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1.
Stem Cell Reports ; 13(1): 193-206, 2019 07 09.
Article in English | MEDLINE | ID: mdl-31231024

ABSTRACT

The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties.


Subject(s)
Cellular Reprogramming/genetics , Chromatin/genetics , DNA Replication Timing , Stem Cells/metabolism , Transcription, Genetic , Cell Cycle/genetics , Cell Differentiation/genetics , Cell Lineage/genetics , Chromatin/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Humans , Models, Biological , Stem Cells/cytology
2.
Genome Res ; 25(8): 1091-103, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26055160

ABSTRACT

Duplication of the genome in mammalian cells occurs in a defined temporal order referred to as its replication-timing (RT) program. RT changes dynamically during development, regulated in units of 400-800 kb referred to as replication domains (RDs). Changes in RT are generally coordinated with transcriptional competence and changes in subnuclear position. We generated genome-wide RT profiles for 26 distinct human cell types, including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm, and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures) and confirmed global consolidation of the genome into larger synchronously replicating segments during differentiation. Surprisingly, transcriptome data revealed that the well-accepted correlation between early replication and transcriptional activity was restricted to RT-constitutive genes, whereas two-thirds of the genes that switched RT during differentiation were strongly expressed when late replicating in one or more cell types. Closer inspection revealed that transcription of this class of genes was frequently restricted to the lineage in which the RT switch occurred, but was induced prior to a late-to-early RT switch and/or down-regulated after an early-to-late RT switch. Analysis of transcriptional regulatory networks showed that this class of genes contains strong regulators of genes that were only expressed when early replicating. These results provide intriguing new insight into the complex relationship between transcription and RT regulation during human development.


Subject(s)
Cell Lineage , DNA Replication Timing , Gene Expression Profiling/methods , Pluripotent Stem Cells/physiology , Cell Differentiation , Cells, Cultured , Cluster Analysis , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Genome, Human , Humans , Pluripotent Stem Cells/cytology
3.
Proteomics ; 15(2-3): 554-66, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25367160

ABSTRACT

Human embryonic stem cells (hESCs) have received considerable attention due to their therapeutic potential and usefulness in understanding early development and cell fate commitment. In order to appreciate the unique properties of these pluripotent, self-renewing cells, we have performed an in-depth multidimensional fractionation followed by LC-MS/MS analysis of the hESCs harvested from defined media to elucidate expressed, phosphorylated, O-linked ß-N-acetylglucosamine (O-GlcNAc) modified, and secreted proteins. From the triplicate analysis, we were able to assign more than 3000 proteins with less than 1% false-discovery rate. This analysis also allowed us to identify nearly 500 phosphorylation sites and 68 sites of O-GlcNAc modification with the same high confidence. Investigation of the phosphorylation sites allowed us to deduce the set of kinases that are likely active in these cells. We also identified more than 100 secreted proteins of hESCs that likely play a role in extracellular matrix formation and remodeling, as well as autocrine signaling for self-renewal and maintenance of the undifferentiated state. Finally, by performing in-depth analysis in triplicate, spectral counts were obtained for these proteins and posttranslationally modified peptides, which will allow us to perform relative quantitative analysis between these cells and any derived cell type in the future.


Subject(s)
Embryonic Stem Cells/metabolism , Proteome/analysis , Acetylglucosamine/analysis , Acetylglucosamine/metabolism , Cell Fractionation , Cell Line , Embryonic Stem Cells/chemistry , Humans , Phosphorylation , Protein Processing, Post-Translational , Proteome/metabolism , Proteomics , Tandem Mass Spectrometry
4.
Cell Stem Cell ; 12(2): 224-37, 2013 Feb 07.
Article in English | MEDLINE | ID: mdl-23318056

ABSTRACT

Embryonic development is characterized by dynamic changes in gene expression, yet the role of chromatin remodeling in these cellular transitions remains elusive. To address this question, we profiled the transcriptome and select chromatin modifications at defined stages during pancreatic endocrine differentiation of human embryonic stem cells. We identify removal of Polycomb group (PcG)-mediated repression on stage-specific genes as a key mechanism for the induction of developmental regulators. Furthermore, we discover that silencing of transitory genes during lineage progression associates with reinstatement of PcG-dependent repression. Significantly, in vivo- but not in vitro-differentiated endocrine cells exhibit close similarity to primary human islets in regard to transcriptome and chromatin structure. We further demonstrate that endocrine cells produced in vitro do not fully eliminate PcG-mediated repression on endocrine-specific genes, probably contributing to their malfunction. These studies reveal dynamic chromatin remodeling during developmental lineage progression and identify possible strategies for improving cell differentiation in culture.


