On-Line Updating of Spatial Information Druing Locomotion Without Vision

Journal of Motor Behavior. Mar, 1999  |  Pubmed ID: 11177618

Two experiments are reported in which the control of locomotion without vision was investigated. In Experiment 1, subjects (N = 10) made similar, although less functional, locomotor adjustments when walking without vision to a target than they did when walking with vision. That result suggests that while walking without vision, the subjects updated their positions on-line with respect to a representation of the target rather than operating from a preformulated action plan. In Experiment 2, there was a significant weakening and loss of functionality of the locomotor adjustments when subjects (N = 10) had to walk without vision the correct distance to the target but in a direction opposite to its true location, as compared with when they walked without vision directly to the target. That finding suggests that the subjects were nonvisually updating their positions not with respect to an abstract representation of the target's distance but with respect to a representation of its relative location within the task environment.

Efficient Method for Expressing Transgenes in Nonhuman Primate Embryos Using a Stable Episomal Vector

Molecular Reproduction and Development. May, 2002  |  Pubmed ID: 11933162

Transgenesis in the nonhuman primate can enhance the study of human biology by providing animal models for the study of primate-specific physiology, pathophysiology, and embryonic development. Progress with this technology has been hindered by the inherent inefficiency of transgenesis, transgene silencing, and practical restrictions on the production of sufficient pronuclear stage nonhuman primate zygotes. We have developed a novel technique using an Epstein Barr virus (EBV)-based episomal vector to produce rhesus monkey (Macaca mulatta) embryos expressing a transgene. Plasmid DNA containing the latent origin of replication, oriP, and Epstein Barr Nuclear Antigen-1 (EBNA-1) of EBV, as well as a CMV IE-enhanced green fluorescent protein (eGFP) expression cassette, was introduced into rhesus embryos by direct pronuclear microinjection. We detected eGFP in early cleavage stage embryos (4-8 cell) and throughout the duration of culture (day 8-9 blastocysts) by epifluorescent microscopy. A 50% transduction rate was obtained with the EBV-based vector. Microinjected embryos expressed eGFP and retained their developmental capacity as evidenced by development to the blastocyst stage. EBV-based vectors present a novel and efficient means of delivering transgenes for the study of the molecular control of primate embryonic development.

Surface Antigens of Human Embryonic Stem Cells: Changes Upon Differentiation in Culture

Journal of Anatomy. Mar, 2002  |  Pubmed ID: 12033729

We have analysed the surface antigen phenotype of a human embryonic stem (hES) cell line (H7) and the changes that occur upon differentiation induced by retinoic acid, hexamethylene bisacetamide and dimethylsulphoxide. The undifferentiated stem cells expressed Stage Specific Embryonic Antigen-3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-8 but not SSEA1. In these characteristics they closely resemble human embryonal carcinoma (EC) cells derived from testicular teratocarcinomas, and are distinct from murine EC and ES cells. The undifferentiated cells also expressed the liver/bone/kidney isozyme of alkaline phosphatase detected by antibody TRA-2-54, the class 1 major histocompatability antigens, HLA-ABC, and the human Thy1 antigen. Differentiation of hES cells was induced by retinoic acid, HMBA and DMSO with the appearance of various cell types including neurons and muscle cells. The surface antigens characteristically expressed by hES cells were down-regulated following induction of differentiation and other antigens appeared, notably several ganglioside glycolipids detected by antibodies VIN-IS-56 (GD3 and GD2), VIN-2PB-22 (GD2), A2B5 (GT3) and ME311 (9-O-acetyl-GD3). Whereas the expression of HLA was slightly down-regulated upon differentiation, its expression was strongly induced by interferon-y in both the undifferentiated and the differentiated cells, although the induction in the differentiated cultures was considerably stronger than in the stem cells. In all of these features the human ES cells, and their pattern of differentiation, resembled the pluripotent human EC cell line NTERA-2 although clearly the range of cells generated by the hES cells was considerably greater.

BMP4 Initiates Human Embryonic Stem Cell Differentiation to Trophoblast

Nature Biotechnology. Dec, 2002  |  Pubmed ID: 12426580

The excitement and controversy surrounding the potential role of human embryonic stem (ES) cells in transplantation therapy have often overshadowed their potentially more important use as a basic research tool for understanding the development and function of human tissues. Human ES cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we show that bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor-beta (TGF-beta) superfamily, induces the differentiation of human ES cells to trophoblast. DNA microarray, RT-PCR, and immunoassay analyses demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). These results underscore fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.

High-level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors

Stem Cells (Dayton, Ohio). 2003  |  Pubmed ID: 12529558

Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor 1alpha promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-beta gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5' end of the chicken beta-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34(+) hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.

Homologous Recombination in Human Embryonic Stem Cells

Nature Biotechnology. Mar, 2003  |  Pubmed ID: 12577066

Homologous recombination applied to mouse embryonic stem (ES) cells has revolutionized the study of gene function in mammals. Although most often used to generate knockout mice, homologous recombination has also been applied in mouse ES cells allowed to differentiate in vitro. Homologous recombination is an essential technique if human ES cells are to fulfill their promise as a basic research tool. It also has important implications for ES cell-based transplantation and gene therapies. Significant differences between mouse and human ES cells have hampered the development of homologous recombination in human ES cells. High, stable transfection efficiencies in human ES cells have been difficult to achieve, and, in particular, electroporation protocols established for mouse ES cells work poorly in human ES cells. Also, in contrast to their murine counterparts, human ES cells cannot be cloned efficiently from single cells, making it difficult to screen for rare recombination events. Here we report an electroporation approach, based on the physical characteristics of human ES cells, that we used to successfully target HPRT1, the gene encoding hypoxanthine phosphoribosyltransferase-1 (HPRT1), and POU5F1, the gene encoding octamer-binding transcription factor 4 (Oct4; also known as POU domain, class 5, transcription factor 1 (POU5F1)).

Human Embryonic Stem Cells Develop into Multiple Types of Cardiac Myocytes: Action Potential Characterization

Circulation Research. Jul, 2003  |  Pubmed ID: 12791707

Human embryonic stem (hES) cells can differentiate in vitro, forming embryoid bodies (EBs) composed of derivatives of all three embryonic germ layers. Spontaneously contracting outgrowths from these EBs contain cardiomyocytes (CMs); however, the types of human CMs and their functional properties are unknown. This study characterizes the contractions and action potentials (APs) from beating EB outgrowths cultured for 40 to 95 days. Spontaneous and electrical field-stimulated contractions were measured with video edge-detection microscopy. beta-Adrenergic stimulation with 1.0 micromol/L isoproterenol resulted in a significant increase in contraction magnitude. Intracellular electrical recordings using sharp KCl microelectrodes in beating EB outgrowths revealed three distinct classes of APs: nodal-like, embryonic atrial-like, and embryonic ventricular-like. The APs were described as embryonic based on the relatively depolarized resting membrane potential and slow AP upstroke. Repeated impalements of an individual beating outgrowth revealed a reproducible AP morphology recorded from different cells, suggesting that each outgrowth is composed of a predominant cell type. Complex functional properties typical of cardiac muscle were observed in the hES cell-derived CMs including rate adaptation of AP duration and provoked early and delayed afterdepolarizations. Repolarization of the AP showed a significant role for IKr based on E-4031 induced prolongation of AP duration as anticipated for human CMs. In conclusion, hES cells can differentiate into multiple types of CMs displaying functional properties characteristic of embryonic human cardiac muscle. Thus, hES provide a renewable source of distinct types of human cardiac myocytes for basic research, pharmacological testing, and potentially therapeutic applications.

