A Novel Automated High-Content Analysis Workflow Capturing Cell Population Dynamics from Induced Pluripotent Stem Cell Live Imaging Data

The twisted path to pluripotency.

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Embryonic stem (ES) cells have generated enormous interest because of their capacity to self-renew and differentiate into various cell types in vitro. Although numerous problems are encountered in the use of ES cells for regenerative medicine, such as ethical issues associated with the use of stem cells established from terminated human embryos and immunorejection due to transplantation of allogenic ES cell-derived cells into patients, recent technologies to generate induced pluripotent stem (iPS) cells from adult somatic cells have provided alternative ways to access pluripotent stem cells (Takahashi et al., 2007). However, the practical application of these pluripotent stem cells has yet to emerge, and regulatory mechanisms are not well known. Moreover, precise differentiation methodologies of ES and iPS cells have not been developed. These problems cause difficulties in the manipulation of pluripotent stem cells and derivation of functionally differentiated cells. Detailed analysis of the transcriptome has allowed elucidation of transcription networks that regulate the pluripotency of these stem cells. However, the specific nuclear infrastructures that maintain the pluripotent stem cell-specific transcription network have not yet been elucidated. We used proteomics to analyze the nuclear protein machinery in stem cells and identified some crucial components for the maintenance of pluripotent stem cells. In addition, various growth factors and extracellular matrix components regulate the pluripotency and differentiation of stem cells. Therefore, the cell surface receptors that bind these regulatory factors are important for the precise regulation of stem cells. We have also explored stem cell-specific cellsurface markers by proteomic analysis of mouse ES cells. These cell-surface membrane proteins can be useful to manipulate pluripotent stem cells. In this chapter, we describe some examples of new findings elucidated by proteomic analysis of ES cells.

To bring the notion of cardiac regenerative medicine to fruition, researchers have tried to determine which stem cells, embryonic stem (ES) cells or somatic stem cells, are most suitable. Thus far, there is no clear indication which is better, because both have their own advantages and disadvantages. In 2006, murine induced pluripotent stem (iPS) cells were first established. Since then, basic research into the properties of iPS cells has continued apace. Originally, human iPS cells were generated from dermal fibroblasts by retrovirus-mediated gene transfer. Although this technique is sophisticated and easy to perform, the skin biopsy is accompanied by some bleeding and pain, and there may be some damage to the host genome because of retrovirus-mediated transgene integration. However, methods of producing iPS cells have improved steadily. For clinical application in the cardiovascular field, efficient methods that produce pluripotent stem cells that can differentiate into cardiomyocytes need to be developed. Existing methods for ES cells can be applied to iPS cells to obtain cardiomyocyte differentiation. In addition, existing purification methods can be used to obtain pure cardiomyocytes from a population of mixed cells. These techniques have themselves been the subject of extensive research, and continued advances are now making the clinical application of pluripotent stem cells a reality. We are at the forefront of medical innovations in the cardiovascular field based on the use of pluripotent stem cells.KeywordsES cellsiPS cellsCardiomyocyteDifferentiation

Although the field of induced pluripotent stem (iPS) cells is a very new, hundreds of research papers regarding them have been published over the past three years. This chapter concentrates on the medical relevance of iPS cells and where the research regarding iPS cells has reached in such a short period time. The reprogramming of cells using the “stemness” genes and the resultant populations similarity to human embryonic stem (ES) cells has allowed for another source of pluripotent stem cells to be generated which have fewer ethical ramifications then ES cells. We have compared other forms of reprogramming somatic cells to pluripotent cells and explain that even though generating iPS cell lines using the “stemness” factors is slow and inefficient it is far superior in generating pluripotent stem cells then the other methods. This relatively new technology has enabled pluripotent cell lines to be generated from various animal species such as pig, which as yet has a no counterpart in ES cell lines. One of the biggest advantages to using iPS cells is the ability to generate patient specific cells that can be used to treat patients without the complications of rejection and immunosuppression associated with using allogeneic ES cells. However, the ability to generate the correct cell type appropriate for treating the disease and, in the case of patients with genetic disorders, generating iPS cells that do not contain the mutation, are problems that must be overcome for the technology to be useful. On the other hand, using iPS cells generated from various disease types could help unfold the stages of development of the disease and enable drug testing on the diseased cells, which ultimately could be applied to treat the disease in patients. There are still some hurdles that need to be overcome; the most crucial is the safety issues associated with the generation of iPS cell lines. At the moment somatic cells are reprogrammed with vectors that integrate the DNA into the host genome in a manner not fully controlled, which could result in unfavorable insertion sites. In addition, there is the fear that the transgene might reactivate oncogenes; MYC, for instance, one of the reprogramming factors, is also known to be an oncogenes. Overall, the ability to reprogram somatic cells using stemness genes to generated iPS cells is a breakthrough whose full potential is still hard to estimate.

