Thomas C. Schulz

Genomic alterations in cultured human embryonic stem cells

Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10−9 per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.

Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum‐Free Suspension Culture

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinsons disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine‐producing neurons in a simple serum‐free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detecz after 2–4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)‐4, but a pulse of BMP‐4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real‐time polymerase chain reaction and whole‐mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum‐free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6‐hydroxydopamine (6‐OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum‐free suspension cultures, were detected 8 weeks after transplantation into 6‐OHDA–lesioned rat brains. This work suggests that hESCs can differentiate in simple serum‐free suspension cultures to produce the large number of cells required for transplantation studies.

Histone variant H2A.Z is required for early mammalian development

Fundamental to the process of mammalian development is the timed and coordinated regulation of gene expression. This requires transcription of a precise subset of the total complement of genes. It is clear that chromatin architecture plays a fundamental role in this process by either facilitating or restricting transcription factor binding [1]. How such specialized chromatin structures are established to regulate gene expression is poorly understood. All eukaryotic organisms contain specialized histone variants with distinctly different amino acid sequences that are even more conserved than the major core histones [2]. On the basis of their highly conserved sequence, histone variants have been assumed critical for the function of mammalian chromatin; however, a requirement for a histone variant has not been shown in mammalian cells. Mice with a deletion of H1 degrees have been generated by gene targeting in ES cells, but these mice show no phenotypic consequences, perhaps due to redundancy of function [3]. Here we show for the first time that a mammalian histone variant, H2A.Z, plays a critical role in early development, and we conclude that this histone variant plays a pivotal role in establishing the chromatin structures required for the complex patterns of gene expression essential for normal mammalian development.

A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

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.

The Cell Surface Glycosphingolipids SSEA‐3 and SSEA‐4 Are Not Essential for Human ESC Pluripotency

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.

Characterization of a New NIH‐Registered Variant Human Embryonic Stem Cell Line, BG01V: A Tool for Human Embryonic Stem Cell Research

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

Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells

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.

A large-scale proteomic analysis of human embryonic stem cells

BackgroundMuch 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.ResultsHere 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.ConclusionOur 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.

Bicyclic C21 terpenoids from the marine sponge Clathria compressa.

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.

A family of RS domain proteins with novel subcellular localization and trafficking

We report the sequence, conservation and cell biology of a novel protein, Psc1, which is expressed and regulated within the embryonic pluripotent cell population of the mouse. The Psc1 sequence includes an RS domain and an RNA recognition motif (RRM), and a sequential arrangement of protein motifs that has not been demonstrated for other RS domain proteins. This arrangement was conserved in a second mouse protein (BAC34721). The identification of Psc1 and BAC34721 homologues in vertebrates and related proteins, more widely throughout evolution, defines a new family of RS domain proteins termed acidic rich RS (ARRS) domain proteins. Psc1 incorporated into the nuclear speckles, but demonstrated novel aspects of subcellular distribution including localization to speckles proximal to the nuclear periphery and localization to punctate structures in the cytoplasm termed cytospeckles. Integration of Psc1 into cytospeckles was dependent on the RRM. Cytospeckles were dynamic within the cytoplasm and appeared to traffic into the nucleus. These observations suggest a novel role in RNA metabolism for ARRS proteins.