Crystallographic Identification of a Noncompetitive Inhibitor Binding Site on the Hepatitis C Virus NS5B RNA Polymerase Enzyme

Journal of Virology. Jul, 2003  |  Pubmed ID: 12805457

The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the "thumb" domain, about 30 A from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization.

Gene Expression Patterns in Human Embryonic Stem Cells and Human Pluripotent Germ Cell Tumors

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2003  |  Pubmed ID: 14595015

Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.

Recurrent Gain of Chromosomes 17q and 12 in Cultured Human Embryonic Stem Cells

Nature Biotechnology. Jan, 2004  |  Pubmed ID: 14661028

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.

Trophoblast Differentiation in Embryoid Bodies Derived from Human Embryonic Stem Cells

Endocrinology. Apr, 2004  |  Pubmed ID: 14684604

Trophoblast differentiation and early placental development are essential for the establishment of pregnancy, yet these critical events are not readily investigated in human pregnancy. We used embryoid bodies (EBs) prepared from human embryonic stem (hES) cells as an in vitro model of early human development. The levels of human chorionic gonadotropin (hCG), progesterone, and estradiol-17beta in medium from hES cell-derived EBs grown in suspension culture for 1 wk were higher than unconditioned culture medium or medium from undifferentiated hES cells or spontaneously differentiated hES cell colonies. EBs were explanted into Matrigel (MG) "rafts" and cultured for up to 53 d. During the first 7-10 d of three-dimensional growth in MG, small protrusions appeared on the outer surface of EBs, some of which subsequently extended into multicellular outgrowths. The secretion of hCG, progesterone, and estradiol-17beta began to increase on approximately d 20 of MG culture and remained dramatically elevated over the next 30 d. EBs maintained in suspension culture failed to demonstrate this elevation in hormone secretion. Suspension-cultured and MG-embedded EBs exhibited widespread expression of cytokeratins 7/8, demonstrating extensive epithelial differentiation as well as consistent hCG expression. We propose that hES cell-derived EBs may be a useful model for investigation of human trophoblast differentiation and placental morphogenesis.

Arrays for the Combinatorial Exploration of Cell Adhesion

Journal of the American Chemical Society. Sep, 2004  |  Pubmed ID: 15339142

A new method for the fabrication of arrays of self-assembled monolayers (SAMs) of alkane thiols (ATs) on gold to combinatorially assay surfaces for cell adhesion is reported. A fluorous SAM, which is both cytophobic and solvophobic, was used as the background between the array features. The resulting solvophobic background permits the application of an assembly after conjugation strategy for fabrication. SAMs containing mixtures of ATs and peptide-terminated ATs were generated. Multiple cell types demonstrated differential and specific binding to these surfaces. Additionally, pluripotent human embryonic stem cells proliferated on surfaces generated by this method.

Crystal Structure of Human ABAD/HSD10 with a Bound Inhibitor: Implications for Design of Alzheimer's Disease Therapeutics

Journal of Molecular Biology. Sep, 2004  |  Pubmed ID: 15342248

The enzyme 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), also known as amyloid beta-peptide-binding alcohol dehydrogenase (ABAD), has been implicated in the development of Alzheimer's disease. This protein, a member of the short-chain dehydrogenase/reductase family of enzymes, has been shown to bind beta-amyloid and to participate in beta-amyloid neurotoxicity. We have determined the crystal structure of human ABAD/HSD10 complexed with NAD(+) and an inhibitory small molecule. The inhibitor occupies the substrate-binding site and forms a covalent adduct with the NAD(+) cofactor. The crystal structure provides a basis for the design of potent, highly specific ABAD/HSD10 inhibitors with potential application in the treatment of Alzheimer's disease.

Functional Endothelial Cells Derived from Rhesus Monkey Embryonic Stem Cells

Blood. Feb, 2004  |  Pubmed ID: 14563647

We have used rhesus monkey embryonic stem (ES) cells to study endothelial cell development. Rhesus ES cells (R366.4 cell line) exposed to medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF) assumed a relatively uniform endothelial cell morphology and could be propagated and expanded with a consistent phenotype and normal karyotype. When placed in Matrigel, these rhesus ES cell-derived endothelial cells (RESDECs) formed capillary-like structures characteristic of endothelial cells. Immunohistochemical and flow cytometric analysis of RESDECs showed that they take up acetylated low-density lipoprotein (LDL), express CD146, von Willebrand factor, and the integrin alpha v beta 3, and bind the lectin ulex europaeus agglutinin-1. These cells also express the VEGF receptor Flk-1 and secrete VEGF. When introduced in a Matrigel plug implanted subcutaneously in mice, RESDECs formed intact vessels and recruited new endothelial cell growth. In vivo function was demonstrated by coinjection of RESDECs with murine tumor cells subcutaneously into immunocompromised adult mice. RESDECs injected alone did not form measurable tumors. Tumor cells grew more rapidly and had increased vascularization when coinjected with the RESDECs. Immunohistochemical staining demonstrated that the RESDECs participated in forming the tumor neovasculature. RESDECs provide a novel means to examine the mechanisms of endothelial cell development, and may open up new therapeutic strategies.

Human Embryonic Stem Cell-derived CD34+ Cells: Efficient Production in the Coculture with OP9 Stromal Cells and Analysis of Lymphohematopoietic Potential

Blood. Jan, 2005  |  Pubmed ID: 15374881

Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 10(7) CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell-derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase-positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.

A Germ Cell Origin of Embryonic Stem Cells?

Development (Cambridge, England). Jan, 2005  |  Pubmed ID: 15623802

Because embryonic stem (ES) cells are generally derived by the culture of inner cell mass (ICM) cells, they are often assumed to be the equivalent of ICM cells. However, various evidence indicates that ICM cells transition to a different cell type during ES-cell derivation. Historically, ES cells have been believed to most closely resemble pluripotent primitive ectoderm cells derived directly from the ICM. However, differences between ES cells and primitive ectoderm cells have caused developmental biologists to question whether ES cells really have an in vivo equivalent, or whether their properties merely reflect their tissue culture environment. Here, we review recent evidence that the closest in vivo equivalent of an ES cell is an early germ cell.

Differentiation of Human Embryonic Stem Cells Occurs Through Symmetric Cell Division

Stem Cells (Dayton, Ohio). Feb, 2005  |  Pubmed ID: 15671139

Embryonic (ES) stem cells can be expanded indefinitely, yet retain the ability to form all cell types of the body. Here we report that human ES cells differentiate exclusively by symmetric cell division in each of four distinct differentiation conditions examined. This suggests that, in some respects, ES cells more closely resemble precursor or transit amplifying cells rather than adult stem cells.

