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PubMed2004

CD34+CD38- hematopoietic precursors derived from human embryonic stem cells exhibit an embryonic gene expression pattern.

Gene expression patterns of CD34(+)CD38(-) cells derived from human embryonic stem cells (ESCs) were compared with those of cells isolated from adult human bone marrow (BM) using microarrays; 1692 and 1494 genes were expressed at levels at least 3-fold above background in cells from BM and ESCs, respectively. Of these, 494 showed similar levels of expression in cells from both sources, 791 genes were overexpressed in cells from BM (BM versus ESCs, at least 2-fold), and 803 genes were preferentially expressed in cells from ESCs (ESCs versus BM, at least 2-fold). The message of the flt-3 gene was markedly decreased in cells from ESCs, whereas there was substantial flt-3 expression in cells from BM. High levels of embryonic epsilon-globin expression were observed-but no adult beta-globin message-in CD34(+)CD38(-) cells from ESCs, whereas high levels of beta-globin expression-but no embryonic epsilon-globin message-could be detected in cells from BM. Furthermore, high levels of major histocompatibility complex (MHC) gene expression were demonstrated in cells from BM but very low levels of MHC message in corresponding cells from ESCs. These observations demonstrate that CD34(+)CD38(-) cells derived from ESCs correspond consistently to an early developmental stage at which the yolk sac and fetal liver are the primary sites of hematopoiesis.

ADP-ribosyl Cyclase↗
PubMed2005

Human embryonic stem cell stability.

Human embryonic stem cells (hESCs) are derived from human preimplantation embryos, and exhibit the defining characteristics of immortality and pluripotency. Indeed, these cell populations can be maintained for several years in continuous culture, and undergo hundreds of population doublings. hESCs are thus likely candidates for source of cells for cell replacement therapies. Although hESC lines appear stable in their expression of cytokine markers, expression of telomerase, ability to differentiate, and maintenance of a stable karyotype, several other aspects of stability have not yet been addressed, including mitochondrial sequencing, methylation patterns, and fine resolution cytogenetic analysis. Because of the potential utility of hESCs, it will be of utmost importance to evaluate the stability of these aspects of ESC biology.

Animals↗
PubMed2005

Human blastocyst culture and derivation of embryonic stem cell lines.

Human embryonic stem cell (hESC) biology is expected to revolutionize the future of medicine by the provision of cell-based therapies for the treatment of a variety of deliberatig diseases. The tremendous versatility of hESCs has reinforced this hope. To understand the biology of these mysterious cells and attempt to differentiate them into desirable tissues, bona fide hESCs that maintain their stability with time are required for research and clinical application. This review discusses the various protocols to derive and propagate hESCs from high quality embryos. The nature and properties of hESCs are also described together with unanswered questions that need to be addressed if this science is to be taken to the bedside.

Blastocyst↗
PubMed2006

Derivation and characterization of hematopoietic cells from human embryonic stem cells.

In vitro, the aggregation of pluripotent human embryonic stem cells (hESC) into cell clusters termed embryoid bodies (EB) allows for the spontaneous differentiation of hESC into progeny representing endoderm, mesoderm, and ectoderm lineages. During human EB (hEB) differentiation, stochastic emergence of hematopoietic cells can be enhanced by a combination of hematopoietic cytokines and the ventral mesoderm inducer bone morphogenetic protein (BMP)-4. Dependent on the presence of hematopoietic cytokines and BMP-4, vascular endothelial growth factor (VEGF-A165) selectively promotes erythropoietic development toward the primitive lineage. The effects of VEGF-A165 can be augmented by erythropoietin (EPO). Hematopoietic cells are derived from a rare subpopulation of hemogenic precursors during hEB development. These hemogenic precursors lack CD45, but express PECAM-1, Flk-1, and VE-cadherin (hereinafter CD45(neg)PFV) and are solely responsible for hematopoietic cell fate. Human ESC-derived hematopoietic cells have similar colony and cellular morphologies to those derived from committed adult hematopoietic tissues, and also show repopulating capacity in immune deficient mice after intrabone marrow transplantation. In this chapter, we describe methods that have been successfully applied in our laboratory, including (1) generation of hematopoietic cells by EB formation; (2) augmentation of hematopoiesis by use of hematopoietic cytokines and BMP-4; (3) promotion of erythropoietic development by addition of VEGF-A165 and EPO; (4) isolation of CD45(neg)PFV hemogenic precursors and generation of hematopoietic cells from these precursors; and (5) characterization of hESC-derived hematopoietic cells in vitro and in vivo.