Subject(s)
Chromatin Assembly and Disassembly/physiology , Embryonic Stem Cells/cytology , Pancreas/cytology , Polycomb-Group Proteins/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Chromatin Assembly and Disassembly/genetics , Embryonic Stem Cells/metabolism , Endocrine Cells/cytology , Endocrine Cells/metabolism , Endoderm/cytology , Endoderm/metabolism , Humans , Mice , Polycomb-Group Proteins/genetics
5.
Mar Drugs ; 10(8): 1662-1670, 2012 Aug.
Article in English | MEDLINE | ID: mdl-23015768

ABSTRACT

Two new briarane diterpenoids briareolate esters J (1) and K (2) were isolated from the methanolic extract of the octocoral Briareum asbestinum collected off the coast of Boca Raton, Florida. The structures of briaranes 1 and 2 were elucidated by interpretation of spectroscopic data. Briareolate ester K (2) showed weak growth inhibition activity against human embryonic stem cells (BG02).


Subject(s)
Anthozoa/chemistry , Diterpenes/pharmacology , Embryonic Stem Cells/drug effects , Esters/pharmacology , Animals , Diterpenes/isolation & purification , Embryonic Stem Cells/metabolism , Esters/isolation & purification , Florida , Humans , Spectrum Analysis
6.
PLoS One ; 7(5): e37004, 2012.
Article in English | MEDLINE | ID: mdl-22623968

ABSTRACT

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.


Subject(s)
Batch Cell Culture Techniques/methods , Cell Differentiation/physiology , Diabetes Mellitus, Type 1/therapy , Embryonic Stem Cells/cytology , Embryonic Stem Cells/transplantation , Insulin-Secreting Cells/cytology , Analysis of Variance , Animals , Cryopreservation/methods , Embryonic Stem Cells/physiology , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Profiling , Humans , Male , Mice , Mice, SCID , Streptozocin
7.
J Nat Prod ; 75(6): 1223-7, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22607472

ABSTRACT

Three new bicyclic C(21) terpenoids, clathric acid (1) and two N-acyl taurine derivatives, clathrimides A (2) and B (3), were isolated from the marine sponge Clathria compressa. The structures of these compounds were elucidated by interpretation of spectroscopic data. Clathric acid showed mild antibacterial activity against several Gram-positive bacteria.


Subject(s)
Anti-Bacterial Agents/isolation & purification , Porifera/chemistry , Terpenes/isolation & purification , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Gram-Positive Bacteria/drug effects , Marine Biology , Microbial Sensitivity Tests , Molecular Structure , Oceans and Seas , Terpenes/chemistry , Terpenes/pharmacology
8.
Org Lett ; 13(15): 3920-3, 2011 Aug 05.
Article in English | MEDLINE | ID: mdl-21749084

ABSTRACT

Three new briarane diterpenoids, briareolate esters L-N (1-3), have been isolated from a gorgonian Briareum asbestinum. Briareolate esters L (1) and M (2) are the first natural products possessing a 10-membered macrocyclic ring with a (E,Z)-dieneone and exhibit growth inhibition activity against both human embryonic stem cells (BG02) and a pancreatic cancer cell line (BxPC-3). Briareolate ester L (1) was found to contain a "spring-loaded" (E,Z)-dieneone Michael acceptor group that can form a reversible covalent bond to model sulfur-based nucleophiles.