Basic FGF and Suppression of BMP Signaling Sustain Undifferentiated Proliferation of Human ES Cells

Nature Methods. Mar, 2005  |  Pubmed ID: 15782187

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.

Directed Differentiation of Dopaminergic Neuronal Subtypes from Human Embryonic Stem Cells

Stem Cells (Dayton, Ohio). Jun-Jul, 2005  |  Pubmed ID: 15917474

How dopamine (DA) neuronal subtypes are specified remains unknown. In this study we show a robust generation of functional DA neurons from human embryonic stem cells (hESCs) through a specific sequence of application of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH). Treatment of hESC-derived Sox1+ neuroepithelial cells with FGF8 and SHH resulted in production of tyrosine hydroxylase (TH)-positive neurons that were mostly bipolar cells, coexpression with gamma-aminobutyric acid, and lack of midbrain marker engrailed 1 (En1) expression. However, FGF8 treatment of precursor cells before Sox1 expression led to the generation of a similar proportion of TH+ neurons characteristic of midbrain projection DA neurons with large cell bodies and complex processes and coexpression of En1. This suggests that one mechanism of generating neuronal subtypes is temporal availability of morphogens to a specific group of precursors. The in vitro-generated DA neurons were electrophysiologically active and released DA in an activity-dependent manner. They may thus provide a renewable source of functional human DA neurons for drug screening and development of sustainable therapeutics for disorders affecting the DA system.

The Effects of Adult Guidance and Peer Discussion on the Development of Children's Representations: Evidence from the Training of Pedestrian Skills

British Journal of Psychology (London, England : 1953). May, 2005  |  Pubmed ID: 15969830

It was hypothesized that practical training is effective in improving children's pedestrian skills because adult scaffolding and peer discussion during training specifically promote E3 level representation (linguistically-encoded, experientially-grounded, generalizable knowledge), as defined by Karmiloff-Smith's (1992) representational redescription (RR) model. Two studies were conducted to examine in detail the impact of this social input in the context of simulation-based training in roadside search skills. A group of 5-8-year-olds were pre-tested on ability to detect relevant road-crossing features. They then participated in four training sessions designed to promote attunement to these, under peer discussion versus adult guidance conditions (Study 1), and adult-child versus adult-group conditions (Study 2). Performance at post-test was compared with that of controls who underwent no training. Study 1 found that children in the adult guidance condition improved significantly more than those in the peer discussion or control conditions, and this improvement was directly attributable to appropriation of E3 level representations from adult dialogue. Study 2 found that progress was greater still when adult scaffolding was supplemented by peer discussion, with E3 level representation attributable to the children's exploration of conflicting ideas. The implications of these findings for the RR model and for practical road safety education are discussed.

Human ERas Gene Has an Upstream Premature Polyadenylation Signal That Results in a Truncated, Noncoding Transcript

Stem Cells (Dayton, Ohio). Nov-Dec, 2005  |  Pubmed ID: 16081664

The ERas gene is expressed in mouse embryonic stem (ES) cells and promotes their in vitro proliferation and tumorigenicity. We analyzed the expression of the human ERas gene in human ES cells by reverse transcription-polymerase chain reaction (RT-PCR) and serial analysis of gene expression but could not detect a full-length coding transcript. Sequence analysis predicted a premature polyadenylation signal for the human ERas transcript, which we confirmed by 3' RACE analysis. By RT-PCR, we identified a truncated noncoding transcript in human ES cells that is downregulated during differentiation, suggesting conserved tissue specificity of the promoter region. Previous reports and expressed sequence tag databases indicate that orthologues of this gene are expressed in other mammals, including the mouse, dog, and cow, which suggests that it became a silenced pseudogene relatively recently in mammalian evolution. In addition to the premature polyadenylation site, both the human and chimpanzee ERas genes include typical Alu-S retrotransposon insertions that could also influence expression at this locus. The lack of ERas expression in human ES cells suggests that they could have significantly different tumorigenic properties than mouse ES cells.

Influence of Virtual Reality Training on the Roadside Crossing Judgments of Child Pedestrians

Journal of Experimental Psychology. Applied. Sep, 2005  |  Pubmed ID: 16221036

The roadside crossing judgments of children aged 7, 9, and 11 years were assessed relative to controls before and after training with a computer-simulated traffic environment. Trained children crossed more quickly, and their estimated crossing times became better aligned with actual crossing times. They crossed more promptly, missed fewer safe opportunities to cross, accepted smaller traffic gaps without increasing the number of risky crossings, and showed better conceptual understanding of the factors to be considered when making crossing judgments. All age groups improved to the same extent, and there was no deterioration when children were retested 8 months later. The results are discussed in relation to theoretical arguments concerning the extent to which children's pedestrian judgments are amenable to training.

Human Embryonic Stem Cell-derived Hematopoietic Cells Are Capable of Engrafting Primary As Well As Secondary Fetal Sheep Recipients

Blood. Mar, 2006  |  Pubmed ID: 16278307

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.

Basic Fibroblast Growth Factor Support of Human Embryonic Stem Cell Self-renewal

Stem Cells (Dayton, Ohio). Mar, 2006  |  Pubmed ID: 16282444

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibro-blast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.

Derivation of Human Embryonic Stem Cells in Defined Conditions

Nature Biotechnology. Feb, 2006  |  Pubmed ID: 16388305

We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

Directed Differentiation of Human Embryonic Stem Cells into Functional Dendritic Cells Through the Myeloid Pathway

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2006  |  Pubmed ID: 16493050

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step, we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then, expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of Ag processing, triggering naive T cells in MLR, and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, because ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance.

Leukosialin (CD43) Defines Hematopoietic Progenitors in Human Embryonic Stem Cell Differentiation Cultures

Blood. Sep, 2006  |  Pubmed ID: 16757688

During hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile.

Feeder-independent Culture of Human Embryonic Stem Cells

Nature Methods. Aug, 2006  |  Pubmed ID: 16862139

A Severe De Novo Methylation of Episomal Vectors by Human ES Cells

Biochemical and Biophysical Research Communications. Nov, 2006  |  Pubmed ID: 16973130

Episomal vectors can allow efficient genetic modification of cells and have the potential advantage of avoiding chromosomal position of integration effects. Here we explore the use of an Epstein-Barr virus-based episomal vector with human embryonic stem (ES) cells, and find high initial transfection rates, but a rapid loss of reporter gene expression. Similar to mouse ES cells, human ES cells express high levels of the de novo DNA methyltransferases, and we detected dramatic CpG methylation and minor non-CpG methylation on the episomes recovered from the human ES cells 7 days after the transfection, which was not present on the same episome recovered from 293 cells. Interestingly, the oriP region of the episomes was relatively excluded from this methylation. These findings define some of the limitations of using episomal vectors with human ES cells and offer a unique platform for analyzing epigenetic gene silencing in human ES cells.

Microarray Analysis of Stem Cells and Differentiation

Methods in Enzymology. 2006  |  Pubmed ID: 17161699

Microarrays have revolutionized molecular biology and enabled biologists to perform global analysis on the expression of tens of thousands of genes simultaneously. They have been widely used in gene discovery, biomarker determination, disease classification, and studies of gene regulation. Microarrays have been applied in stem cell research to identify major features or expression signatures that define stem cells and characterize their differentiation programs toward specific lineages. Here we provide a review of the microarray technology, including the introduction of array platforms, experimental designs, RNA isolation and amplification, array hybridization, and data analysis. We also detail examples that apply microarray technology to address several of the main questions in stem cell biology.