Animals↗
PubMed1996

Differentiation and delayed cell death in embryonal stem cells exposed to low doses of ionising radiation.

Embryonal stem cells have been used to study the effects of environmentally relevant doses of radiation on cell death and differentation. The ES cells were found to have a greater than 60% chance of surviving the traversal of a single alpha-particle, the lowest possible dose of high linear energy transfer radiation a cell may receive. The ES cells appeared to possess the cell cycle checkpoints believed to prevent the transmission of the radiation damage. However, delayed effects were observed in the progeny. An increased incidence of apoptosis and haempoietic differentiation capacity was found to persist in the ES cell population over many cell divisions. Since both cell death and differentiation are known to play a key role in tissue kinetics, an ES cell model will provide a valuable and versatile cell system for studying the role of cell death and differentiation in the pathology of radiogenic diseases.

Journal Article↗
PubMed2006

Germ cells from mouse and human embryonic stem cells.

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts - recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.

Animals↗
PubMed2003

A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.

BACKGROUND: Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells. METHODS: We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment. RESULTS: Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24-36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing. CONCLUSIONS: We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.

Animals↗
PubMed2006

Establishment of the gene-inducible system in primate embryonic stem cell lines.

Human embryonic stem cells (ESCs) would provide a potentially unlimited source for cell replacement therapies. However, the molecular mechanisms involved in the maintenance of "stemness" are not fully understood. Monkey ESCs are much more similar in character to human ESCs than are mouse ESCs. Therefore, studies using monkey ESCs can give conclusions that are more relevant and may be readily applicable to both basic research and clinical applications for future regenerative medicine. For such studies, generation of a gene-inducible system regulatable in primate ESCs would serve as a powerful tool. Here, we established a Tet-Off gene-inducible system in monkey ESC lines. Such manipulated cells maintained ESC characteristics, and inducible gene expression in both the stem cells and differentiated cells could be reliably controlled by doxycycline administration.

Animals↗
PubMed2003

HNF-6-independent differentiation of mouse embryonic stem cells into insulin-producing cells.

AIMS/HYPOTHESIS: Embryonic stem cells, when grown as embryoid bodies, spontaneously generate insulin-producing cells which could be used in therapy of diabetes mellitus, provided that their selection and differentiation are optimized. To achieve such optimization, one needs to know whether the differentiation of cells in embryoid bodies mimicks that of pancreatic beta cells in embryos. To address this question we verified if the differentiation of the insulin-producing cells in embryoid bodies requires Hepatocyte Nuclear Factor-6 (HNF-6), a transcription factor known to control pancreatic endocrine differentiation in embryos. METHODS: We generated mouse Hnf6-/- embryonic stem cells and grew them as embryoid bodies. The expression of HNF-6, insulin, and transcription factors that are regulated by HNF-6 in developing pancreas was compared in wild-type and Hnf6-/- embryoid bodies. RESULTS: No difference was observed in the expression of insulin between wild-type and Hnf6-/-embryoid bodies. In both cases insulin was expressed in the outer layer of cells, which is similar to the visceral endoderm. In wild-type embryoid bodies HNF-6 was transiently expressed in the outer layer of cells, but was not co-expressed with insulin. The expression of genes that are targets of HNF-6 in developing pancreas was unaffected in Hnf6-/-embryoid bodies. CONCLUSION/INTERPRETATION: In contrast to the development of pancreatic beta cells, the differentiation of insulin-producing cells in embryoid bodies did not require HNF-6. Thus, the differentiation mechanism of insulin-producing cells in embryoid bodies differs from that of the beta cells and it is likely to resemble that of insulin-producing cells in the visceral endoderm.

Animals↗
PubMed1997

The MS-5 murine stromal cell line and hematopoietic growth factors synergize to support the megakaryocytic differentiation of embryonic stem cells.

