The formation of proximal and distal definitive endoderm populations in culture requires p38 MAPK activity.

Endoderm formation in the mammal is a complex process with two lineages forming during the first weeks of development, the primitive (or extraembryonic) endoderm, which is specified in the blastocyst, and the definitive endoderm that forms later, at gastrulation, as one of the germ layers of the embryo proper. Fate mapping evidence suggests that the definitive endoderm arises as two waves, which potentially reflect two distinct cell populations. Early primitive ectoderm-like (EPL) cell differentiation has been used successfully to identify and characterise mechanisms regulating molecular gastrulation and lineage choice during differentiation. The roles of the p38 MAPK family in the formation of definitive endoderm were investigated using EPL cells and chemical inhibitors of p38 MAPK activity. These approaches define a role for p38 MAPK activity in the formation of the primitive streak and a second role in the formation of the definitive endoderm. Characterisation of the definitive endoderm populations formed from EPL cells demonstrates the formation of two distinct populations, defined by gene expression and ontogeny, that were analogous to the proximal and distal definitive endoderm populations of the embryo. Formation of the proximal definitive endoderm was found to require p38 MAPK activity and is correlated with molecular gastrulation, defined by the expression of brachyury (T). Distal definitive endoderm formation also requires p38 MAPK activity but can form when T expression is inhibited. Understanding lineage complexity will be a prerequisite for the generation of endoderm derivatives for commercial and clinical use.

Oxygen modulates human embryonic stem cell metabolism in the absence of changes in self-renewal.

Human embryonic stem (ES) cells are routinely cultured under atmospheric oxygen (~20%), a concentration that is known to impair embryo development in vitro and is likely to be suboptimal for maintaining human ES cells compared with physiological (~5%) oxygen conditions. Conflicting reports exist on the effect of oxygen during human ES cell culture and studies have been largely limited to characterisation of typical stem cell markers or analysis of global expression changes. This study aimed to identify physiological markers that could be used to evaluate the metabolic impact of oxygen on the MEL-2 human ES cell line after adaptation to either 5% or 20% oxygen in extended culture. ES cells cultured under atmospheric oxygen displayed decreased glucose consumption and lactate production when compared with those cultured under 5% oxygen, indicating an overall higher flux of glucose through glycolysis under physiological conditions. Higher glucose utilisation at 5% oxygen was accompanied by significantly increased expression of all glycolytic genes analysed. Analysis of amino acid turnover highlighted differences in the consumption of glutamine and threonine and in the production of proline. The expression of pluripotency and differentiation markers was, however, unaltered by oxygen and no observable difference in proliferation between cells cultured in 5% and 20% oxygen was seen. Apoptosis was elevated under 5% oxygen conditions. Collectively these data suggest that culture conditions, including oxygen concentration, can significantly alter human ES cell physiology with coordinated changes in gene expression, in the absence of detectable alterations in undifferentiated marker expression.

Distinct profiles of human embryonic stem cell metabolism and mitochondria identified by oxygen.

Oxygen is a powerful regulator of cell function and embryonic development. It has previously been determined that oxygen regulates human embryonic stem (hES) cell glycolytic and amino acid metabolism, but the effects on mitochondria are as yet unknown. Two hES cell lines (MEL1, MEL2) were analyzed to determine the role of 5% (physiological) and 20% (atmospheric) oxygen in regulating mitochondrial activity. In response to extended physiological oxygen culture, MEL2 hES cells displayed reduced mtDNA content, mitochondrial mass and expression of metabolic genes TFAM, NRF1, PPARa and MT-ND4. Furthermore, MEL2 hES cell glucose consumption, lactate production and amino acid turnover were elevated under physiological oxygen. In stark contrast, MEL1 hES cell amino acid and carbohydrate use and mitochondrial function were relatively unaltered in response to oxygen. Furthermore, differentiation kinetics were delayed in the MEL1 hES cell line following BMP4 treatment. Here we report the first incidence of metabolic dysfunction in a hES cell population, defined as a failure to respond to oxygen concentration through the modulation of metabolism, demonstrating that hES cells can be perturbed during culture despite exhibiting the defining characteristics of pluripotent cells. Collectively, these data reveal a central role for oxygen in the regulation of hES cell metabolism and mitochondrial function, whereby physiological oxygen promotes glucose flux and suppresses mitochondrial biogenesis and gene expression.