Human Embryonic Stem Cells Reprogram Myeloid Precursors Following Cell-cell Fusion

Stem Cells (Dayton, Ohio). Jan, 2006  |  Pubmed ID: 16210403

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.

Nanog and Transcriptional Networks in Embryonic Stem Cell Pluripotency

Cell Research. Jan, 2007  |  Pubmed ID: 17211451

Several extrinsic signals such as LIF, BMP and Wnt can support the self-renewal and pluripotency of embryonic stem (ES) cells through regulating the "pluripotent genes." A unique homeobox transcription factor, Nanog, is one of the key downstream effectors of these signals. Elevated level of Nanog can maintain the mouse ES cell self-renewal independent of LIF and enable human ES cell growth without feeder cells. In addition to the external signal pathways, intrinsic transcription factors such as FoxD3, P53 and Oct4 are also involved in regulating the expression of Nanog. Functionally, Nanog works together with other key pluripotent factors such as Oct4 and Sox2 to control a set of target genes that have important functions in ES cell pluripotency. These key factors form a regulatory network to support or limit each other's expression level, which maintains the properties of ES cells.

Differential Requirements for Hematopoietic Commitment Between Human and Rhesus Embryonic Stem Cells

Stem Cells (Dayton, Ohio). Feb, 2007  |  Pubmed ID: 17284653

Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34(+)CD45(-) cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34(+) and detectable levels of CD45(+) cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.

Defined Substrates for Human Embryonic Stem Cell Growth Identified from Surface Arrays

ACS Chemical Biology. May, 2007  |  Pubmed ID: 17480050

Methods for the rapid identification of defined cell growth conditions are lacking. This deficiency is a major barrier to the investigation and application of human embryonic stem (ES) cells. To address this problem, we developed a method for generating arrays of self-assembled monolayers (SAMs) in which each element constitutes a defined surface. By screening surface arrays, we identified peptidic surfaces that support ES cell growth and self-renewal. The ability of the active surface array elements to support ES cell growth depends on their composition: both the density of the peptide presented and its sequence are critical. These findings support a role for specific surface-cell interactions. Moreover, the data from the surface arrays are portable. They can be used to design an effective 3D synthetic scaffold that supports the growth of undifferentiated human ES cells. Our results demonstrate that synthetic substrates for promoting and probing human ES cell self-renewal can be discovered through SAM surface arrays.

LongSAGE Profiling of Nine Human Embryonic Stem Cell Lines

Genome Biology. 2007  |  Pubmed ID: 17570852

To facilitate discovery of novel human embryonic stem cell (ESC) transcripts, we generated 2.5 million LongSAGE tags from 9 human ESC lines. Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells, and identified novel ESC transcripts, at least one of which may represent a marker of the pluripotent state.

A Microarray Analysis of the Emergence of Embryonic Definitive Hematopoiesis

Experimental Hematology. Sep, 2007  |  Pubmed ID: 17761287

Human embryonic stem (ES) cells provide a unique model for studying the development and function of human tissues and have proven utility in a number of areas. However, results from ES cell-based studies have been limited by the paucity of information available about early human hematopoietic development.

Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells

Science (New York, N.Y.). Dec, 2007  |  Pubmed ID: 18029452

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

Conserved Regulation of MAP Kinase Expression by PUF RNA-binding Proteins

PLoS Genetics. Dec, 2007  |  Pubmed ID: 18166083

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3' untranslated region (3' UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3'UTR elements in both Erk2 and p38alpha mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38alpha 3' UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.

Defined, Feeder-independent Medium for Human Embryonic Stem Cell Culture

Current Protocols in Stem Cell Biology. Sep, 2007  |  Pubmed ID: 18785163

The developmental potential of human ES cells makes them an important tool in developmental, pharmacological, and clinical research. For human ES cell technology to be fully exploited, however, culture efficiency must be improved, large-scale culture enabled, and safety ensured. Traditional human ES cell culture systems have relied on serum products and mouse feeder layers, which limit the scale, present biological variability, and expose the cells to potential contaminants. Defined, feeder-independent culture systems improve the safety and efficiency of ES cell technology, enabling translational research. The protocols herein are designed with the standard research laboratory in mind. They contain recipes for the formulation of mTeSR (a defined medium for human ES cell culture) and detailed protocols for the culture, transfer, and passage of cells grown in these feeder-independent conditions. They provide a basis for routine feeder-independent culture, and a starting point for additional optimization of culture conditions.

Whole-genome Analysis of Histone H3 Lysine 4 and Lysine 27 Methylation in Human Embryonic Stem Cells

Cell Stem Cell. Sep, 2007  |  Pubmed ID: 18371364

We mapped Polycomb-associated H3K27 trimethylation (H3K27me3) and Trithorax-associated H3K4 trimethylation (H3K4me3) across the whole genome in human embryonic stem (ES) cells. The vast majority of H3K27me3 colocalized on genes modified with H3K4me3. These commodified genes displayed low expression levels and were enriched in developmental function. Another significant set of genes lacked both modifications and was also expressed at low levels in ES cells but was enriched for gene function in physiological responses rather than development. Commodified genes could change expression levels rapidly during differentiation, but so could a substantial number of genes in other modification categories. SOX2, POU5F1, and NANOG, pluripotency-associated genes, shifted from modification by H3K4me3 alone to colocalization of both modifications as they were repressed during differentiation. Our results demonstrate that H3K27me3 modifications change during early differentiation, both relieving existing repressive domains and imparting new ones, and that colocalization with H3K4me3 is not restricted to pluripotent cells.

A Study of the Relationships Between Oligonucleotide Properties and Hybridization Signal Intensities from NimbleGen Microarray Datasets

Nucleic Acids Research. May, 2008  |  Pubmed ID: 18385155

Well-defined relationships between oligonucleotide properties and hybridization signal intensities (HSI) can aid chip design, data normalization and true biological knowledge discovery. We clarify these relationships using the data from two microarray experiments containing over three million probes from 48 high-density chips. We find that melting temperature (T(m)) has the most significant effect on HSI while length for the long oligonucleotides studied has very little effect. Analysis of positional effect using a linear model provides evidence that the protruding ends of probes contribute more than tethered ends to HSI, which is further validated by specifically designed match fragment sliding and extension experiments. The impact of sequence similarity (SeqS) on HSI is not significant in comparison with other oligonucleotide properties. Using regression and regression tree analysis, we prioritize these oligonucleotide properties based on their effects on HSI. The implications of our discoveries for the design of unbiased oligonucleotides are discussed. We propose that isothermal probes designed by varying the length is a viable strategy to reduce sequence bias, though imposing selection constraints on other oligonucleotide properties is also essential.

