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PubMed2004

Spontaneous differentiation of germ cells from human embryonic stem cells in vitro.

Little is known of molecular requirements for specification of human germ cells. However, it is likely that they are specified through the action of sequentially expressed genes just as in model organisms. We sought to determine whether human embryonic stem (ES) cell lines, like those of mice, might be capable of forming germ cells in vitro. We compared transcriptional profiles of three pluripotent human ES cells to those of isolated inner cell mass (ICM) cells. We found that ICM cells expressed NANOS1, STELLAR and OCT4, whereas undifferentiated human ES cells expressed these genes along with the germ cell-specific gene, DAZL. Upon ES cell differentiation into embryoid bodies (EBs), we observed a shift in expression from RNA and protein markers of immature germ cells to those indicative of mature germ cells, including expression of VASA, BOL, SCP1, SCP3, GDF9 and TEKT1. Although ability to test the function of these putative VASA positive germ cells is limited, these results demonstrate that differentiation of human ES cells into EBs in vitro results in formation of cells that express markers specific to gonocytes.

Animals↗
PubMed2006

Characterization and in vitro differentiation potential of a new human embryonic stem cell line, ReliCellhES1.

Human embryonic stem cells (hESCs) are an exceptionally useful tool for studies of human development and represent a potential source for transplantation therapies. At present, only a limited number of hESCs lines representing a very small sample of genetic diversity of the human populations are available. Here, we report the derivation and characterization of a new hESC line, ReliCellhES1. These cells, established from the inner cell mass (ICM) on mouse embryonic feeder (MEF) layer, satisfy the criteria that characterize pluripotent hESCs: The cell line expresses high levels of cell surface markers (such as SSEA-3, SSAEA-4, TRA-1-60 and TRA-1-81), transcription factor Oct-4, alkaline phosphatase (AP) and telomerase. The cell line retains normal karyotype in long-term culture and has a distinct identity as revealed by DNA fingerprinting by short tandem repeat (STR) analysis. Further, upon examination of the in vitro differentiation potential, ReliCellhES1 was found to be capable of giving rise to dopaminergic neurons, cardiomyocytes, pancreatic islets, and hepatocyte-like cells belonging to ectoderm, mesoderm, and endoderm lineages, respectively. To our knowledge, this is the first report of a well-characterized hES cell line from the Indian subcontinent.

Animals↗
PubMed2003

Pancreatic precursors and differentiated islet cell types from murine embryonic stem cells: an in vitro model to study islet differentiation.

Embryonic stem (ES) cells differentiating in vitro reproduce many facets of early embryonic development, including the expression of developmentally regulated transcription factors and the differentiation of multipotent precursor cells. ES cells were evaluated for their ability to differentiate into pancreatic and islet lineage-restricted stages including pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic precursor cells, early endocrine cell progenitors, and islet hormone-producing cells. Following growth and differentiation in nonselective medium containing serum, murine ES cells spontaneously differentiated into cells individually expressing each of the four major islet hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. PDX1 immunostaining cells appeared first, before hormone-positive cells had emerged. Hormone-positive cells appeared within focal clusters of cells coexpressing PDX1 and the nonclassical hormone markers peptide YY (YY) and islet amyloid polypeptide (IAPP) in combination with the definitive hormones, characteristic of endocrine cells appearing during early pancreaticogenesis. This system allows the investigation of many facets of islet development since it promotes the appearance of the complete range of islet phenotypes and reproduces important developmental stages of normal islet cytodifferentiation in differentiating ES cell cultures.

Amyloid↗
PubMed2005

A modified method for generation of neural precursor cells from cultured mouse embryonic stem cells.

The pluripotency and high proliferative capacity of embryonic stem (ES) cells make them an attractive source of different cell types for biomedical research and cell replacement therapies. It has been demonstrated that ES cells can be induced into neural precursor cells (NPCs) under conditions. NPCs can be expanded in large numbers for significant periods of time to provide a reliable source of cells for transplantation in neurodegenerative disorders and injury of the central nervous system. This study describes a modified method for generation of NPCs from cultured mouse ES cells.

