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PubMed2005

Derivation and growing human embryonic stem cells on feeders derived from themselves.

Human embryonic stem cells (hESCs) are pluripotent. They have the potential to differentiate into every cell type of an organism. Since many human somatic cell types have the ability to support the growth of hESCs, cells differentiated from hESCs may also be able to support the growth of themselves. We tested this hypothesis by growing hESCs on feeders derived from themselves and demonstrated that such feeders did constitute an environment suitable for the derivation and long-term growth of hESCs. hESCs maintained in this system expressed all the markers indicative of the undifferentiated state and gave rise to cell types representative of all three primary germ layers upon differentiation. By modifying the genome of hESCs, feeders with special features can be derived and mass produced. The system will facilitate large-scale production of hESCs in a standardized animal pathogen-free environment.

Animals↗
PubMed2005

Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells.

Embryonic stem (ES) cells have the ability to self-replicate and differentiate into cells from all three germ layers, holding great promise for tissue regeneration applications. However, controlling the differentiation of ES cells and obtaining homogenous cell populations still remains a challenge. We hypothesize that a supportive three-dimensional (3D) environment provides ES cell-derived cells an environment that more closely mimics chondrogenesis in vivo. In the present study, the chondrogenic differentiation capability of ES cell-derived embryoid bodies (EBs) encapsulated in poly(ethylene glycol)-based (PEG) hydrogels was examined and compared with the chondrogenic potential of EBs in conventional monolayer culture. PEG hydrogel-encapsulated EBs and EBs in monolayer were cultured in vitro for up to 17 days in chondrogenic differentiation medium in the presence of transforming growth factor (TGF)-beta1 or bone morphogenic protein-2. Gene expression and protein analyses indicated that EB-PEG hydrogel culture upregulated cartilage-relevant markers compared with a monolayer environment and induction of chondrocytic phenotype was stimulated with TGF-beta1. Histology of EBs in PEG hydrogel culture with TGF-beta1 demonstrated basophilic extracellular matrix deposition characteristic of neocartilage. These findings suggest that EB-PEG hydrogel culture, with an appropriate growth factor, may provide a suitable environment for chondrogenic differentiation of intact ES cell-derived EBs.

Animals↗
PubMed2005

Microarray analysis of LIF/Stat3 transcriptional targets in embryonic stem cells.

Mouse embryonic stem (ES) cells can be propagated in vitro while retaining their properties of pluripotency and self-renewal under the continuous presence of leukemia inhibitor factor (LIF). An essential role has been attributed to subsequent activation of the Stat3 transcription factor in mediating LIF self-renewal response. To date, however, downstream target genes of Stat3 in ES cells are still unknown. To isolate these genes, we performed a microarray-based kinetic comparison of LIF-stimulated (undifferentiated) ES cells versus ES cells induced to differentiate by shutting down Stat3 activity through either LIF deprivation or, more specifically, expression of a Stat3 dominant-negative mutant. In each case, we chose the earliest time at which ES cells lose their self-renewal properties, as illustrated by a decrease in the number of embryoid bodies and blast cell colony formation as well as germ layer marker expression. Comparison of the two independent approaches revealed similarly regulated genes that are likely to be involved in the Stat3 effects on ES cell self-renewal. For instance, upregulation of growth factors such as the transforming growth factor-beta relative Lefty1 or transcriptional regulators such as Id1 and Id2 and down-regulation of the groucho-like protein Aes1 (grg5) were found. Promoter analysis of the aes1 gene revealed three functional Stat3 consensus sites, as shown by luciferase assays. Furthermore, chromatin immunoprecipitation experiment demonstrated that Stat3 is recruited to the promoter of aes1 in ES cells. These data demonstrated that the aes1 gene is a direct transcriptional target of Stat3 in ES cells.

Animals↗
PubMed2005

Human embryonic stem cells reprogram myeloid precursors following cell-cell fusion.