Caspase Activity Mediates the Differentiation of Embryonic Stem Cells

Cell Stem Cell. Jun, 2008  |  Pubmed ID: 18522852

Embryonic stem cells (ESCs) are capable of indefinite self-renewal while retaining the ability to differentiate to any of the three germ layers that give rise to all somatic cell types. An emerging view is that a core set of transcription factors, including Oct4, Sox2, and Nanog, form a robust autoregulatory circuit that maintains ESCs in a self-renewing state. To accommodate the capacity of such cells to undergo germ layer-specific differentiation, we predicted a posttranslational mechanism that could negatively regulate these core self-renewal factors. Here we report caspase-induced cleavage of Nanog in differentiating ESCs. Stem cells lacking the Casp3 gene showed marked defects in differentiation, while forced expression of a caspase cleavage-resistant Nanog mutant in ESCs strongly promoted self-renewal. These results link a major component of the programmed cell-death pathway to the regulation of ESC development.

Ronin is Essential for Embryogenesis and the Pluripotency of Mouse Embryonic Stem Cells

Cell. Jun, 2008  |  Pubmed ID: 18585351

Pluripotency is a unique biological state that allows cells to differentiate into any tissue type. Here we describe a candidate pluripotency factor, Ronin, that possesses a THAP domain, which is associated with sequence-specific DNA binding and epigenetic silencing of gene expression. Ronin is expressed primarily during the earliest stages of murine embryonic development, and its deficiency in mice produces periimplantational lethality and defects in the inner cell mass. Conditional knockout of Ronin prevents the growth of ES cells while forced expression of Ronin allows ES cells to proliferate without differentiation under conditions that normally do not promote self-renewal. Ectopic expression also partly compensates for the effects of Oct4 knockdown. We demonstrate that Ronin binds directly to HCF-1, a key transcriptional regulator. Our findings identify Ronin as an essential factor underlying embryogenesis and ES cell pluripotency. Its association with HCF-1 suggests an epigenetic mechanism of gene repression in pluripotent cells.

Optical Mapping Discerns Genome Wide DNA Methylation Profiles

BMC Molecular Biology. 2008  |  Pubmed ID: 18667073

Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification.

Pluripotent Stem Cell Lines

Genes & Development. Aug, 2008  |  Pubmed ID: 18676805

The derivation of human embryonic stem cells 10 years ago ignited an explosion of public interest in stem cells, yet this achievement depended on prior decades of research on mouse embryonic carcinoma cells and embryonic stem cells. In turn, the recent derivation of mouse and human induced pluripotent stem cells depended on the prior studies on mouse and human embryonic stem cells. Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in vitro while maintaining the ability to differentiate into advanced derivatives of all three germ layers, features very useful for understanding the differentiation and function of human tissues, for drug screen and toxicity testing, and for cellular transplantation therapies. Here we review the family of pluripotent cell lines derived from early embryos and from germ cells, and compare them with the more recently described induced pluripotent stem cells.

NANOG is a Direct Target of TGFbeta/activin-mediated SMAD Signaling in Human ESCs

Cell Stem Cell. Aug, 2008  |  Pubmed ID: 18682241

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling, and differentiation is promoted by BMP signaling, but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium, we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG, OCT4, and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal.

Cell Type-specific DNA Methylation Patterns in the Human Breast

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2008  |  Pubmed ID: 18780791

Cellular identity and differentiation are determined by epigenetic programs. The characteristics of these programs in normal human mammary epithelium and their similarity to those in stem cells are unknown. To begin investigating these issues, we analyzed the DNA methylation and gene expression profiles of distinct subpopulations of mammary epithelial cells by using MSDK (methylation-specific digital karyotyping) and SAGE (serial analysis of gene expression). We identified discrete cell-type and differentiation state-specific DNA methylation and gene expression patterns that were maintained in a subset of breast carcinomas and correlated with clinically relevant tumor subtypes. CD44+ cells were the most hypomethylated and highly expressed several transcription factors with known stem cell function including HOXA10 and TCF3. Many of these genes were also hypomethylated in BMP4-treated compared with undifferentiated human embryonic stem (ES) cells that we analyzed by MSDK for comparison. Further highlighting the similarity of epigenetic programs of embryonic and mammary epithelial cells, genes highly expressed in CD44+ relative to more differentiated CD24+ cells were significantly enriched for Suz12 targets in ES cells. The expression of FOXC1, one of the transcription factors hypomethylated and highly expressed in CD44+ cells, induced a progenitor-like phenotype in differentiated mammary epithelial cells. These data suggest that epigenetically controlled transcription factors play a key role in regulating mammary epithelial cell phenotypes and imply similarities among epigenetic programs that define progenitor cell characteristics.

Secreted Proteoglycans Directly Mediate Human Embryonic Stem Cell-basic Fibroblast Growth Factor 2 Interactions Critical for Proliferation

Stem Cells (Dayton, Ohio). Dec, 2008  |  Pubmed ID: 18802039

Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.

Steady-state and Pre-steady-state Kinetic Evaluation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CLpro Cysteine Protease: Development of an Ion-pair Model for Catalysis

Biochemistry. Feb, 2008  |  Pubmed ID: 18237196

Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CLpro) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CLpro to characterize the reaction mechanism. We show that 3CLpro has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CLpro-mediated ester hydrolysis, including the individual rate constants, is consistent with a simple double displacement mechanism. The pre-steady-state burst rate was independent of ester substrate concentration indicating a high commitment to catalysis. When homologous peptidic amide and ester substrates were compared, a series of interesting observations emerged. Despite a 2000-fold difference in nonenzymatic reactivity, highly related amide and ester substrates were found to have similar kinetic parameters in both the steady-state and pre-steady-state. Steady-state solvent isotope effect (SIE) studies showed an inverse SIE for the amide but not ester substrates. Evaluation of the SIE in the pre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studies on amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition state while the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivation profile of 3CLpro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CLpro mechanism that utilizes an "electrostatic" trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P1, and a general base-catalyzed deacylation reaction.

Dielectrophoretic Separation of Platelets from Diluted Whole Blood in Microfluidic Channels

Electrophoresis. Mar, 2008  |  Pubmed ID: 18288670

The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform size-based fractionation of blood samples and continuously enrich the platelets in a label-free manner. Cytometry analysis revealed that a single pass through the two-stage DACS device yields a high purity of platelets (approximately 95%) at a throughput of approximately 2.2 x 10(4) cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free dielectrophoretic separation of delicate cells from complex samples and such a separation approach may open a path toward continuous screening of blood products by integrated microfluidic devices.

Mass Spectrometry Identifies and Quantifies 74 Unique Histone H4 Isoforms in Differentiating Human Embryonic Stem Cells

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2008  |  Pubmed ID: 18326628

Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.

Induced Pluripotent Stem Cells from a Spinal Muscular Atrophy Patient

Nature. Jan, 2009  |  Pubmed ID: 19098894

Spinal muscular atrophy is one of the most common inherited forms of neurological disease leading to infant mortality. Patients have selective loss of lower motor neurons resulting in muscle weakness, paralysis and often death. Although patient fibroblasts have been used extensively to study spinal muscular atrophy, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem cells from skin fibroblast samples taken from a child with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show that human induced pluripotent stem cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.

