem cells at the University of Wisconsin, Madison in 1998, there has been high levels of both interest and controversy in this area of research. This book provides a concise overview of an exciting field, covering the characteristics of both human embryonic stem cells and pluripotent stem cells from other human cell lineages. The following chapters describe state-of-the-art differentiation and characterization of specific ectoderm, mesoderm and endoderm-derived lineages from human embryonic stem cells, emphasizing how these can be used to study human developmental mechanisms. A further chapter discusses genetic manipulation of human ES cells. The concluding section covers therapeutic applications of human ES cells, as well as addressing the ethical and legal issues that this research have raised.

This dissertation, "Quantitative Characterization of Mouse Embryonic Stem Cell State Transition" by Xibin, Lu, 盧希彬, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: It is known that on-and-off transcription factors (TFs) regulations play crucial roles in differentiation and reprogramming. Oct4, Sox2 and Nanog as well as their constituted gene networks cooperatively maintain ES cell pluripotency. Recently, TFs such as Nanog, Stella and Rex1 show heterogeneous expression in ES cell populations and such heterogeneity lead to different differentiation potential indicating that heterogeneous gene expression may control cell fate. Besides gene regulation triggered by TFs, intrinsic noise was also found to be a potential source in heterogeneity and stochastic cell fate determination. For safe clinical application of stem cell-based therapy, it is crucial to quantify these dynamic processes and transition rate during research and development. However, in most cases, the transition rate is too low to be measured and is often entangled with cell proliferation. In order to disentangle these processes, we established a Nanog-EGFP reporter ES cell line and quantified the transition rates between two subpopulations. We also developed a mathematical model involving cell state transition, proliferation and noise to facilitate deciphering this process. We first generated a dual reporter ES cell line (Nanog-EGFP and EF1α-H2B-mCherry) based on PiggyBac system. Using a simple ES cell differentiation process, differentiation and dedifferentiation could be obviously observed based on Nanog ES cell reporter. In order to quantify the transition and proliferation rate, we performed multiple batches of ES cell differentiation and dedifferentiation based with this reporter ES cell line. Mathematical models (drift-diffusion-growth equation) are also introduced to fit the data. The quantitative and nontrivial model predictions lead to new experiments and hypotheses. Combined the flow cytometry data with mathematical models, we are able to directly derive the most likely potential landscape (Waddington's landscape) based on Nanog distribution. Our results and approaches developed would provide more clues for future ES cell differentiation studies. In another project, we generated multiple ES cell reporters in which Nanog, Oct4 and Rex1 were all labeled with different fluorescence proteins. We differentiated the reporter ES cell line specifically into epiblast stem cell (EpiSC) based on previous protocols. Based on flow cytometry analysis, we found that, during the transition from ES cell to EpiSC state, all the three marker genes showed heterogeneous expression pattern, and three sub-populations can be identified at the EpiSC steady (later) state. The cell fate identity and potential converting ability into other identities like ES cell or inter-converting ability among three populations were being conducted based on chemical factors. In addition, we quantitatively characterized PiggyBac mediated multiplex gene transfer in mouse embryonic stem cell which lay a good foundation in above mentioned ES cell reporter generation. HS4 insulator was also characterized on the effect of protecting from promoter interference and gene silencing as well as transposition efficiency. Our results suggest that, even after the development of CRISPR and TALEN, transposon technique is still a powerful tool to study synthetic gene circuit in mammalian systems, especially for prototyping and complex circuits with many parts. DOI: 10.5353/th_b5351003 Subjects: Stem cells Ce

This volume provides readers with a comprehensive collection of methods to guide them on how to generate, characterize, and use naïve human pluripotent stem cells (hPSCs). The chapters in this book cover topics such as three predominant routes to generate naïve hPSC lines; methods to differentiate naïve hPSCs into specialized cell types; and techniques to characterize naïve hPSCs using key molecular landmarks that benchmark and quality control the cell lines. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Human Naïve Pluripotent Stem Cells: Methods and Protocols is a valuable resource for novice and expert researchers who are looking to learn more or expand their research in this developing field.

Recent scientific breakthroughs, celebrity patient advocates, and conflicting religious beliefs have come together to bring the state of stem cell researchâ€"specifically embryonic stem cell researchâ€"into the political crosshairs. President Bush's watershed policy statement allows federal funding for embryonic stem cell research but only on a limited number of stem cell lines. Millions of Americans could be affected by the continuing political debate among policymakers and the public. Stem Cells and the Future of Regenerative Medicine provides a deeper exploration of the biological, ethical, and funding questions prompted by the therapeutic potential of undifferentiated human cells. In terms accessible to lay readers, the book summarizes what we know about adult and embryonic stem cells and discusses how to go about the transition from mouse studies to research that has therapeutic implications for people. Perhaps most important, Stem Cells and the Future of Regenerative Medicine also provides an overview of the moral and ethical problems that arise from the use of embryonic stem cells. This timely book compares the impact of public and private research funding and discusses approaches to appropriate research oversight. Based on the insights of leading scientists, ethicists, and other authorities, the book offers authoritative recommendations regarding the use of existing stem cell lines versus new lines in research, the important role of the federal government in this field of research, and other fundamental issues.