Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos

The first differentiation event of the mammalian embryo is thought to occur during blastulation and results in two populations of cells, the inner cell mass (ICM) and the trophectoderm. Most embryonic stem (ES) cell lines have been derived from the ICM or a further subset of ICM cells known as the epiblast. There appears to be a limited period of embryonic development during which pluripotent ES cells can be adapted from the cells of the blastocyst to culture. A method is presented here that allows ES cell lines to be isolated from preblastocyst mouse embryos. These lines were derived from 129S2/SvHsd mouse morulae and earlier cleavage stages with high efficiency. The lines expressed genes and antigens characteristic of pluripotent ES cells. XY cell lines remained karyotypically stable through extensive passaging and produced germ-line-competent chimeras upon blastocyst injection. These results suggest that true ES cells can be derived from embryos explanted at any stage of preimplantation development in the mouse. This finding raises the interesting question of whether ES cell lines derived from embryos at different stages of preimplantation development possess the same potential.     

Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells

OBJECTIVE: We have previously shown the simultaneous generation of CD73(+) mesenchymal stromal cells (MSCs) along with CD34(+) hematopoietic cells from human embryonic stem cells (ESCs) when they are cocultured with OP9 murine stromal cells. We investigated whether MSCs can be derived from human ESCs without coculturing with OP9 cells, and if such cells exhibit immunological properties similar to MSCs derived from adult human bone marrow (BM). MATERIALS AND METHODS: Our starting populations were undifferentiated human ESCs cultured on Matrigel-coated plates without feeder cells. The differentiated fibroblast-looking cells were tested for expression of MSC markers and their potential for multilineage differentiation. We investigated surface expression of human leukocyte antigen (HLA) molecules on these MSCs before and after treatment with interferon-gamma (IFN-gamma). We also tested the proliferative response of T-lymphocytes toward MSCs and the effects of MSCs in mixed lymphocyte reaction (MLR) assays. RESULTS: We derived populations of MSCs from human ESCs with morphology, cell surface marker characteristics, and differentiation potential similar to adult BM-derived MSCs. Similar to BM-derived MSCs, human ESC-derived MSCs express cell surface HLA class I (HLA-ABC) but not HLA class II (HLA-DR) molecules. However, stimulation with IFN-gamma induced the expression of HLD-DR molecules. Human ESC-derived MSCs did not induce proliferation of T-lymphocytes when cocultured with peripheral blood mononuclear cells. Furthermore, ESC-derived MSCs suppressed proliferation of responder T-lymphocytes in MLR assays. CONCLUSIONS: MSCs can be derived from human ESCs without feeder cells. These human ESC-derived MSCs have cell surface markers, differentiation potentials, and immunological properties in vitro that are similar to adult BM-derived MSCs.     

Derivation and potential applications of human embryonic stem cells

Embryonic stem cells are pluripotent cell lines that are derived from the blastocyst-stage early mammalian embryo. These unique cells are characterized by their capacity for prolonged undifferentiated proliferation in culture while maintaining the potential to differentiate into derivatives of all three germ layers. During in vitro differentiation, embryonic stem cells can develop into specialized somatic cells, including cardiomyocytes, and have been shown to recapitulate many processes of early embryonic development. The present review describes the derivation and unique properties of the recently described human embryonic stem cells as well as the properties of cardiomyocytes derived using this unique differentiating system. The possible applications of this system in several cardiac research areas, including developmental biology, functional genomics, pharmacological testing, cell therapy, and tissue engineering, are discussed. Because of their combined ability to proliferate indefinitely and to differentiate to mature tissue types, human embryonic stem cells can potentially provide an unlimited supply of cardiomyocytes for cell therapy procedures aiming to regenerate functional myocardium. However, many obstacles must still be overcome on the way to successful clinical utilization of these cells.     

In vitro generation of hematopoietic stem cells from an embryonic stem cell line.

Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC.     