Human Embryonic Stem Cell Phosphoproteome Revealed by Electron Transfer Dissociation Tandem Mass Spectrometry

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2009  |  Pubmed ID: 19144917

Protein phosphorylation is central to the understanding of cellular signaling, and cellular signaling is suggested to play a major role in the regulation of human embryonic stem (ES) cell pluripotency. Here, we describe the use of conventional tandem mass spectrometry-based sequencing technology--collision-activated dissociation (CAD)--and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome. In total, these experiments resulted in the identification of 11,995 unique phosphopeptides, corresponding to 10,844 nonredundant phosphorylation sites, at a 1% false discovery rate (FDR). Among these phosphorylation sites are 5 localized to 2 pluripotency critical transcription factors--OCT4 and SOX2. From these experiments, we conclude that ETD identifies a larger number of unique phosphopeptides than CAD (8,087 to 3,868), more frequently localizes the phosphorylation site to a specific residue (49.8% compared with 29.6%), and sequences whole classes of phosphopeptides previously unobserved.

Functional Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

Circulation Research. Feb, 2009  |  Pubmed ID: 19213953

Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines because of residual transgene expression. Proliferation of iPS and ES cell-derived cardiomyocytes based on 5-bromodeoxyuridine labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to beta-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.

Histone Modifications at Human Enhancers Reflect Global Cell-type-specific Gene Expression

Nature. May, 2009  |  Pubmed ID: 19295514

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast, enhancers are marked with highly cell-type-specific histone modification patterns, strongly correlate to cell-type-specific gene expression programs on a global scale, and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression.

Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences

Science (New York, N.Y.). May, 2009  |  Pubmed ID: 19325077

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. We describe the derivation of human iPS cells with the use of nonintegrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors and removes one obstacle to the clinical application of human iPS cells.

ProbeMatch: Rapid Alignment of Oligonucleotides to Genome Allowing Both Gaps and Mismatches

Bioinformatics (Oxford, England). Jun, 2009  |  Pubmed ID: 19351619

SUMMARY: We have developed a tool, called ProbeMatch, for matching a large set of oligonucleotide sequences against a genome database using gapped alignments. Unlike most of the existing tools such as ELAND which only perform ungapped alignments allowing at most two mismatches, ProbeMatch generates both ungapped and gapped alignments allowing up to three errors including insertion, deletion and mismatch. To speedup sequence alignment, ProbeMatch uses gapped q-grams and q-grams of various patterns to identify target hits to a query sequence. This approach results in fewer initial sequences to examine with no loss in sensitivity. ProbeMatch has been used to align 169,095 Illumina GAII reads against the human genome, which could not be mapped by ELAND, and found alignments for 28,625 reads of the 169,095 reads in less than 3 h. AVAILABILITY: Source code is freely available at (http://www.cs.wisc.edu/~jignesh/probematch/).

MicroRNA-145 Regulates OCT4, SOX2, and KLF4 and Represses Pluripotency in Human Embryonic Stem Cells

Cell. May, 2009  |  Pubmed ID: 19409607

MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression and play an important role in many developmental processes. We report here that expression of microRNA-145 (miR-145) is low in self-renewing human embryonic stem cells (hESCs) but highly upregulated during differentiation. We identify the pluripotency factors OCT4, SOX2, and KLF4 as direct targets of miR-145 and show that endogenous miR-145 represses the 3' untranslated regions of OCT4, SOX2, and KLF4. Increased miR-145 expression inhibits hESC self-renewal, represses expression of pluripotency genes, and induces lineage-restricted differentiation. Loss of miR-145 impairs differentiation and elevates OCT4, SOX2, and KLF4. Furthermore, we find that the miR-145 promoter is bound and repressed by OCT4 in hESCs. This work reveals a direct link between the core reprogramming factors and miR-145 and uncovers a double-negative feedback loop involving OCT4, SOX2, KLF4, and miR-145.

MicroRNA 92b Controls the G1/S Checkpoint Gene P57 in Human Embryonic Stem Cells

Stem Cells (Dayton, Ohio). Jul, 2009  |  Pubmed ID: 19544458

Human embryonic stem (ES) cells exhibit a shorter G(1) cell cycle phase than most somatic cells. Here, we examine the role of an abundant, human ES cell-enriched microRNA, miR-92b, in cell cycle distribution. Inhibition of miR-92b in human ES cells results in a greater number of cells in the G(1) phase and a lower number in the S phase. Conversely, overexpression of miR-92b in differentiated cells results in a decreased number of cells in G1 phase and an increased number in S-phase. p57, a gene whose product inhibits G(1) to S-phase progression, is one of the predicted targets of miR-92b. Inhibition of miR-92b in human ES cells increases p57 protein levels, and miR-92b overexpression in differentiated cells decreases p57 protein levels. Furthermore, miR-92b inhibits a luciferase reporter construct that includes part of the 3' untranslated region of the p57 gene containing the predicted target of the miR-92b seed sequence. Thus, we show that the miRNA miR-92b directly downregulates protein levels of the G(1)/S checkpoint gene p57. STEM CELLS 2009;27:1524-1528.

Human DNA Methylomes at Base Resolution Show Widespread Epigenomic Differences

Nature. Nov, 2009  |  Pubmed ID: 19829295

DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA-protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.

The Microwell Control of Embryoid Body Size in Order to Regulate Cardiac Differentiation of Human Embryonic Stem Cells

Biomaterials. Mar, 2010  |  Pubmed ID: 19945747

The differentiation of human embryonic stem cells (hESCs) into cardiomyocytes (CMs) using embryoid bodies (EBs) is relatively inefficient and highly variable. Formation of EBs using standard enzymatic disaggregation techniques results in a wide range of sizes and geometries of EBs. Use of a 3-D cuboidal microwell system to culture hESCs in colonies of defined dimensions, 100-500 microm in lateral dimensions and 120 microm in depth, enabled formation of more uniform-sized EBs. The 300 microm microwells produced highest percentage of contracting EBs, but flow cytometry for myosin light chain 2A (MLC2a) expressing cells revealed a similar percentage (approximately 3%) of cardiomyocytes formed in EBs from 100 microm to 300 microm microwells. These data, and immunolabeling with anti-MF20 and MLC2a, suggest that the smaller EBs are less likely to form contracting EBs, but those contracting EBs are relatively enriched in cardiomyocytes compared to larger EB sizes where CMs make up a proportionately smaller fraction of the total cells. We conclude that microwell-engineered EB size regulates cardiogenesis and can be used for more efficient and reproducible formation of hESC-CMs needed for research and therapeutic applications.

RNA-Seq Gene Expression Estimation with Read Mapping Uncertainty

Bioinformatics (Oxford, England). Feb, 2010  |  Pubmed ID: 20022975

MOTIVATION: RNA-Seq is a promising new technology for accurately measuring gene expression levels. Expression estimation with RNA-Seq requires the mapping of relatively short sequencing reads to a reference genome or transcript set. Because reads are generally shorter than transcripts from which they are derived, a single read may map to multiple genes and isoforms, complicating expression analyses. Previous computational methods either discard reads that map to multiple locations or allocate them to genes heuristically. RESULTS: We present a generative statistical model and associated inference methods that handle read mapping uncertainty in a principled manner. Through simulations parameterized by real RNA-Seq data, we show that our method is more accurate than previous methods. Our improved accuracy is the result of handling read mapping uncertainty with a statistical model and the estimation of gene expression levels as the sum of isoform expression levels. Unlike previous methods, our method is capable of modeling non-uniform read distributions. Simulations with our method indicate that a read length of 20-25 bases is optimal for gene-level expression estimation from mouse and maize RNA-Seq data when sequencing throughput is fixed.