Here, we examine the ability of undifferentiated human embryonic stem cells (hESCs) to reprogram the nuclei of hESC-derived myeloid precursors following cell-cell fusion. Using an OP9 coculture system, we produced CD45+ CD33+ myeloperoxidase+ myeloid precursors from an Oct4-enhanced green fluorescent protein (EGFP) knock-in hESC line and demonstrated that Oct4-EGFP expression was extinguished in these precursors. Upon fusion with undifferentiated hESCs, EGFP expression from the endogenous Oct4 promoter/regulatory region was re-established, ESC-specific surface antigens and marker genes were expressed, and myeloid precursor-specific antigens were no longer detectable. When the hybrid cells were formed into embryoid bodies, upregulation of genes characteristic of the three germ layers and extraembryonic tissues occurred, indicating that the hybrid cells had the potential to differentiate into multiple lineages. Interestingly, the hybrid cells were capable of redifferentiating into myeloid precursors with efficiency comparable with that of diploid hESCs despite their neartetraploid chromosome complement. These results indicate that hESCs are capable of reprogramming nuclei from differentiated cells and that hESC hybrid cells provide a new model system for studying the mechanisms of nuclear reprogramming.

Animals↗
PubMed2006

Notch signaling is inactive but inducible in human embryonic stem cells.

The NOTCH signaling pathway performs a wide range of critical functions in a number of different cell types during development and differentiation. The role of NOTCH signals in human embryonic stem cells (hESCs) has not been tested. We measured the activity of canonical NOTCH signaling in undifferentiated embryonic stem (ES) cells and tested the requirement for NOTCH activity in hESC self-renewal or differentiation by growing hESCs in the presence of gamma-secretase inhibitors. Our results suggest that NOTCH signaling is not required for the propagation of undifferentiated human ES cells but instead is required for the maintenance of the differentiating cell types that accumulate in human ES cell cultures. Our studies suggest that NOTCH signaling is not required in human embryonic differentiation until the formation of extraembryonic, germ layer, or tissue-specific stem cells and progenitors.

Amyloid Precursor Protein Secretases↗
PubMed2005

Characterization of a new NIH-registered variant human embryonic stem cell line, BG01V: a tool for human embryonic stem cell research.

Human embryonic stem cells (hESCs) offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent manipulations. For example, identifying genes expressed during culture, as well as their temporal expression order after passaging and conditions influencing the formation of all three germ layers may be helpful for the production of functional beta islet cells used in treating type I diabetes. Although several hESC lines have demonstrated karyotypic instability during extended time in culture, select variant lines exhibit characteristics similar to their normal parental lines. Such variant lines may be excellent tools and abundant sources of cells for pilot studies and in vitro differentiation research in which chromosome number is not a concern, similar to the role currently played by embryonal carcinoma cell lines. It is crucial that the cells be surveyed at a genetic and proteomic level during extensive propagation, expansion, and manipulation in vitro. Here we describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Our characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination.

Cell Culture Techniques↗
PubMed2006

Derivation of distal lung epithelial progenitors from murine embryonic stem cells using a novel three-step differentiation protocol.

Embryonic stem cells (ESCs) are a potential source for the cell-based therapy of a wide variety of lung diseases for which the only current treatment is transplantation. However, distal lung epithelium, like many other endodermally derived somatic cell lineages, is proving difficult to obtain from both murine and human ESCs. We have previously obtained alveolar epithelium from ESCs, although final cell yield remained extremely low. Here, we present an optimized three-step protocol for the derivation of distal lung epithelial cells from murine ESCs. This protocol incorporates (a) treatment of early differentiating embryoid bodies with activin A to enhance the specification of the endodermal germ layer, followed by (b) adherent culture in serum-free medium and (c) the final application of a commercial, lung-specific medium. As well as enhancing the specification of distal lung epithelium, this protocol was found to yield cells with a phenotype most closely resembling that of lung-committed progenitor cells present in the foregut endoderm and the early lung buds during embryonic development. This is in contrast to our previous differentiation method, which drives differentiation through to mature type II alveolar epithelial cells. The derivation of a committed lung progenitor cell type from ESCs is particularly significant for regenerative medicine because the therapeutic implantation of progenitor cells has several clear advantages over the transplantation of mature, terminally differentiated somatic cells.

Activins↗
PubMed2006

Ablation of undifferentiated human embryonic stem cells: exploiting innate immunity against the Gal alpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) epitope.