Murine embryonic stem (ES) cells are able to differentiate into erythroid, mast, and granulomonocytic cells by using appropriate culture conditions. Because we were interested in the regulation of tissue-specific expression of the platelet glycoprotein IIb gene, we studied the culture conditions, aiming at the reproducible production of myeloid cells that included megakaryocytes (MKs) from ES cells. We showed that even a complex cocktail of HGFs (stem cell factor, interleukin 3, IL6, IL11, granulocyte colony-stimulating factor, and erythropoietin) is unable to induce significant myeloid differentiation in day 12 embryoid bodies. Cocultures of MS-5 stromal cells with ES cells were slightly more productive than HGFs. A strong synergistic effect was observed on the growth of myeloid colonies and MKs when we used a combination of MS-5 cells plus the HGF cocktail. Conditioned medium from MS-5 cells also synergized with the HGF cocktail to produce a substantial number of mixed colonies containing MKs. The addition of fibroblast growth factor-2 (FGF-2) to the HGF cocktail plus MS-5 nearly doubled the number of myeloid progenitors, including those with MKs. Thrombopoietin (TPO) alone or in any combination with MS-5 or HGFs, did not increase the number of MK-containing colonies. However, when TPO was added to the HGF cocktail + FGF-2 + MS-5, the number of MKs in liquid cultures and mixed colonies increased, and many exhibited a "hairy" appearance resembling pseudopodial proplatelet formation. Having defined the culture conditions of ES cells that allow the production of all the myeloid lineages including MKs, we conclude that the hematopoietic differentiation model of ES cells is especially useful for studying the regulation of expression of any gene important in early hematopoiesis.

Animals↗
PubMed2006

[Signaling pathways regulating self-renewal of mouse embryonic stem cells--review].

Mouse embryonic stem cells (ES cells) are pluripotent in that they can give rise to almost all the cell types in vitro and in vivo. Also, they can sustain self-renewal in vitro owing to symmetrical mitosis, i.e., only the cell number increases while the daughter cells remain pluripotent. Self-renewal and pluripotency of ES cells are under stringent regulation of several signaling pathways. Activation of either JAK-STAT3 or PI3K, the downstream cascade of gp130, can maintain the self-renewal of ES cells, while phosphorylation of another gp130-related branch, SHP2-Ras-ERK, drives the differentiation. BMP2/4-mediated signaling is capable of suppressing the differentiation of ES cells in collaboration with activated JAK-STAT3 under serum free culture conditions. Other signaling such as Wnt also contributes to the self-renewal of ES cells. Generally, the network, which is composed of various signaling pathways, modulates the self-renewal and differentiation of mouse ES cells precisely. This review focuses on the role of gp130 in proliferation of mouse ES cells including inhibitory effect of JAK-STAT3 pathway activation on differentiation of mouse ES cells, maintenance effect of PI3K pathway activation on self-renewal of ES cells, promotive effect of SHP-2-Ras-ERK pathway activation on differentiation of ES cells, and influence of other signaling pathways on self-renewal of mouse ES cells, including maintenance effect of BMP combination with LIF under serum free culture conditions on self-renewal of ES cells and promotive effect of Wnt pathway activation on self-renewal of ES cells.

Animals↗
PubMed2006

Derivation of human embryonic stem cell lines.

Human embryonic stem cells (hESC) are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely, while retaining their capability to differentiate into specialized somatic cell types. Over the last decade there has been great interest in derivation and culture of these cells, as they can potentially provide a supply of readily available differentiated cells and tissues of all types to be used for therapeutic purposes in cell transplantation in humans, as well as for other medical uses such as drug discovery. The source of hESC lines is usually excess human embryos from in vitro fertilization treatments, although novel ways of producing hESCs have been suggested recently. The actual methods of hESC derivation have not changed greatly since the first report by Thomson et al. in 1998 . However, the main emphasis over the last several years has been in finding defined conditions for derivation and culture of hESCs, because to enable the clinical use of hESC for cell transplantation, the use of animal derived biological components is no longer acceptable. For basic research, the aim is to replace even human derived materials with completely defined systems. In this paper we describe methods utilized in our laboratory for hESC derivation and describe two studies conducted in an attempt to improve derivation efficiency and to enable research outcomes to be achieved using fewer embryos.

Animals↗
PubMed2006

[Embryonic stem cells. Future perspectives].

Embryonic stem cells (ES cells) are able to differentiate into any cell type, and therefore represent an excellent source for cellular replacement therapies in the case of widespread diseases, for example heart failure, diabetes, Parkinson's disease and spinal cord injury. A major prerequisite for their efficient and safe clinical application is the availability of pure populations for direct cell transplantation or tissue engineering as well as the immunological compatibility of the transplanted cells. The expression of human surface markers under the control of cell type specific promoters represents a promising approach for the selection of cardiomyocytes and other cell types for therapeutic applications. The first human clinical trial using ES cells will start in the United States this year.

