Embryonic stem cells: prospects for developmental biology and cell therapy

Stem cells represent natural units of embryonic development and tissue regeneration. Embryonic stem (ES) cells, in particular, possess a nearly unlimited self-renewal capacity and developmental potential to differentiate into virtually any cell type of an organism. Mouse ES cells, which are established as permanent cell lines from early embryos, can be regarded as a versatile biological system that has led to major advances in cell and developmental biology. Human ES cell lines, which have recently been derived, may additionally serve as an unlimited source of cells for regenerative medicine. Before therapeutic applications can be realized, important problems must be resolved. Ethical issues surround the derivation of human ES cells from in vitro fertilized blastocysts. Current techniques for directed differentiation into somatic cell populations remain inefficient and yield heterogeneous cell populations. Transplanted ES cell progeny may not function normally in organs, might retain tumorigenic potential, and could be rejected immunologically. The number of human ES cell lines available for research may also be insufficient to adequately determine their therapeutic potential. Recent molecular and cellular advances with mouse ES cells, however, portend the successful use of these cells in therapeutics. This review therefore focuses both on mouse and human ES cells with respect to in vitro propagation and differentiation as well as their use in basic cell and developmental biology and toxicology and presents prospects for human ES cells in tissue regeneration and transplantation.     

Neural differentiation from embryonic stem cells: which way?

Embryonic stem (ES) cells can in theory produce all cell types of a living organism while renewing themselves with a stable genetic background. These unique features make ES cells a favorable tool for biomedical researches as well as a potential source for therapeutic application. A first step for approaching to ES cells is the directed differentiation to cells of interest, such as the neural cell lineage. Here, we summarize the up and down sides of each category of neural differentiation protocols that have so far been used in mouse and human ES cells, and introduce an efficient and plausible method used in our laboratory for derivation of neuroectodermal cells from human ES cells. This synthesis has led to our suggestions on issues for future design of neural differentiation protocols.     

Efficient derivation of embryonic stem cells by inhibition of glycogen synthase kinase-3

Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocysts. The use of ES cells as a source of differentiated cells holds great promise for cell transplantation therapy. The efficiency of ES cell derivation is affected by genetic variation in mice; that is, some mouse strains, such as C57BL/6, are amenable to ES cell derivation, whereas others, such as BALB/c, are refractory. Developing an efficient method to establish ES cells from strains of various genetic backgrounds should be valuable for derivation of ES cells in various mammalian species, including human. Although it is well-established that various signaling pathways, including phosphoinositide 3-kinase (PI3K)/Akt and Wnt/beta-catenin, regulate the maintenance of ES cell pluripotency, little is known about the signaling pathways involved in the derivation of ES cells from ICMs. In this study, we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3), one of the crucial molecules in the regulation of the Wnt/beta-catenin, Hedgehog, and Notch signaling pathways, dramatically augmented ES cell derivation from both C57BL/6 and BALB/c mouse strains. In contrast, Akt signaling activation enhanced the growth of ICM but did not increase the efficiency of ES cell derivation. Our study establishes an efficient means for ES cell derivation by pharmacological inhibition of GSK-3.     

Establishment of human embryonic stem cell lines and their therapeutic application

Embryonic stem (ES) cell lines are pluripotent stem cell lines that can be propagated indefinitely in culture, retaining their potency to differentiate into every type of cell and tissue in the body. ES cell lines were first established from mouse blastocysts, and have been used for research in developmental biology. ES cells have been proven to be very valuable in the genetic modification of the mouse, especially in producing knockout mice. Since establishment of human ES cell lines was reported, their use in cell replacement therapies has been enthusiastically expected. There have been reports of the differentiation of several useful cell types from human ES cell lines, and clinical use of functional tissues and cells from human ES cells is anticipated. In Japan, there have also been many demands for the use of human ES cells in basic and pre-clinical research. We obtained governmental permission to establish human ES cell lines in April 2002 and started research using donated frozen embryos in January 2003. We successfully established three ES cell line from three blastocysts. These cell lines will be distributed at cost to researchers who have governmental permission to use human ES cells.     

胚胎干细胞蛋白质组学

胚胎干细胞（embryonic stem cells，ES细胞）具有自我复制和无限增殖的潜能，在发育生物学和再生医学中具有重要的研究价值，ES细胞蛋白质组学研究对揭示ES细胞增殖、分化的机制具有重大意义。     

Effects of Akt signaling on nuclear reprogramming

Reprogramming of the epigenetic state from differentiated to pluripotent cells can be attained by cell fusion of differentiated somatic cells with embryonic stem (ES) cells or transfer of the nucleus of a differentiated cell into an enucleated oocyte. Activation of Akt signaling is sufficient to maintain pluripotency of ES cells and promotes derivation of embryonic germ (EG) cells from primordial germ cells (PGCs). Here we analyzed the effects of Akt signaling on somatic cell nuclear reprogramming after cell fusion and nuclear transfer. We found that forced activation of Akt signaling stimulated reprogramming after cell fusion of ES cells with thymocytes or mouse embryonic fibroblasts. These hybrid cells showed ES cell characteristics, including in vitro and in vivo differentiation capacity. In contrast, Akt signaling significantly reduced the efficiency of reprogramming with nuclear transfer. Our results demonstrate that Akt signaling plays important roles on the nuclear reprogramming of somatic cells.     