Subject(s)
Anthozoa/chemistry , Diterpenes/chemistry , Animals , Biological Products/chemistry , Biological Products/isolation & purification , Cell Line , Diterpenes/isolation & purification , Humans , Models, Molecular , Molecular Structure
9.
Genome Res ; 20(6): 761-70, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20430782

ABSTRACT

To identify evolutionarily conserved features of replication timing and their relationship to epigenetic properties, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two human induced pluripotent stem cell lines (hiPSCs), and compared them with related mouse cell types. Results confirm the conservation of coordinately replicated megabase-sized "replication domains" punctuated by origin-suppressed regions. Differentiation-induced replication timing changes in both species occur in 400- to 800-kb units and are similarly coordinated with transcription changes. A surprising degree of cell-type-specific conservation in replication timing was observed across regions of conserved synteny, despite considerable species variation in the alignment of replication timing to isochore GC/LINE-1 content. Notably, hESC replication timing profiles were significantly more aligned to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Comparison with epigenetic marks revealed a signature of chromatin modifications at the boundaries of early replicating domains and a remarkably strong link between replication timing and spatial proximity of chromatin as measured by Hi-C analysis. Thus, early and late initiation of replication occurs in spatially separate nuclear compartments, but rarely within the intervening chromatin. Moreover, cell-type-specific conservation of the replication program implies conserved developmental changes in spatial organization of chromatin. Together, our results reveal evolutionarily conserved aspects of developmentally regulated replication programs in mammals, demonstrate the power of replication profiling to distinguish closely related cell types, and strongly support the hypothesis that replication timing domains are spatially compartmentalized structural and functional units of three-dimensional chromosomal architecture.


Subject(s)
Biological Evolution , Chromatin/genetics , DNA Replication , Animals , Cell Line , Embryonic Stem Cells/metabolism , Humans , Mice
10.
BMC Genomics ; 8: 478, 2007 Dec 27.
Article in English | MEDLINE | ID: mdl-18162134

ABSTRACT

BACKGROUND: Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs) is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells. RESULTS: Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins. CONCLUSION: Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.


Subject(s)
Embryonic Stem Cells/metabolism , Proteome/metabolism , Proteomics/methods , Blotting, Western , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Occludin , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Processing, Post-Translational , Proteome/classification , Proteome/genetics , Transcription, Genetic , Zonula Occludens-1 Protein
11.
Blood ; 110(12): 4111-9, 2007 Dec 01.
Article in English | MEDLINE | ID: mdl-17761519

ABSTRACT

Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.


Subject(s)
Cell Proliferation , Embryonic Stem Cells/metabolism , Pluripotent Stem Cells/metabolism , Receptor, ErbB-2/metabolism , Receptor, IGF Type 2/metabolism , Signal Transduction/physiology , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Benzothiazoles/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line , Cell Proliferation/drug effects , Culture Media, Conditioned , Embryonic Stem Cells/cytology , Fibroblast Growth Factor 2/pharmacology , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mice , Neuregulin-1/pharmacology , Phosphorylation/drug effects , Pluripotent Stem Cells/cytology , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-3/antagonists & inhibitors , Receptor, ErbB-3/metabolism , Receptor, IGF Type 2/antagonists & inhibitors , Receptor, Insulin/antagonists & inhibitors , Receptor, Insulin/metabolism , Signal Transduction/drug effects , Tyrphostins/pharmacology
12.
Stem Cells ; 25(1): 54-62, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17008424

ABSTRACT

Pluripotent cells can be isolated from the human blastocyst and maintained in culture as self-renewing, undifferentiated, human ESCs (hESCs). These cells are a valuable model of human development in vitro and are the focus of substantial research aimed at generating differentiated populations for cellular therapies. The extracellular markers that have been used to characterize hESCs are primarily carbohydrate epitopes on proteoglycans or sphingolipids, such as stage-specific embryonic antigen (SSEA)-3 and -4. The expression of SSEA-3 and -4 is tightly regulated during preimplantation development and on hESCs. Although this might imply a molecular function in undifferentiated cells, it has not yet been tested experimentally. We used inhibitors of sphingolipid and glycosphingolipid (GSL) biosynthesis to block the generation of SSEA-3 and -4 in hESCs. Depletion of these antigens and their precursors was confirmed using immunostaining, flow cytometry, and tandem mass spectroscopy. Transcriptional analysis, immunostaining, and differentiation in vitro and in teratomas indicated that other properties of pluripotency were not noticeably affected by GSL depletion. These experiments demonstrated that the GSLs recognized as SSEA-3 and -4 do not play critical functional roles in maintaining the pluripotency of hESCs, but instead suggested roles for this class of molecules during cellular differentiation.