Alkaline Phosphatase↗
PubMedDate not supplied

In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells.

The first report of the derivation of embryonic stem (ES) cell lines from human blastocysts had major implications for research into developmental biology and regenerative medicine. Finding efficient and reproducible methods to derive therapeutically useful cells from an ES cell source is a key feature of many regenerative medicine strategies. We have previously demonstrated that it is possible to induce osteogenic differentiation of murine ES cells by supplementing the culture medium with ascorbic acid, beta-glycerophosphate, and dexamethasone. This study investigated whether methods for driving osteogenic differentiation developed with murine ES cells could be applied successfully to human ES cells. The H1 line was propagated in vitro on murine feeder layers and shown to be pluripotent by expression of the markers Oct-4 and SSEA-4. Subsequently, differentiation was initiated via embryoid body (EB) formation and, after 5 days in suspension culture, cells harvested from EBs were replated in a medium containing osteogenic supplements. We found that the treatment regimen previously identified as optimal for murine ES cells, and in particular the addition of dexamethasone at specific time points, also induced the greatest osteogenic response from human ES cells. We identified mineralizing cells in vitro that immunostained positively for osteocalcin and found an increase in expression of an essential bone transcription factor, Runx2. When implanted into SCID mice on a poly-D, L-lactide (PDLLA) scaffold, the cells had the capacity to give rise to mineralized tissue in vivo. After 35 days of implantation, regions of mineralized tissue could be identified within the scaffold by von Kossa staining and immunoexpression of the human form of osteocalcin. We did not see any evidence of teratoma formation. These data therefore demonstrate the derivation of osteoblasts from pluripotent human ES cells with the capacity to form mineralized tissue both in vitro and in vivo. We have also shown that a culture methodology established for differentiation of murine ES cells was entirely transferable to human ES cells. Further development of this technology will result in the capacity to generate sufficient yields of osteogenic cells for use in skeletal tissue repair.

Animals↗
PubMed2005

Embryonic stem cells: understanding their history, cell biology and signalling.

Embryonic stem cells offer enormous potential as a source of a variety of differentiated cells for cell therapy, drug discovery and toxicology screening. With the creation of human embryonic stem cell lines we now have a resource with the potential to differentiate into every tissue of the body. To fully harness this resource it is necessary to understand their biology. Here we give a background to their history, describe interesting elements of their cell biology and introduce the underlying signalling mechanisms that control their ability to self-renew and differentiate.

Animals↗
PubMed2004

Three-dimensional porous alginate scaffolds provide a conducive environment for generation of well-vascularized embryoid bodies from human embryonic stem cells.

Differentiation of human embryonic stem cells (hESCs) can be instigated through the formation of embryo-like aggregates in suspension, termed human embryoid bodies (hEBs). Controlling cell aggregation and agglomeration during hEBs formation has a profound effect on the extent of cell proliferation and differentiation. In a previous work, we showed that control over hEBs formation and differentiation can be achieved via cultivation of hESC suspensions in a rotating bioreactor system. We now report that hEBs can be generated directly from hESC suspensions within three-dimensional (3D) porous alginate scaffolds. The confining environments of the alginate scaffold pores enabled efficient formation of hEBs with a relatively high degree of cell proliferation and differentiation; encouraged round, small-sized hEBs; and induced vasculogenesis in the forming hEBs to a greater extent than in static or rotating cultures. We therefore conclude that differentiation of hEBs can be induced and directed by physical constraints in addition to chemical cues.

Alginates↗
PubMed2005

[Establishment and characteristics of clonal human embryonic stem cell lines].