Although undifferentiated human embryonic stem cells (hESCs) are tumorigenic, this capacity is lost after differentiation, and hESCs are being widely investigated for applications in regenerative medicine. To engineer protection against the unintentional transplantation of undifferentiated cells, we generated hESCs carrying a construct in which the alpha1,3-galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitope. The low-variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti-Neu5Gc, were not observed.

ABO Blood-Group System↗
PubMed2006

In vitro differentiation of human placenta-derived multipotent cells into hepatocyte-like cells.

Multipotent cells isolated from human term placenta (placenta-derived multipotent cells [PDMCs]) have been known to be able to differentiate into mesodermal lineage cells, including adipocytes and osteoclasts. The low infection rate and young age of placenta compared with other tissue origins of adult stem cells make theses cells attractive target for cell-based therapy. However, the differentiation potential of PDMCs toward hepatic cells has not been evaluated yet. In this study, we cultivated PDMCs with hepatic differentiation medium to evaluate the ability of these cells in differentiating toward hepatic cells. After treatment, the morphologies of differentiated PDMCs changed to polygonal epithelial cell-like. The differentiated cells not only show the hepatocyte-like morphologies but also express hepatocyte-specific markers, including albumin and cytokeratin 18. The bioactivity assays revealed that these hepatocyte-like cells could uptake lipoprotein and store glycogen. Furthermore, the addition of rifampicin increased the gene expression of CYP3A4, which is similar with the activities of human liver cells. According to our previous results, PDMCs were capable of differentiating into mesodermal and ectodermal lineage cells. Our results indicate that PDMCs can differentiate into three germ layer cells, which is similar to embryonic stem cells. In conclusion, placenta might be an easily accessible source for progenitor cells that are capable of differentiating toward hepatocyte-like cells in vitro.

Albumins↗
PubMed2006

A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation.

Human embryonic stem cells (hESCs) exhibit pluripotency and indefinite proliferation and are a potential source of cells for transplantation therapies and drug discovery. These applications will require large amounts of hESCs. However, hESCs are difficult to culture and maintain at larger scales, in part because of their low resistance to dissociation during passaging. To circumvent this, we developed a simple and easy method for establishing hESC sublines tolerant of complete dissociation. These cells exhibit high replating efficiency and also high cloning efficiency, and they maintain their ability to differentiate into the three germ layers. Several sublines have no detectable abnormalities in their karyotypes, and they retained their characteristics under feeder-free culture conditions and after freeze-thawing. Thus, these hESC sublines would be valuable for hESC applications.

Biomarkers↗
PubMed2006

Molecular analysis of LEFTY-expressing cells in early human embryoid bodies.

Human ESCs (HESCs) are self-renewing pluripotent cell lines that are derived from the inner cell mass of blastocyst-stage embryos. These cells can produce terminally differentiated cells representing the three embryonic germ layers. We thus hypothesized that during the course of in vitro differentiation of HESCs, progenitor-like cells are transiently formed. We demonstrated that LEFTY proteins, which are known to play a major role during mouse gastrulation, are transiently expressed during HESC differentiation. Moreover, LEFTY proteins seemed to be exclusively expressed by a certain population of cells in the early human embryoid bodies that does not overlap with the population expressing the ESC marker OCT4. We also showed that LEFTY expression is regulated at the cellular transcription level by molecular labeling of LEFTY-positive cells. A DNA microarray analysis of LEFTY-overexpressing cells revealed a signature of cell surface markers such as CADHERIN 2 and 11. Expression of LEFTY controlled by NODAL appears to have a substantial role in mesodermal origin cell population establishment, since inhibition of NODAL activity downregulated expression not only of LEFTY A and LEFTY B but also of BRACHYURY, an early mesodermal marker. In addition, other mesodermal lineage-related genes were downregulated, and this was accompanied by an upregulation in ectoderm-related genes. We propose that during the initial step of HESC differentiation, mesoderm progenitor-like cells appear via activation of the NODAL pathway. Our analysis suggests that in vitro differentiation of HESCs can model early events in human development.

Animals↗
PubMed2006

Derivation of human embryonic stem cells from developing and arrested embryos.

Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. However, usage of embryos and derivation of hESC for research and potential medical application has resulted in polarized ethical debates since the process involves destruction of viable developing human embryos. Here we describe that not only developing embryos (morulae and blastocysts) of both good and poor quality but also arrested embryos could be used for the derivation of hESC. Analysis of arrested embryos demonstrated that these embryos express pluripotency marker genes such OCT4, NANOG, and REX1. Derived hESC lines also expressed specific pluripotency markers (TRA-1-60, TRA-1-81, SSEA4, alkaline phosphatase, OCT4, NANOG, TERT, and REX1) and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. All of the new lines, including lines derived from late arrested embryos, have normal karyotypes. These results demonstrate that arrested embryos are additional valuable resources to surplus and donated developing embryos and should be used to study early human development or derive pluripotent hESC.

Embryo, Mammalian↗
PubMed2006

Concise review: embryonic stem cells: a new tool to study osteoblast and osteoclast differentiation.

Bone remodeling involves synthesis of organic matrix by osteoblasts and bone resorption by osteoclasts. A tight collaboration between these two cell types is essential to maintain a physiological bone homeostasis. Thus, osteoblasts control bone-resorbing activities and are also involved in osteoclast differentiation. Any disturbance between these effectors leads to the development of skeletal abnormalities and/or bone diseases. In this context, the determination of key genes involved in bone cell differentiation is a new challenge to treat any skeletal disorders. Different models are used to study the differentiation process of these cells, but all of them use pre-engaged progenitor cells, allowing us to study only the latest stages of the differentiation. Embryonic stem (ES) cells come from the inner mass of the blastocyst prior its implantation to the uterine wall. Because of their capacity to differentiate into all germ layers, and so into all tissues of the body, ES cells represent the best model by which to study earliest stages of bone cell differentiation. Osteoblasts are generated by two methods, one including the generation of embryoid body, the other not. Mineralizing cells are obtained after 2 weeks of culture and express all the specific osteoblastic markers (alkaline phosphatase, type I collagen, osteocalcin, and others). Osteoclasts are generated from a single-cell suspension of ES cells seeded on a feeder monolayer, and bone-resorbing cells expressing osteoclastic markers such as tartrate-resistant alkaline phosphatase or receptor activator of nuclear factor kappaB are obtained within 11 days. The aim of this review is to present recent discoveries and advances in the differentiation of both osteoblasts and osteoclasts from ES cells.

Bone Diseases↗
PubMed2006

Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability.

Although all human ESC (hESC) lines have similar morphology, express key pluripotency markers, and can differentiate toward primitive germ layers in vitro, the lineage-specific developmental potential may vary between individual lines. In the current study, four hESC lines were cultured in the same feeder-free conditions to provide a standardized platform for interline analysis. A high-throughput, forced-aggregation system involving centrifugation of defined numbers of hESCs in V-96 plates (V-96FA) was developed to examine formation, growth, and subsequent cardiomyocyte differentiation from >22,000 EBs. Homogeneity of EBs formed by V-96FA in mouse embryo fibroblast-conditioned medium was significantly improved compared with formation in mass culture (p < .02; Levene's test). V-96FA EB formation was successful in all four lines, although significant differences in EB growth were observed during the first 6 days of differentiation (p = .044 to .001; one-way analysis of variance [ANOVA]). Cardiomyocyte differentiation potential also varied; 9.5% +/- 0.9%, 6.6% +/- 2.4%, 5.2% +/- 3.1%, and 1.6% +/- 1.0% beating EBs were identified for HUES-7, NOTT2, NOTT1, and BG01, respectively (p = .008; one-way ANOVA). Formation of HUES-7 V-96FA EBs in defined medium containing activin A and basic fibroblast growth factor resulted in 23.6% +/- 3.6% beating EBs, representing a 13.1-fold increase relative to mass culture (1.8% +/- 0.7%), consistent with an observed 14.8-fold increase in MYH6 (alphaMHC) expression by real-time polymerase chain reaction. In contrast, no beating areas were derived from NOTT1-EBs and BG01-EBs formed in defined medium. Thus, the V-96FA system highlighted interline variability in EB growth and cardiomyocyte differentiation but, under the test conditions described, identified HUES-7 as a line that can respond to cardiomyogenic stimulation.

Animals↗
PubMed2007

Concise review: regulation of embryonic stem cell lineage commitment by mitogen-activated protein kinases.