Subject(s)
Antigens, Tumor-Associated, Carbohydrate/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Glycosphingolipids/physiology , Pluripotent Stem Cells/physiology , Cells, Cultured , Flow Cytometry , Gene Deletion , Gene Expression Regulation, Developmental , Glycosphingolipids/deficiency , Glycosphingolipids/genetics , Humans , Pluripotent Stem Cells/cytology , Stage-Specific Embryonic Antigens
13.
Restor Neurol Neurosci ; 22(6): 421-8, 2004.
Article in English | MEDLINE | ID: mdl-15798361

ABSTRACT

PURPOSE: To explore a karyotypically abnormal variant human embryonic stem cell (hESC) line, BG01V, as a potential model for studies of dopaminergic neuronal differentiation. METHODS: The properties of BG01V cells were compared to those of normal BG01 cells using immunocytochemistry, RT-PCR, focused microarrays and in vitro differentiation, including dopaminergic differentiation, by culturing with the stromal cell line PA6. RESULTS: Despite the karyotypic abnormality (49, +12, +17 and XXY), undifferentiated BG01V cells expressed pluripotent ESC markers similar to BG01 cells, and retained the ability to differentiate into cell types characteristic of all three germ layers. When co-cultured with the stromal cell line PA6, BG01V cells differentiated into dopaminergic cells which exhibited properties similar to those of mature dopaminergic neurons. CONCLUSIONS: BG01V cells were easier to maintain in culture than karyotypically normal BG01 cells and can be used as an alternative pluripotent hESC type for in vitro developmental studies.


Subject(s)
Cell Differentiation/physiology , Cell Proliferation , Dopamine/metabolism , Stem Cells/physiology , Blotting, Northern/methods , Cell Line , Chromosome Aberrations , Dopamine/genetics , Embryo, Mammalian , Gene Expression Regulation/physiology , Humans , Immunohistochemistry/methods , Neurons/metabolism , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , Tyrosine 3-Monooxygenase/metabolism
14.
Stem Cells Dev ; 13(6): 585-97, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15684826

ABSTRACT

The number of human embryonic stem cell (hESC) lines available to federally funded U.S. researchers is currently limited. Thus, determining their basic characteristics and disseminating these lines is important. In this report, we recovered and expanded the earliest available cryopreserved stocks of the BG01, BG02, and BG03 hESC lines. These cultures exhibited multiple definitive characteristics of undifferentiated cells, including long-term self-renewal, expression of markers of pluripotency, maintenance of a normal karyotype, and differentiation to mesoderm, endoderm, and ectoderm. Each cell line exhibited a unique genotype and human leukocyte antigen (HLA) isotype, confirming that they were isolated independently. BG01, BG02, and BG03 maintained in feederfree conditions demonstrated self-renewal, maintenance of normal karyotype, and gene expression indicative of undifferentiated pluripotent stem cells. A survey of gene expression in BG02 cells using massively parallel signature sequencing generated a digital read-out of transcript abundance and showed that this line was similar to other hESC lines. BG01, BG02, and BG03 hESCs are therefore independent, undifferentiated, and pluripotent lines that can be maintained without accumulation of karyotypic abnormalities.


Subject(s)
Cell Culture Techniques , Cell Line , Embryo, Mammalian/cytology , Genotype , Karyotyping , Pluripotent Stem Cells/cytology , Stem Cells/cytology , Alkaline Phosphatase/metabolism , Cell Differentiation , Cell Lineage , Cryopreservation , Cytogenetics/methods , DNA, Complementary/metabolism , Gene Expression Regulation , Histocompatibility Testing , Humans , Microscopy, Fluorescence , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Signal Transduction
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