Genomic Approaches to Deconstruct 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.

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.

In the first part of this chapter the different types of pluripotent stem cells are described in general: embryonal carcinoma cells, mouse and human embryonic stem cells, germ cells, epiblast stem cells and induced pluripotent cells. The methods used for isolation of rabbit embryonic stem like cells and rabbit primordial germ cells are detailed in the second part, including the species specific factors playing role in the maintenance of pluripotency. Detection of pluripotency markers both at mRNA and protein levels is an important tool in embryonic stem cell characterization. In vitro differentiation, teratoma formation and chimera forming ability are all inevitable tools to characterize embryonic stem cells. Finally the future potential of a truly validated widely available rabbit embryonic stem cell line is highlighted.

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.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

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.

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2, and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of basic FGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4, and NANOG, whereas the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm, and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state, showing high similarity to their embryonic stem cell counterparts we generated recently. However, their global gene expression analysis revealed a slight but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and embryonic stem cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans.

Keywords: : mesenchymal stem cellspluripotency transcription factorOct4NanogDnmt1self-renewalproliferationspontaneous differentiation

Germline development from human pluripotent stem cells toward disease modeling of infertility

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.

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

Recent studies have demonstrated that somatic stem cells have a flexible potential greater than previously expected when they are transplanted into different tissues. On the other hand, recent studies also have revealed that these potentials might occur because of spontaneous cell fusion with recipient cells. The nuclei of somatic cells could have been reprogrammed when they were artificially or spontaneously fused with mouse embryonic stem (ES) cells. The resultant hybrid cells acquired a developmental pluripotency that the original somatic cells did not have but that ES cells did. LaBarge and Blau (Cell 2002; 111:589-601) demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This means that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage to the mouse, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells. Considered together, the differentiation potential of the somatic stem cell nucleus itself remains unclear. Although the pluripotency of somatic stem cell populations has been evaluated, the developmental totipotency of the nuclei of somatic stem cells, whether or not they fused with other cells, has not been shown, except in only one study concerning fetal neural cells (never in adult stem cells). Here, we showed the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer.

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.