Embryonic stem (ES) cells can give rise, in vivo, to the ectodermal, endodermal, and mesodermal germ layers and, in vitro, can differentiate into multiple cell lineages, offering broad perspectives in regenerative medicine. Understanding the molecular mechanisms governing ES cell commitment is an essential challenge in this field. The mitogen-activated protein kinase (MAPK) pathways extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38MAPK are able to regulate ES commitment from early steps of the process to mature differentiated cells. Whereas the ERK pathway inhibits the self-renewal of ES cells, upon commitment this pathway is involved in the development of extraembryonic tissues, in early mesoderm differentiation, and in the formation of mature adipocytes; p38MAPK displays a large spectrum of action from neurons to adipocytes, and JNK is involved in both ectoderm and primitive endoderm differentiations. Furthermore, for a given pathway, several of these effects are isoform-dependent, revealing the complexity of the cellular response to activation of MAPK pathways. Regarding tissue regeneration, the potential outcome of systematic analysis of the function of different MAPKs in different ES cell differentiation programs is discussed. Disclosure of potential conflicts of interest is found at the end of this article.

Animals↗
PubMed2003

Multilineage potential of homozygous stem cells derived from metaphase II oocytes.

Human stem cells derived from human fertilized oocytes, fetal primordial germ cells, umbilical cord blood, and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However, the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach, employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D), we established stem cell lines homozygous for H-2-B and H-2-D, respectively. The undifferentiated cells retained a normal karyotype, expressed stage-specific embryonic antigen-1 and Oct4, and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition, these cells demonstrated the potential for in vitro differentiation into endoderm, neuronal, and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes, and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible "homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation, while decreasing the ethical concerns surrounding human embryonic stem cell research.

Animals↗
PubMed2003

Selective ablation of human embryonic stem cells expressing a "suicide" gene.

Over the past few years, technological procedures have been developed for utilizing stem cells in transplantation medicine. Human embryonic stem (ES) cells can produce an unlimited number of differentiated cells and are, therefore, considered a potential source of cellular material for use in transplantation medicine. However, serious clinical problems can arise when uncontrolled cell proliferation occurs following transplantation. To avoid these potential problems, we genetically engineered human ES cell lines to express the herpes simplex virus thymidine kinase (HSV-tk) gene. Expression of the HSV-tk protein renders the ES cells sensitive to the U.S. Food and Drug Administration-approved drug ganciclovir, inducing destruction of HSV-tk(+) cells at ganciclovir concentrations that are nonlethal to other cell types. The reversion rate of engineered cells was low even under prolonged selection with ganciclovir. The HSV-tk(+) clones retained a normal karyotype and the ability to differentiate to cells from all three germ layers. Most importantly, tumors that arose in mice following subcutaneous injection of HSV-tk(+) human ES cells could be ablated in vivo by administration of ganciclovir. By utilizing these cell lines, safety levels can be improved in transplantations involving tissues derived from human ES cells.

Animals↗
PubMed2004

Differentiation prevents assessment of neural stem cell pluripotency after blastocyst injection.

Earlier studies reported that neural stem (NS) cells injected into blastocysts appeared to be pluripotent, differentiating into cells of all three germ layers. In this study, we followed in vitro green fluorescent protein (GFP)-labeled NS and embryonic stem (ES) cells injected into blastocysts. Forty-eight hours after injection, significantly fewer blastocysts contained GFP-NS cells than GFP-ES cells. By 96 hours, very few GFP-NS cells remained in blastocysts compared with ES cells. Moreover, 48 hours after injection, GFP-NS cells in blastocysts extended long cellular processes, ceased expressing the NS cell marker nestin, and instead expressed the astrocytic marker glial fibrillary acidic protein. GFP-ES cells in blastocysts remained morphologically undifferentiated, continuing to express the pluripotent marker stage-specific embryonic antigen-1. Selecting cells from the NS cell population that preferentially formed neurospheres for injection into blastocysts resulted in identical results. Consistent with this in vitro behavior, none of almost 80 mice resulting from NS cell-injected blastocysts replaced into recipient mothers were chimeric. These results strongly support the idea that NS cells cannot participate in chimera formation because of their rapid differentiation into glia-like cells. Thus, these results raise doubts concerning the pluripotency properties of NS cells.

Animals↗