Formation of a thymus from rat ES cells in xenogeneic nude mouse↔rat ES chimeras

Various conditions for differentiating embryonic stem (ES) cells or induced pluripotent stem (iPS) cells into specific kinds of cell lines are under intensive investigation. However, the production of a functional organ with a three‐dimensional structure from ES or iPS cells is difficult to achieve in vitro. In the present paper, we describe the establishment of a green fluorescent protein‐expressing rat ES cell line and production of mouse?rat ES chimera by injecting rat ES cells into mouse blastocysts. The rat ES cells contributed to various organs in the chimera, including germ cells. When we injected ES cells into blastocysts of nu/nu mice lacking a thymus, the resultant chimeras produced thymus derived from rat ES cells in their bodies. The chimeric animals may provide a method for the derivation of various organs from ES or iPS cells.     

The funGenES consortium

Although the complete sequence of a mammalian genome defines the information content of each cell, understanding the selective usage of this information during the development of specific cell types is limited. The fundamental questions that remain to be answered includes, which are the gene subsets that define the pluripotential self-renewing state of embryonic stem (ES) cells, partially and terminally differentiated developmental states, and how are transitions between these states regulated (lineage commitment)? The FunGenES consortium has been formed to address this challenge by mapping the gene subsets involved in pluripotent, lineage committed, and selected differentiated cell types using gene expression profiling and functional screens. They create an atlas of mammalian genome participating in early and late developmental processes. To fulfil the aim, mouse ES cells were used as an in vitro developmental model system that is very close to the human as they are pluripotent. They can be differentiated through the three major developmental pathways ecto-, meso-, and endoderm into many committed cell types and can be genetically engineered with relative ease. Knowledge of genetic pathways in mouse ES cell differentiation and development might be translated to human ES cells and the potential development of stem cell-based therapies.     

Dynamic changes in the epigenomic state and nuclear organization of differentiating mouse embryonic stem cells

Changes in nuclear organization and the epigenetic state of the genome are important driving forces for developmental gene expression. However, a strategy that allows simultaneous visualization of the dynamics of the epigenomic state and nuclear structure has been lacking to date. We established an experimental system to observe global DNA methylation in living mouse embryonic stem (ES) cells. The methylated DNA binding domain (MBD) and the nuclear localization signal (nls) sequence coding for human methyl CpG-binding domain protein 1 (MBD1) were fused to the enhanced green fluorescent protein (EGFP) reporter gene, and ES cell lines carrying the construct (EGFP-MBD-nls) were established. The EGFP-MBD-nls protein was used to follow DNA methylation in situ under physiological conditions. We also monitored the formation and rearrangement of methylated heterochromatin using EGFP-MBD-nls. Pluripotent mouse ES cells showed unique nuclear organization in that methylated centromeric heterochromatin coalesced to form large clusters around the nucleoli. Upon differentiation, the organization of these heterochromatin clusters changed dramatically. Time-lapse microscopy successfully captured a moment of dramatic change in chromosome positioning during the transition between two differentiation stages. Thus, this experimental system should facilitate studies focusing on relationships between nuclear organization, epigenetic status and cell differentiation.     

Embryonic stem cell differentiation: emergence of a new era in biology and medicine

The discovery of mouse embryonic stem (ES) cells >20 years ago represented a major advance in biology and experimental medicine, as it enabled the routine manipulation of the mouse genome. Along with the capacity to induce genetic modifications, ES cells provided the basis for establishing an in vitro model of early mammalian development and represented a putative new source of differentiated cell types for cell replacement therapy. While ES cells have been used extensively for creating mouse mutants for more than a decade, their application as a model for developmental biology has been limited and their use in cell replacement therapy remains a goal for many in the field. Recent advances in our understanding of ES cell differentiation, detailed in this review, have provided new insights essential for establishing ES cell-based developmental models and for the generation of clinically relevant populations for cell therapy.     

Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines

The optimization of human embryonic stem (hES) cell line derivation methods is challenging because many worldwide laboratories have neither access to spare human embryos nor ethical approval for using supernumerary human embryos for hES cell derivation purposes. Additionally, studies performed directly on human embryos imply a waste of precious human biological material. In this study, we developed a new strategy based on the combination of whole-blastocyst culture followed by laser drilling destruction of the trophoectoderm for improving the efficiency of inner cell mass (ICM) isolation and ES cell derivation using murine embryos. Embryos were divided into good- and poor-quality embryos. We demonstrate that the efficiency of both ICM isolation and ES cell derivation using this strategy is significantly superior to whole-blastocyst culture or laser drilling technology itself. Regardless of the ICM isolation method, the ES cell establishment depends on a feeder cell growth surface. Importantly, this combined methodology can be successfully applied to poor-quality blastocysts that otherwise would not be suitable for laser drilling itself nor immunosurgery in an attempt to derive ES cell lines due to the inability to distinguish the ICM. The ES cell lines derived by this combined method were characterized and shown to maintain a typical morphology, undifferentiated phenotype, and in vitro and in vivo three germ layer differentiation potential. Finally, all ES cell lines established using either technology acquired an aneuploid karyotype after extended culture periods, suggesting that the method used for ES cell derivation does not seem to influence the karyotype of the ES cells after extended culture. This methodology may open up new avenues for further improvements for the derivation of hES cells, the majority of which are derived from frozen, poor-quality human embryos.     

Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens

Cell-surface antigens provide invaluable tools for the identification of cells and for the analysis of cell differentiation. In particular, stage-specific embryonic antigens that are developmentally regulated during early embryogenesis are widely used as markers to monitor the differentiation of both mouse and human embryonic stem (ES) cells and their malignant counterparts, embryonic carcinoma (EC) cells. However, there are notable differences in the expression patterns of some such markers between human and mouse ES/EC cells, and hitherto it has been unclear whether this indicates significant differences between human and mouse embryos, or whether ES/EC cells correspond to distinct cell types within the early embryos of each species. We now show that human ES cells are characterized by the expression of the cell-surface antigens, SSEA3, SSEA4, TRA-1-60, and TRA-1-81, and by the lack of SSEA1, and that inner cell mass cells of the human blastocyst express a similar antigen profile, in contrast to the corresponding cells of the mouse embryo.     

The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells

Islet-like cells derived from embryonic stem (ES) cells may be a promising therapeutic option for future diabetes treatment. Here, we demonstrated a five-stage protocol with adding exendin-4 instead of nicotinamide finally could generate islet-like cells from human embryonic stem (ES) cells. Immunofluorescence analysis revealed a high percentage of c-peptide positive cells in the derivation. However, in addition to insulin/c-peptide, most cells also coexpressed PDX-1 (pancreas duodenum homeobox-1), glucagon, somatostatin or pancreatic polypeptide. Insulin and other pancreatic beta-cell-specific genes were all present in the differentiated cells. Insulin secretion could be detected and increased significantly by adding KCL in high glucose concentration in vitro. Furthermore, subcutaneous transplantation of scaffolds seeded with the islet-like cells or cell transplantation under kidney capsules for further differentiation in vivo could improve 6 h fasted blood glucose levels and diabetic phenotypes in streptozotocin-induced diabetic SCID mice. More interestingly, blood vessels of host origin, characterized by mouse CD31 immunostaining, invaded the cell–scaffold complexes. This work reveals a five-stage protocol with adding exendin-4 may be an effective protocol on the differentiation of human ES cells into islet-like cells, and suggests scaffolds can serve as vehicles for islet-like cell transplantation.     

Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

Embryonic stem (ES) cells display heterogeneous responses upon induction of differentiation. Recent analysis has shown that Hes1 expression oscillates with a period of about 3–5 h in mouse ES cells and that this oscillating expression contributes to the heterogeneous responses: Hes1‐high ES cells are prone to the mesodermal fate, while Hes1‐low ES cells are prone to the neural fate. These outcomes of Hes1‐high and Hes1‐low ES cells are very similar to those of inactivation and activation of Notch signaling, respectively. These results suggest that Hes1 and Notch signaling lead to opposite outcomes in ES cell differentiation, although they work in the same direction in most other cell types. Here, we found that Hes1 acts as an inhibitor but not as an effector of Notch signaling in ES cell differentiation. Our results indicate that sustained Hes1 expression delays the differentiation of ES cells and promotes the preference for the mesodermal rather than the neural fate by suppression of Notch signaling.     

Collagen IV induces trophoectoderm differentiation of mouse embryonic stem cells

The earliest segregation of lineages in the developing embryo is the commitment of cells to the inner cell mass or the trophoectoderm in preimplantation blastocysts. The exogenous signals that control commitment to a particular cell lineage are poorly understood; however, it has been suggested that extracellular "niche" and extracellular matrix, in particular, play an important role in determining the developmental fate of stem cells. Collagen IV (ColIV) has been reported to direct embryonic stem (ES) cell differentiation to mesodermal lineages in both mouse and human ES cells. To define the effects of ColIV on ES cell differentiation and to identify the resulting heterogeneous cell types, we performed microarray analyses and determined global gene expression. We observed that ColIV induced the expression of mesodermal genes specific to hematopoietic, endothelial, and smooth muscle cells and, surprisingly, also a panel of trophoectoderm-restricted markers. This effect was specific to collagen IV, as no trophoblast differentiation was seen on collagen I, laminin, or fibronectin. Stimulation with basic fibroblast growth factor (FGF) or FGF4 increased the number of trophoectodermal cells. These cells were isolated under clonal conditions and successfully differentiated into a variety of trophoblast derivatives. Interestingly, differentiation of ES cells to trophoblastic lineages was only seen in ES cell lines maintained on embryonic feeder layers and was caudal-type homeobox protein 2 (Cdx2)-dependent, consistent with Cdx2's postulated role in trophoectoderm commitment. Our data suggest that, given the appropriate extracellular stimuli, mouse embryonic stem cells can differentiate into trophoectoderm. Disclosure of potential conflicts of interest is found at the end of this article.