Regulation of pluripotent cell differentiation by a small molecule, staurosporine.

Research in the embryo and in culture has resulted in a sophisticated understanding of many regulators of pluripotent cell differentiation. As a consequence, protocols for the differentiation of pluripotent cells generally rely on a combination of exogenous growth factors and endogenous signalling. Little consideration has been given to manipulating other pathways to achieve pluripotent cell differentiation. The integrity of cell:cell contacts has been shown to influence lineage choice during pluripotent cell differentiation, with disruption of cell:cell contacts promoting mesendoderm formation and maintenance of cell:cell contacts resulting in the preferential formation of neurectoderm. Staurosporine is a broad spectrum inhibitor of serine/threonine kinases which has several effects on cell function, including interruption of cell:cell contacts, decreasing focal contact size, inducing epithelial to mesenchyme transition (EMT) and promoting cell differentiation. The possibility that staurosporine could influence lineage choice from pluripotent cells in culture was investigated. The addition of staurosporine to differentiating mouse EPL resulted in preferential formation of mesendoderm and mesoderm populations, and inhibited the formation of neurectoderm. Addition of staurosporine to human ES cells similarly induced primitive streak marker gene expression. These data demonstrate the ability of staurosporine to influence lineage choice during pluripotent cell differentiation and to mimic the effect of disrupting cell:cell contacts. Staurosporine induced mesendoderm in the absence of known inducers of formation, such as serum and BMP4. Staurosporine induced the expression of mesendoderm markers, including markers that were not induced by BMP4, suggesting it acted as a broad spectrum inducer of molecular gastrulation. This approach has identified a small molecule regulator of lineage choice with potential applications in the commercial development of ES cell derivatives, specifically as a method for forming mesendoderm progenitors or as a culture adjunct to prevent the formation of ectoderm progenitors during pluripotent cell differentiation.

Culture environment regulates amino acid turnover and glucose utilisation in human ES cells.

Human embryonic stem (ES) cells have been proposed as a renewable source of pluripotent cells that can be differentiated into various cell types for use in research, drug discovery and in the emerging area of regenerative medicine. Exploitation of this potential will require the development of ES cell culture conditions that promote pluripotency and a normal cell metabolism, and quality control parameters that measure these outcomes. There is, however, relatively little known about the metabolism of pluripotent cells or the impact of culture environment and differentiation on their metabolic pathways. The effect of two commonly used medium supplements and cell differentiation on metabolic indicators in human ES cells were examined. Medium modifications and differentiation were compared in a chemically defined and feeder-independent culture system. Adding serum increased glucose utilisation and altered amino acid turnover by the cells, as well as inducing a small proportion of the cells to differentiate. Cell differentiation could be mitigated by inhibiting p38 mitogen-activated protein kinase (p38 MAPK activity). The addition of Knockout Serum Replacer also increased glucose uptake and changed amino acid turnover by the cells. These changes were distinct from those induced by serum and occurred in the absence of detectable differentiation. Induction of differentiation by bone morphogenetic protein 4 (BMP4), in contrast, did not alter metabolite turnover. Deviations from metabolite turnover by ES cells in fully defined medium demonstrated that culture environment can alter metabolite use. The challenge remains to understand the impact of metabolic changes on long-term cell maintenance and the functionality of derived cell populations.

Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis.