OBJECTIVE: To establish clonal human embryonic stem cell lines and investigate their biological characteristics. METHODS: Cells were derived from one inner cell mass of human blastocyst, multiplied for 20 passages, and then dissociated into single cell suspension by digestion with 0.5% trypsin. Single cell was picked up and plated into individual well of a 96-well plate containing feeder-layers directly under a dissection microscope. The outgrowth clones were passed by treatment with collagenase. Surface markers were detected by cytochemistry and histoimmunochemistry. Karyotypes were tested using standard G-banding techniques. The pluripotency was analyzed by inoculating cells into severe combined immunodeficient (SCID) mice. RESULTS: Two clonal human embryonic stem cell lines were established. Cells of these two lines possess the characteristics and differentiating potencies: normal 46 XX karyotypes; expressing a series of surface markers such as: alkaline phosphotase, stage-specific embryonic antigen (SSEA)-4, tumor recognition antigen (TRA)-1-60, TRA-1-81 etc; and forming teratomas comprising derivatives of three embryonic germ layers such as neural tissue, cartilage, squamous epithelium and columnar epithelium when injected into SCID mice. CONCLUSIONS: The two single cell-cloned human embryonic stem (hES) cell lines were derived in our laboratory. The cells possess stable biological characteristics of undifferentiated hES cells.

Cell Culture Techniques↗
PubMed2004

Artifactual insulin release from differentiated embryonic stem cells.

Several recent reports claim the generation of insulin-producing cells from embryonic stem cells via the differentiation of progenitors that express nestin. Here, we investigate further the properties of these insulin-containing cells. We find that although differentiated cells contain immunoreactive insulin, they do not contain proinsulin-derived C-peptide. Furthermore, we find variable insulin release from these cells upon glucose addition, but C-peptide release is never detected. In addition, many of the insulin-immunoreactive cells are undergoing apoptosis or necrosis. We further show that cells cultured in the presence of a phosphoinositide 3-kinase inhibitor, which previously was reported to facilitate the differentiation of insulin(+) cells, are not C-peptide immunoreactive but take up fluorescein isothiocyanate-labeled insulin from the culture medium. Together, these data suggest that nestin(+) progenitor cells give rise to a population of cells that contain insulin, not as a result of biosynthesis but from the uptake of exogenous insulin. We conclude that C-peptide biosynthesis and secretion should be demonstrated to claim insulin production from embryonic stem cell progeny.

Animals↗
PubMed2006

Multipotent cell fate of neural crest-like cells derived from embryonic stem cells.

Neural crest cells migrate throughout the embryo and differentiate into diverse derivatives: the peripheral neurons, cranial mesenchymal cells, and melanocytes. Because the neural crest cells have critical roles in organogenesis, detailed elucidation of neural crest cell differentiation is important in developmental biology. We recently reported that melanocytes could be induced from mouse ESCs. Here, we improved the culture system and showed the existence of neural crest-like precursors. The addition of retinoic acid to the culture medium reduced the hematopoiesis and promoted the expression of the neural crest marker genes. The colonies formed contained neural crest cell derivatives: neurons and glial cells, together with melanocytes. This suggested that neural crest-like cells assuming multiple cell fates had been generated in these present cultures. To isolate the neural crest-like cells, we analyzed the expression of c-Kit, a cell-surface protein expressed in the early stage of neural crest cells in vivo. The c-Kit-positive (c-Kit(+)) cells appeared as early as day 9 of the culture period and expressed the transcriptional factors Sox10 and Snail, which are expressed in neural crest cells. When the c-Kit(+) cells were separated from the cultures and recultured, they frequently formed colonies containing neurons, glial cells, and melanocytes. Even a single c-Kit(+) cell formed colonies that contained these three cell types, confirming their multipotential cell fate. The c-Kit(+) cells were also capable of migrating along neural crest migratory pathways in vivo. These results indicate that the c-Kit(+) cells isolated from melanocyte-differentiating cultures of ESCs are closely related to neural crest cells.

Animals↗
PubMed2003

Derivation of oocytes from mouse embryonic stem cells.

Continuation of mammalian species requires the formation and development of the sexually dimorphic germ cells. Cultured embryonic stem cells are generally considered pluripotent rather than totipotent because of the failure to detect germline cells under differentiating conditions. Here we show that mouse embryonic stem cells in culture can develop into oogonia that enter meiosis, recruit adjacent cells to form follicle-like structures, and later develop into blastocysts. Oogenesis in culture should contribute to various areas, including nuclear transfer and manipulation of the germ line, and advance studies on fertility treatment and germ and somatic cell interaction and differentiation.