A wealth of evidence attests that the organs of developing embryos, particularly the developing brain, are acutely sensitive to chemical perturbations. However, scientists know very little about how exposures to specific endogenous chemicals actually impact human development or children’s ability to learn. And there are almost no data on how the vast majority of the 84,000 chemicals currently listed in the Toxic Substances Control Act (TSCA) Inventory1—including most of the 201 compounds known to be neurotoxic to adults and the 1,000 chemicals shown to be neurotoxic to animals2—may affect developing infants. It is also unclear whether testing with animals always provides accurate insights into human developmental susceptibility. A new line of research based on human stem cells is providing important insights into how chemicals may affect neonatal development. Stem cells are the master cells capable of producing some or all of the 200-plus different types of cells in the human body. In time, some researchers believe stem cells may enable scientists to amass far more data on how exposure to environmental chemicals affects human development, particularly the development of the brain. Now is a “critical time to be talking about stem cell research in the environmental health context,” says Tracey Woodruff, director of the Program on Reproductive Health and the Environment at the University of California, San Francisco (UCSF) Medical School.

Pluripotent stem cells (PSCs), characterized by being able to differentiate into various types of cells, are generally regarded as the most promising sources for cell replacement therapies. However, as typical PSCs, embryonic stem cells (ESCs) are still far away from human clinics so far due to ethical issues and immune rejection response. One way to avoid such problems is to use stem cells derived from autologous somatic cells. Up to date, PSCs could be obtained by reprogramming somatic cells to pluripotent state with approaches including somatic cell nuclear transfer (SCNT), fusion with stem cells, coculture with cells’ extracts, and induction with defined factors. Among these, through reprogramming somatic cells directly by retroviral transduction of transcription factors, induced pluripotent stem (iPS) cells have been successfully generated in both mouse and human recently. These iPS cells shared similar morphology and growth properties to ESCs, could express ESCs marker genes, and could produce adult or germline-competent chimaeras and differentiate into a variety of cell types, including germ cells. Moreover, with iPS technique, patient specific PSCs could be derived more easily from handful somatic cells in human without immune rejection responses innately connected to ESCs. Consequently, generation of iPS cells would be of great help to further understand disease mechanisms, drug screening, and cell transplantation therapies as well. In summary, the recent progress in the study of cell reprogramming for the creation of patientspecific pluripotent stem cells, some existing problems, and research perspectives were suggested.

Induced pluripotent stem (iPS) cells, are a type of pluripotent stem cell derived from adult somatic cells. They have been reprogrammed through inducing genes and factors to be pluripotent. iPS cells are similar to embryonic stem (ES) cells in many aspects. This review summarizes the recent progresses in iPS cell reprogramming and iPS cell based therapy, and describe patient specific iPS cells as a disease model at length in the light of the literature. This review also analyzes and discusses the problems and considerations of iPS cell therapy in the clinical perspective for the treatment of disease.

Induced pluripotent stem (iPS) cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell—typically an adult somatic cell—by inducing a “forced” expression of specific genes. Typically, iPS cells are generated by retroviral induction of transcription factors, OCT (octamer-binding transcription factor)-4, SRY (sex determining region Y)-box 2 also known as SOX2, kruppel-like factor (KLF)-4, and c-MYC, in fibroblasts. The development of iPS cells could be a strategy to overcome the limitations of human embryonic stem cells. iPS cells can replace animal and ES experiments in drug development and toxicity tests and for testing mechanicistic hypotheses of diseases. Although the creation of multiple lineages with iPS cells can seem limitless, a number of challenges need to be addressed in order to effectively use these cell lines for disease modeling. These include the low efficiency of iPS cell generation without genetic alterations, the possibility of tumor formation in vivo, the random integration of retroviral-based delivery vectors into the genome, and unregulated growth of the remaining cells that are partially reprogrammed and refractory to differentiation. The establishment of protein or RNA-based reprogramming strategies will help generate human iPS cells without permanent genetic alterations for future development of personalized medicine.