Neural Differentiation of Human Induced Pluripotent Stem Cells Follows Developmental Principles but with Variable Potency

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2010  |  Pubmed ID: 20160098

For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.

No Evidence for Clonal Selection Due to Lentiviral Integration Sites in Human Induced Pluripotent Stem Cells

Stem Cells (Dayton, Ohio). Apr, 2010  |  Pubmed ID: 20166152

Derivation of induced pluripotent stem (iPS) cells requires the expression of defined transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28) in the targeted cells. Lentiviral or standard retroviral gene transfer remains the most robust and commonly used approach. Low reprogramming frequency overall, and the higher efficiency of derivation utilizing integrating vectors compared to more recent nonviral approaches, suggests that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. We provide for the first time an extensive analysis of the lentiviral integration profile in human iPS cells. We identified a total of 78 independent IS in eight recently established iPS cell lines derived from either human fetal fibroblasts or newborn foreskin fibroblasts after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28. The number of IS ranged from 5 to 15 IS per individual iPS clone, and 75 IS could be assigned to a unique chromosomal location. The different iPS clones had no IS in common. Expression analysis as well as extensive bioinformatic analysis did not reveal functional concordance of the lentiviral targeted genes between the different clones. Interestingly, in six of the eight iPS clones, some of the IS were found in pairs, integrated into the same chromosomal location within six base pairs of each other or in very close proximity. Our study supports recent reports that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes.

Distinct Epigenomic Landscapes of Pluripotent and Lineage-committed Human Cells

Cell Stem Cell. May, 2010  |  Pubmed ID: 20452322

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells, yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes, but how much epigenomes differ remains unclear. Here, we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts, we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks, which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally, we observe novel, context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment.

Actin-myosin Contractility is Responsible for the Reduced Viability of Dissociated Human Embryonic Stem Cells

Cell Stem Cell. Aug, 2010  |  Pubmed ID: 20682449

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.

Quantitative Selection of DNA Aptamers Through Microfluidic Selection and High-throughput Sequencing

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2010  |  Pubmed ID: 20705898

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with K(d) < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have approximately 3-8-fold higher affinity and approximately 2-4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.

The Social Cognitive Determinants of Offending Drivers' Speeding Behaviour

Accident; Analysis and Prevention. Nov, 2010  |  Pubmed ID: 20728608

The efficacy of an extended theory of planned behaviour (TPB) was tested in relation to offending drivers' (N=1403) speeding behaviour. Postal questionnaires were issued at Time 1 to measure intention, instrumental and affective attitude, subjective and descriptive norm, self-efficacy, perceived controllability, moral norm, anticipated regret, self-identity, and past speeding behaviour. At Time 2 (6 months later), subsequent speeding behaviour was measured, again using self-completion postal questionnaires. The extended TPB accounted for 68% of the variation in intention and 51% of the variation in subsequent behaviour. The independent predictors of intention were instrumental attitude, affective attitude, self-efficacy, moral norm, anticipated regret and past behaviour. The independent predictors of behaviour were intention, self-efficacy, anticipated regret and past behaviour. Theoretical and practical implications of the findings are discussed in relation to targeting road safety interventions.

The NIH Roadmap Epigenomics Mapping Consortium

Nature Biotechnology. Oct, 2010  |  Pubmed ID: 20944595

The NIH Roadmap Epigenomics Mapping Consortium aims to produce a public resource of epigenomic maps for stem cells and primary ex vivo tissues selected to represent the normal counterparts of tissues and organ systems frequently involved in human disease.

A Mesoderm-derived Precursor for Mesenchymal Stem and Endothelial Cells

Cell Stem Cell. Dec, 2010  |  Pubmed ID: 21112566

Among the three embryonic germ layers, the mesoderm is a major source of the mesenchymal precursors giving rise to skeletal and connective tissues, but these precursors have not previously been identified and characterized. Using human embryonic stem cells directed toward mesendodermal differentiation, we show that mesenchymal stem/stromal cells (MSCs) originate from a population of mesodermal cells identified by expression of apelin receptor. In semisolid medium, these precursors form FGF2-dependent compact spheroid colonies containing mesenchymal cells with a transcriptional profile representative of mesoderm-derived embryonic mesenchyme. When transferred to adherent cultures, individual colonies give rise to MSC lines with chondro-, osteo-, and adipogenic differentiation potentials. Although the MSC lines lacked endothelial potential, endothelial cells could be derived from the mesenchymal colonies, suggesting that, similar to hematopoietic cells, MSCs arise from precursors with angiogenic potential. Together, these studies identified a common precursor of mesenchymal and endothelial cells, mesenchymoangioblast, as the source of mesoderm-derived MSCs.

Hotspots of Aberrant Epigenomic Reprogramming in Human Induced Pluripotent Stem Cells

Nature. Mar, 2011  |  Pubmed ID: 21289626

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

Efficient Generation of Transgene-free Induced Pluripotent Stem Cells from Normal and Neoplastic Bone Marrow and Cord Blood Mononuclear Cells

Blood. Apr, 2011  |  Pubmed ID: 21296996

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9, blood-derived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods.

FGF2 Sustains NANOG and Switches the Outcome of BMP4-induced Human Embryonic Stem Cell Differentiation

Cell Stem Cell. Mar, 2011  |  Pubmed ID: 21362572

Here, we show that as human embryonic stem cells (ESCs) exit the pluripotent state, NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ESCs into extraembryonic lineages. Here, we find that FGF2, acting through the MEK-ERK pathway, switches BMP4-induced human ESC differentiation outcome to mesendoderm, characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation, that forced NANOG expression results in FGF-independent BMP4 induction of mesendoderm, and that knockdown of NANOG greatly reduces T induction. Together, our results demonstrate that FGF2 signaling switches the outcome of BMP4-induced differentiation of human ESCs by maintaining NANOG levels through the MEK-ERK pathway.

Somatic Coding Mutations in Human Induced Pluripotent Stem Cells

Nature. Mar, 2011  |  Pubmed ID: 21368825

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.

Generation of Red Blood Cells from Human Induced Pluripotent Stem Cells

Stem Cells and Development. Sep, 2011  |  Pubmed ID: 21434814

Differentiation of human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into the erythroid lineage of cells offers a novel opportunity to study erythroid development, regulation of globin switching, drug testing, and modeling of red blood cell (RBC) diseases in vitro. Here we describe an approach for the efficient generation of RBCs from hiPSC/hESCs using an OP9 coculture system to induce hematopoietic differentiation followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. We showed that fibroblast-derived transgenic hiPSCs generated using lentivirus-based vectors and transgene-free hiPSCs generated using episomal vectors can be differentiated into RBCs with an efficiency similar to that of H1 hESCs. Erythroid cultures established with this approach consisted of an essentially pure population of CD235a(+)CD45(-) leukocyte-free RBCs with robust expansion potential and long life span (up to 90 days). Similar to hESCs, hiPSC-derived RBCs expressed predominately fetal γ and embryonic ɛ globins, indicating complete reprogramming of β-globin locus following transition of fibroblasts to the pluripotent state. Although β-globin expression was detected in hiPSC/hESC-derived erythroid cells, its expression was substantially lower than the embryonic and fetal globins. Overall, these results demonstrate the feasibility of large-scale production of erythroid cells from fibroblast-derived hiPSCs, as has been described for hESCs. Since RBCs generated from transgene-free hiPSCs lack genomic integration and background expression of reprogramming genes, they would be a preferable cell source for modeling of diseases and for gene function studies.