Animals↗
PubMed2007

A murine respiratory-inducing niche displays variable efficiency across human and mouse embryonic stem cell species.

Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differentiating hESCs into respiratory lineages may benefit respiratory therapeutic programs. We previously demonstrated that 24% of all mouse embryonic stem cell (mESC) derivatives cocultured with embryonic day 11.5 (E11.5) mouse lung rudiments display immunoreactivity to the pneumonocyte II specific marker surfactant-associated protein C (Sftpc). Here we further investigate the effects of this inductive niche in terms of its competence to induce hESC derivative SFTPC immunoreactivity and the expression of other markers of terminal lung secretory units. When hESCs were cocultured as single cells, clumps of approximately 10 cells or embryoid bodies (EBs), hESC derivatives formed pan-keratin-positive epithelial tubules at high frequency (>30% of all hESC derivatives). However, human-specific SFTPC immunoreactivity associated with tubule formation only at low frequency (<0.1% of all hESC derivatives). Human-specific SFTPD and secretoglobin family 1A member 1 (SCGB1A1, also known as CC10) transcripts were detected by PCR after prolonged culture. Expression of other terminal lung secretory unit markers (TITF1, SFTPA, and SFTPB) was not detected at any time point analyzed. On the other hand, hESC derivatives cultured as plated EBs in media previously demonstrated to induce Sftpc expression in isolated mouse fetal tracheal epithelium expressed all terminal lung secretory unit markers examined. mESCs and hESCs thus display fundamental differences in their response to the E11.5 mouse lung inductive niche, and these data provide an important step in the delineation of signaling mechanisms capable of efficiently inducing hESC differentiation into terminal secretory units of the lung.

Animals↗
PubMedDate not supplied

Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test.

The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a reliable, scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of "beating areas" and to adapt the EST to applications in high-throughput screening systems we improved and expanded the EST protocol by establishing molecular endpoints of differentiation. The quantitative expression of sarcomeric myosin heavy chain (MHC) and alpha-actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and alpha-actinin and the differentiation into beating cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days, maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, alpha-actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data. Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to developmental toxicity using stem cell technology and technological advances in the field of gene expression analysis.

Actinin↗
PubMed2005

Human embryonic stem cell lines derived from the Chinese population.

Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

Animals↗
PubMed2002

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro.

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.

Animals↗
PubMed2001

Step-wise divergence of primitive and definitive haematopoietic and endothelial cell lineages during embryonic stem cell differentiation.

BACKGROUND: The developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage. RESULTS: We found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA-1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA-1- cells give rise to VE-cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE-cadherin+ cells. CONCLUSIONS: These observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE-cadherin+ endothelial cells in the development of haematopoietic and endothelial cells.

Animals↗
PubMedDate not supplied

Differentiation of embryonic stem cells after transplantation into peritoneal cavity of irradiated mice and expression of specific germ cell genes in pluripotent cells.

Permanent embryonic stem cell lines (ES cells) are considered as one of the most promising cellular sources for regenerative medicine. ES cells have a high proliferative potency and ability to differentiate into all kinds of somatic and germ cells. However, transplantation of undifferentiated ES cells into adult recipient tissue results in the formation of teratomas. To understand the mechanisms underlying self-renewal and determination of pluripotent cells, we investigated differentiation potencies of undifferentiated ES cells and differentiating embryoid bodies (EB). ES cells and EBs growing on acetate-cellulose membranes were transplanted into the peritoneal cavity of irradiated mice. Behavior and differentiation of transplanted cells were studied within 1, 2, 3, and 6 weeks after transplantation. No differences in the cell composition were found in the teratomas formed by ES cells and differentiating EBs. The pattern of expression of the genes specific for pluripotent and germ cells was studied in all types of experimental teratomas. The expression of oct4, stella, fragilis was detected in the teratomas, but nanog was not expressed. We conclude that pluripotent cells are retained in the experimental teratomas formed after transplantation of ES cells and EBs but the pattern of expression of the studied genes underwent changes.

Animals↗