Introduction: Induced pluripotent stem (iPS) cells share key features with embryonic stem (ES) cells, including similar limitations such as the accumulation of (epi)genetic and genomic alterations. However, sex chromosome instability in mouse iPS cells remains poorly understood. This retrospective study aimed to investigate this phenomenon by analyzing mouse iPS cell clones. Specifically, we examined whether factors such as passage number, cell sex, founder cell type, or reprogramming method influence the presence or absence of sex chromosome abnormalities. Methods: Sex chromosome stability was evaluated in 26 independent male and female mouse iPS cell clones using standard karyotyping techniques. Additionally, we analyzed an artificially generated XXY iPS cell model, in which an extra X chromosome was introduced into a normal XY cell line. Statistical analyses were conducted on the karyotyping results and correlated with both continuous variables (e.g., passage number) and categorical variables (e.g., cell sex, founder cell type, and reprogramming method). Results: Female iPS cell clones displayed significantly higher levels of sex chromosome instability compared to their male counterparts, regardless of the founder cell type or reprogramming strategy. A similar pattern of X chromosome instability was also observed in the XXY iPS cell model. Conclusion: Our findings demonstrate that sex chromosome instability occurs in mouse iPS cells, as previously reported in mouse ES cells, suggesting a conserved phenomenon across pluripotent stem cell types. Importantly, the presence of multiple X chromosomes in the pluripotent state appears to contribute to this instability. Further studies are required to elucidate the underlying molecular mechanisms.

To generate familial ALS patient specific induced pluripotent stem (iPS) cell lines and motor neurons and provide a cell-based disease model for amyotrophic lateral sclerosis (ALS) in Han Chinese. iPS cells were derived from familial ALS patient by introducing 4 transcription factors OCT3/4, SOX2, KLF4 and c-MYC into fibroblast cells by retroviruses. Karyotypic analysis, immunofluorescence staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR) were used to identify the pluripotency of these iPS cell lines. In addition, motor neurons were derived from these iPS cells by inhibiting SMAD pathway. IPS cell lines were established from ALS patient carrying SOD1-V14M mutation. They had pluripotency and were similar to human ES cells. Furthermore, motor neurons were successfully induced from these iPS cells. SOD1-V14M mutation does not affect the reprogramming of fibroblast cells and pluripotency of iPS cells, nor does it prevent differentiation of motor neurons. Furthermore, the above cell-based disease model can recapitulate key aspects of ALS pathogenesis so that it provides an indispensible resource for further elucidating ALS disease pathogenesis and screening appropriate drug candidates in Han Chinese.

Human embryonic stem (hES) cells are already transforming our vision of regenerative medicine and cell replacement therapies because of their unique ability to maintain themselves indefinitely and to form all cell types in the body. hES cell lines with genomes that are predisposed to disease can be used to develop cellular models of human pathologies. The generation of disease‐specific and/or genotypically diverse human stem cell lines that can differentiate into many cell types will have great value for understanding the biology of the cell types affected in disease and analysing disease mechanisms, screening for drug candidates that can slow or prevent disease‐related degeneration as well as toxicological testing and understanding the variation between patients in their responses to therapeutics. In addition, to prepare for cell‐based therapies, new technologies for the generation of stem cell lines that are histocompatible with or specific to individual patients will provide a strategy to overcome the challenges of immune rejection. Several different methods have been developed to make hES cells; to date, these all require the use of human female egg cells as a starting point. Recently, several laboratories have reported that adult cells from human skin and other sources can be induced to revert back to earlier stages of development and exhibit stem cell‐like properties. The methods for ‘reprogramming’ have been developing rapidly but generally involve putting multiple genes into skin cells and then exposing them to specific chemical environments tailored to hES cell growth. While these cells appear to have a developmental potential that is similar to that of hES cells, they are not derived from human embryos. To distinguish these reprogrammed cells from the embryo‐sourced hES cells, they are termed induced pluripotent stem (iPS) cells. The iPS technology is the source of much optimism; »The iPS technology is the source of much …

Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/β-actin promoter with β-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.

A fundamental goal of human cell therapy is to regenerate ailing organs affected by congenital and acquired disease processes. Pluripotent stem cells such as embryonic stem (ES) cells can be differentiated into progenitor and fully differen­tiated cell types of all adult organs such as the brain, pancreas and the heart and therefore represent a promising source of cells for use in cell therapy for a variety of diseases. Importantly, the recent discovery that terminally differentiated somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells with many of the properties of ES cells including the potential to generate diverse adult cell types bypasses important ethical concerns surrounding the derivation and use of human ES cells. Here, the therapeutic promise and limitations of these pluripotent cell types are discussed with a focus on iPS cells and their possible use in regenerative medicine, disease modeling and the development of pharmacological agents.