Patterning definitive hematopoietic stem cells from embryonic stem cells

Derived from the inner cell mass of blastocysts, embryonic stem cells (ESCs) retain the pluripotent features of early embryonic epiblast cells. In vitro, ESCs undergo spontaneous differentiation into a multitude of tissues, and thus are a powerful tool for the study of early developmental processes and a promising resource for cell-based therapies. We have pursued the derivation of functional, multipotent and engraftable hematopoietic stem cells (HSCs) from ESCs in order to investigate the genetic pathways specifying blood formation, as well as to lay the foundation for hematopoietic cell replacement therapies based on engineered ESCs. Theoretically, the generation of HSCs from patient-specific ESCs derived by nuclear transfer could provide for autologous hematopoietic therapies for the treatment of malignant and genetic bone marrow disorders. Although significant progress has been made in achieving hematopoietic differentiation from both murine and human ESCs, we have only a primitive understanding of the underlying mechanisms that specify hematopoietic cell fate, and a very limited capacity to direct the differentiation of the definitive HSC that would be suitable for clinical engraftment studies. Here we will review the progress to date and the significant problems that remain, and outline a strategy to achieve the directed differentiation of HSCs under conditions that might be appropriate for clinical scale-up and disease applications.     

Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells

Human embryonic stem cells (hESCs) provide an important means to effectively study soluble and cell-bound mediators that regulate development of early blood and endothelial cells in a human model system. Here, several complementary methods are used to demonstrate canonical Wnt signaling is important for development of hESC-derived cells with both hematopoietic and endothelial potential. Analyses using both standard flow cy-tometry, as well the more detailed high-throughput image scanning flow cytometry, characterizes sequential development of distinct early developing CD34(bright)CD31(+)Flk1(+) cells and a later population of CD34(dim)CD45(+) cells. While the CD34(bright)CD31(+)Flk1(+) have a more complex morphology and can develop into both endothelial cells and hematopoietic cells, the CD34(dim)CD45(+) cells have a simpler morphology and give rise to only hematopoietic cells. Treatment with dickkopf1 to inhibit Wnt signaling results in a dramatic decrease in development of cells with hematoendothelial potential. In addition, activation of the canonical Wnt signaling pathway in hESCs by coculture with stromal cells that express Wnt1, but not use of noncanonical Wnt5-expressing stromal cells, results in an accelerated differentiation and higher percentage of CD34(bright)CD31(+)Flk1(+) cells at earlier stages of differentiation. These studies effectively demonstrate the importance of canonical Wnt signaling to mediate development of early hematoendothelial progenitors during human development.     

Development of hematopoietic cells from embryonic stem cells

Embryonic stem cells are pluripotent stem cells that can differentiate into all somatic cell lineages and germ lineage cells in vivo. In vitro differentiation capacity of the cells is rather limited compared with the in vivo pluripotency. However, differentiation into hematopoietic lineages is easily obtained, and it is a powerful tool to investigate hematopoietic development and differentiation. In this article, we describe a differentiation induction method that we established, the OP9 system, a unique method using the macrophage colony-stimulating factor-deficient stromal cell line OP9. The utility of the OP9 system includes hematopoietic development, differentiation, B-cell formation, osteoclast formation, and so on. The usefulness and limits of embryonic stem cell-derived hematopoietic cells in cell therapy are also discussed.     

Mice cloned from embryonic stem cells

Cloning allows the asexual reproduction of selected individuals such that the offspring have an essentially identical nuclear genome. Cloning by nuclear transfer thus far has been reported only with freshly isolated cells and cells from primary cultures. We previously reported a method of cloning mice from adult somatic cells after nuclear transfer by microinjection. Here, we apply this method to clone mice from widely available, established embryonic stem (ES) cell lines at late passage. With the ES cell line R1, 29% of reconstructed oocytes developed in vitro to the morula/blastocyst stage, and 8% of these embryos developed to live-born pups when transferred to surrogate mothers. We thus cloned 26 mice from R1 cells. Nuclei from the ES cell line E14 also were shown to direct development to term. We present evidence that the nuclei of ES cells at G(1)- or G(2)/M-phases are efficiently able to support full development. Our findings demonstrate that late-passage ES cells can be used to produce viable cloned mice and provide a link between the technologies of ES cells and animal cloning. It thus may be possible to clone from a single cell a large number of individuals over an extended period.     

Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei

To assess the extent of abnormal gene expression in clones, we assessed global gene expression by microarray analysis on RNA from the placentas and livers of neonatal cloned mice derived by nuclear transfer (NT) from both cultured embryonic stem cells and freshly isolated cumulus cells. Direct comparison of gene expression profiles of more than 10,000 genes showed that for both donor cell types approximately 4% of the expressed genes in the NT placentas differed dramatically in expression levels from those in controls and that the majority of abnormally expressed genes were common to both types of clones. Importantly, however, the expression of a smaller set of genes differed between the embryonic stem cell- and cumulus cell-derived clones. The livers of the cloned mice also showed abnormal gene expression, although to a lesser extent, and with a different set of affected genes, than seen in the placentas. Our results demonstrate frequent abnormal gene expression in clones, in which most expression abnormalities appear common to the NT procedure whereas others appear to reflect the particular donor nucleus.     

Endothelial cells derived from human embryonic stem cells

Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.     

Characterizing endothelial cells derived from the murine embryonic stem cell line CCE

Embryonic stem cells (ESC) are defined by two main properties of self-renewal and their multipotency to differentiate into virtually all cell types of the body, including endothelial cells. ESCs have been widely regarded as an unlimited source of cells in regeneration medicine and also an ideal in vitro model to investigate complex developmental processes. Here, we report a simple and efficient in vitro model to derive a nearly pure population of endothelial cells from a murine ESC line. CCE ES cells are exposed to alpha-MEM medium containing 10% FBS for 4 days and then cultured in endothelial basal-2 medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and 2% FBS for 42 days. The cells acquired a relatively uniform endothelial cell morphology and were able to propagate and expand in culture. When murine ES cell-derived endothelial cells (MESDECs) were cultured on Matrigel and incubated for 48 h, vessel-like tube structures consisting of CD31 (PECAM-1) or BS-1 immunoreactive cells were developed. Immunocytochemistry and RT-PCR analyses revealed that MESDECs express endothelial cell-specific marker proteins such as Flk-1, PECAM-1, Tie-1, and Tie-2, in which the expressions persist for long periods of time after differentiation. The cells were also capable of taking up acetylated low-density lipoprotein (LDL) in culture. Our data suggest that MESDECs could provide a suitable in vitro model to study molecular events involved in vascular development and open up a new therapeutic strategy in regeneration medicine of cardiovascular disorders.     

Germ layer induction from embryonic stem cells

Embryonic stem (ES) cells have the potential to develop into all cell types of the adult body. This capability provides the basis for considering the ES cell system as a novel and unlimited source of cells for replacement therapies for the treatment of a wide range of diseases. Before the cell-based therapy potential of ES cells can be realized, a better understanding of the pathways regulating lineage-specific differentiation is required. Current studies suggest that the bone morphogenic protein, transforming growth factor-beta, Wnt, and fibroblast growth factor pathways that are required for gastrulation and germ layer induction in the embryo are also essential for differentiation of ES cells in culture. The current understanding of how these factors influence germ layer induction in both the embryo and in the ES cell differentiation system is addressed in this review.     

Production of beta-cells from human embryonic stem cells

The making of functional pancreatic islets in renewable numbers has been a goal of stem cell biologists since early 2000. Since that time, many studies have reported successful creation of glucose-responsive pancreatic beta-cells. Not until the more recent systematic application of developmental principles to stem cell biology systems were breakthroughs achieved on directed specification of the required early developmental intermediates. The most important first step is the formation of the definitive endoderm (DE) lineage which is compulsory for production of the epithelium of the pancreas and the other important endoderm-derived organs such as the liver, intestine and lung. The formation of DE from embryonic stem cells made possible additional experimentation aimed at directing the endoderm to further specified foregut and pancreatic endoderm lineages. With these discoveries came the first production of immature pancreatic endocrine cells. Most recently, the production in vivo of glucose-responsive insulin-producing cells with the capacity to correct Steptozotocin-induced hyperglycaemia in mice has been achieved. The work leading up to this achievement, in relation to the other principle human stem cell studies conducted in this area, will be briefly described. The necessary steps and ideal characteristics of embryonic stem cell-based differentiation to pancreatic beta-cells capable of glucose stimulated insulin secretion will be underscored.