Genetic Correction and Analysis of Induced Pluripotent Stem Cells from a Patient with Gyrate Atrophy

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2011  |  Pubmed ID: 21464322

Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.

Chemically Defined Conditions for Human IPSC Derivation and Culture

Nature Methods. May, 2011  |  Pubmed ID: 21478862

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.

TF-Cluster: a Pipeline for Identifying Functionally Coordinated Transcription Factors Via Network Decomposition of the Shared Coexpression Connectivity Matrix (SCCM)

BMC Systems Biology. 2011  |  Pubmed ID: 21496241

Identifying the key transcription factors (TFs) controlling a biological process is the first step toward a better understanding of underpinning regulatory mechanisms. However, due to the involvement of a large number of genes and complex interactions in gene regulatory networks, identifying TFs involved in a biological process remains particularly difficult. The challenges include: (1) Most eukaryotic genomes encode thousands of TFs, which are organized in gene families of various sizes and in many cases with poor sequence conservation, making it difficult to recognize TFs for a biological process; (2) Transcription usually involves several hundred genes that generate a combination of intrinsic noise from upstream signaling networks and lead to fluctuations in transcription; (3) A TF can function in different cell types or developmental stages. Currently, the methods available for identifying TFs involved in biological processes are still very scarce, and the development of novel, more powerful methods is desperately needed.

Optic Vesicle-like Structures Derived from Human Pluripotent Stem Cells Facilitate a Customized Approach to Retinal Disease Treatment

Stem Cells (Dayton, Ohio). Aug, 2011  |  Pubmed ID: 21678528

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three-dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)-like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease-specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV-like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine.

Human Lymphoblastoid B-cell Lines Reprogrammed to EBV-free Induced Pluripotent Stem Cells

Blood. Aug, 2011  |  Pubmed ID: 21708888

Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations. We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules. LCL-derived iPSCs exhibited normal karyotype, expressed pluripotency markers, lost oriP/EBNA-1 episomal vectors, generated teratomas, retained donor identity, and differentiated in vitro into hematopoietic, cardiac, neural, and hepatocyte-like lineages. Significantly, although the parental LCLs express viral EBNA-1 and other Epstein-Barr virus latency-related elements for their survival, their presence was not detectable in LCL-iPSCs. Thus, reprogramming LCLs could offer an unlimited source for patient-specific iPSCs.

Dynamic Chromatin States in Human ES Cells Reveal Potential Regulatory Sequences and Genes Involved in Pluripotency

Cell Research. Oct, 2011  |  Pubmed ID: 21876557

Pluripotency, the ability of a cell to differentiate and give rise to all embryonic lineages, defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes, accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells, as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency, we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation, except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression, suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers, and observed much greater dynamics in chromatin modifications, especially H3K4me1 and H3K27ac, which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency.

High Purity Human-induced Pluripotent Stem Cell-derived Cardiomyocytes: Electrophysiological Properties of Action Potentials and Ionic Currents

American Journal of Physiology. Heart and Circulatory Physiology. Nov, 2011  |  Pubmed ID: 21890694

Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (I(Na)), L-type calcium (I(Ca)), hyperpolarization-activated pacemaker (I(f)), transient outward potassium (I(to)), inward rectifier potassium (I(K1)), and the rapidly and slowly activating components of delayed rectifier potassium (I(Kr) and I(Ks), respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications.

Proteomic and Phosphoproteomic Comparison of Human ES and IPS Cells

Nature Methods. 2011  |  Pubmed ID: 21983960

Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

Proteomics and Pluripotency

Critical Reviews in Biochemistry and Molecular Biology. Dec, 2011  |  Pubmed ID: 21999516

The fields of mass spectrometry (MS) and stem cell biology have expanded greatly in the past twenty years. Taken alone, these fields occupy entirely different branches of science; however, the points where they overlap provide valuable insight, both in the biological and technical arenas. From a biological perspective, MS-based proteomics offers the capacity to follow post-transcriptional regulation and signaling that are (1) fundamental to pluripotency and differentiation, (2) largely beyond the reach of genomic technologies, and (3) otherwise difficult or impossible to examine on a large scale. At the same time, addressing questions fundamental to stem cell biology has compelled proteomic researchers to pursue more sensitive and creative ways to probe the proteome, both in a targeted and high-throughput manner. Here, we highlight experiments that straddle proteomics and stem cell biology, with an emphasis on studies that apply mass spectrometry to dissect pluripotency and differentiation.

Thermal Stability of FGF Protein is a Determinant Factor in Regulating Self-Renewal, Differentiation and Reprogramming in Human Pluripotent Stem Cells

Stem Cells (Dayton, Ohio). Dec, 2011  |  Pubmed ID: 22213113

FGF, TGF/Nodal, and Insulin/IGF signaling pathways are sufficient to maintain human ES and iPS cells in a proliferative, undifferentiated state. Here we show that only a few FGF family members (FGF2, FGF4, FGF6 and FGF9) are able to sustain strong ERK phosphorylation and NANOG expression levels in human ES cells. Surprisingly, FGF1, which is reported to target the same set of receptors as FGF2, fails to sustain ERK phosphorylation and NANOG expression under standard culture conditions. We find that the failure of FGF1 to sustain ES is due to thermal instability of the wild type protein, not receptor specificity, and that a mutated thermal stable FGF1 sustains human ES cells, and supports both differentiation and reprogramming protocols.

Protein Kinase C Mediated Extraembryonic Endoderm Differentiation of Human Embryonic Stem Cells

Stem Cells (Dayton, Ohio). Mar, 2012  |  Pubmed ID: 22213079

Unlike mouse embryonic stem cells (ESCs), which are closely related to the inner cell mass, human ESCs appear to be more closely related to the later primitive ectoderm. For example, human ESCs and primitive ectoderm share a common epithelial morphology, growth factor requirements, and the potential to differentiate to all three embryonic germ layers. However, it has previously been shown that human ESCs can also differentiate to cells expressing markers of trophoblast, an extraembryonic lineage formed before the formation of primitive ectoderm. Here, we show that phorbol ester 12-O-tetradecanoylphorbol 13-acetate causes human ESCs to undergo an epithelial mesenchymal transition and to differentiate into cells expressing markers of parietal endoderm, another extraembryonic lineage. We further confirmed that this differentiation is through the activation of protein kinase C (PKC) pathway and demonstrated that a particular PKC subtype, PKC-δ, is most responsible for this transition. STEM CELLS 2012;30:461-470.