Cells, Cultured↗
PubMed2006

Improved cardiac function in infarcted mice after treatment with pluripotent embryonic stem cells.

Because pluripotent embryonic stem cells (ESCs) are able to differentiate into any tissue, they are attractive agents for tissue regeneration. Although improvement of cardiac function has been observed after transplantation of pluripotent ESCs, the extent to which these effects reflect ESC-mediated remuscularization, revascularization, or paracrine mechanisms is unknown. Moreover, because ESCs may generate teratomas, the ability to predict the outcome of cellular differentiation, especially when transplanting pluripotent ESCs, is essential; conversely, a requirement to use predifferentiated ESCs would limit their application to highly characterized subsets that are available in limited numbers. In the experiments reported here, we transplanted low numbers of two murine ESC lines, respectively engineered to express a beta-galactosidase gene from either a constitutive (elongation factor) or a cardiac-specific (alpha-myosin heavy chain) promoter, into infarcted mouse myocardium. Although ESC-derived tumors formed within the pericardial space in 21% of injected hearts, lacZ histochemistry revealed that engraftment of ESC was restricted to the ischemic myocardium. Echocardiographic monitoring of ESC-injected hearts that did not form tumors revealed functional improvements by 4 weeks postinfarction, including significant increases in ejection fraction, circumferential fiber shortening velocity, and peak mitral blood flow velocity. These experiments indicate that the infarcted myocardial environment can support engraftment and cardiomyogenic differentiation of pluripotent ESCs, concomitant with partial functional recovery.

Animals↗
PubMed2000

Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro.

The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the beta-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45(+) myelomonocytic progenitors and Ter119(+) erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34(+) cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34(+) CD31(lo) and CD34(+) CD45(-)cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

Antigens, CD34↗
PubMedDate not supplied

Human embryonic stem cells: origin, properties and applications.

Human embryonic stem cells originate from the human preimplantation embryo. The derivation of the first human embryonic stem cells was reported in 1998. Since then we have learnt a great deal about how to isolate and culture these cells. Additionally, their stem cell phenotype and differentiation competence have been determined. Although it is expected that many basic biological properties, such as self-renewal and cell specification, are evolutionary conserved, at least from the mouse, we lack significant knowledge about the molecular events that regulate the unique stem cell features of human embryonic stem cells. The pluripotent nature of human embryonic stem cells has attracted great interest in using them as a source of cells and tissues in cell therapy. Recent progress in human somatic cell nuclear transfer suggests that there may be a solution to the immunotolerance problems associated with the use of human embryonic stem cells in cell-replacement therapy. Thus, human embryonic stem cells supply the research community with unique research tools to study basic biological processes in human cells, model human genetic diseases and develop new cell-replacement therapies.

Animals↗
PubMed2006

Osteogenic differentiation of mouse embryonic stem cells and mouse embryonic fibroblasts in a three-dimensional self-assembling peptide scaffold.

In the present work, we studied the differentiation capacity of mouse embryonic stem cells (mESCs) and mouse embryonic fibroblasts (MEFs) to differentiate into osteoblast-like cells in a 3-dimensional (3D) self-assembling peptide scaffold, a synthetic nanofiber biomaterial with potential applications in regenerative medicine. We demonstrated that 2D and 3D systems promoted differentiation of mESCs into cells with an osteoblast-like phenotype consisting of osteopontin and collagen I marker expression, as well as high alkaline phosphatase (ALP) activity and calcium phosphate deposits. In 3D cultures the frequency of appearance of embryonic stem cell-like colonies was substantially greater, suggesting that the 3D microenvironment promoted the generation of a stem cell-like niche that allows undifferentiated stem cell maintenance. On the other hand, after MEFs were cultured in the 3D system with their regular growth medium, but not in the 2D system, they expressed osteopontin, up-regulated metalloproteinase activities, and acquired a distinct phenotype consisting of small, elongated cells with remaining mitotic activity. Furthermore, only 3D MEF cultures underwent osteoblast differentiation after osteogenic induction, based on matrix mineralization, collagen I synthesis, ALP activity, and expression of the osteoblast transcription factor Runx2, suggesting that the 3D environment promotes differentiation of MEFs into osteoblast-like cells. We propose that the 3D system provides a unique microenvironment that promotes differentiation of mESCs and MEFs into osteoblast-like cells.

Animals↗