Differentiation of mouse embryonic stem cells (mESCs) is accompanied by changes in replication timing. To explore the relationship between replication timing and cell fate transitions, we constructed genome-wide replication-timing profiles of 22 independent mouse cell lines representing 10 stages of early mouse development, and transcription profiles for seven of these stages. Replication profiles were cell-type specific, with 45% of the genome exhibiting significant changes at some point during development that were generally coordinated with changes in transcription. Comparison of early and late epiblast cell culture models revealed a set of early-to-late replication switches completed at a stage equivalent to the post-implantation epiblast, prior to germ layer specification and down-regulation of key pluripotency transcription factors [POU5F1 (also known as OCT4)/NANOG/SOX2] and coinciding with the emergence of compact chromatin near the nuclear periphery. These changes were maintained in all subsequent lineages (lineage-independent) and involved a group of irreversibly down-regulated genes, at least some of which were repositioned closer to the nuclear periphery. Importantly, many genomic regions of partially reprogrammed induced pluripotent stem cells (piPSCs) failed to re-establish ESC-specific replication-timing and transcription programs. These regions were enriched for lineage-independent early-to-late changes, which in female cells included the inactive X chromosome. Together, these results constitute a comprehensive "fate map" of replication-timing changes during early mouse development. Moreover, they support a model in which a distinct set of replication domains undergoes a form of "autosomal Lyonization" in the epiblast that is difficult to reprogram and coincides with an epigenetic commitment to differentiation prior to germ layer specification.

Response to BMP4 signalling during ES cell differentiation defines intermediates of the ectoderm lineage.

The formation and differentiation of multipotent precursors underlies the generation of cell diversity during mammalian development. Recognition and analysis of these transient cell populations has been hampered by technical difficulties in accessing them in vivo. In vitro model systems, based on the differentiation of embryonic stem (ES) cells, provide an alternative means of identifying and characterizing these populations. Using a previously established mouse ES-cell-based system that recapitulates the development of the ectoderm lineage we have identified a transient population that is consistent with definitive ectoderm. This previously unidentified progenitor occurs as a temporally discrete population during ES cell differentiation, and differs from the preceding and succeeding populations in gene expression and differentiation potential, with the unique ability to form surface ectoderm in response to BMP4 signalling.

The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells.

There is an increasing appreciation that amino acids can act as signaling molecules in the regulation of cellular processes through modulation of intracellular cell signaling pathways. In culture, embryonic stem (ES) cells can be differentiated to a second, pluripotent cell population, early primitive ectoderm-like cells in response to biological activities within the conditioned medium MEDII. The amino acid l-proline has been identified as a component of MEDII required for ES cell differentiation. Here, we define the primary l-proline transporter on ES and early primitive ectoderm-like cells as sodium-coupled neutral amino acid transporter 2 (SNAT2). SNAT2 uptake of l-proline can be inhibited by the addition of millimolar concentrations of other substrates. The addition of excess amino acids was used to regulate the uptake of l-proline by ES cells, and the effect on differentiation was analyzed. The ability of SNAT2 substrates, but not other amino acids, to prevent changes in morphology, gene expression, and differentiation kinetics suggested that l-proline uptake through SNAT2 was required for ES cell differentiation. These data reveal an unexpected role for amino acid uptake and the amino acid transporter SNAT2 in regulation of pluripotent cells in culture and provides a number of specific, inexpensive, and nontoxic culture additives with the potential to improve the quality of ES cell culture.

L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture.

The development of cell therapeutics from embryonic stem (ES) cells will require technologies that direct cell differentiation to specific somatic cell lineages in response to defined factors. The initial step in formation of the somatic lineages from ES cells, differentiation to an intermediate, pluripotent primitive ectoderm-like cell, can be achieved in vitro by formation of early primitive ectoderm-like (EPL) cells in response to a biological activity contained within the conditioned medium MEDII. Fractionation of MEDII has identified two activities required for EPL cell formation, an activity with a molecular mass of <3 kDa and a second, much larger species. Here, we have identified the low-molecular-weight activity as l-proline. An inhibitor of l-proline uptake, glycine, prevented the differentiation of ES cells in response to MEDII. Supplementation of the culture medium of ES cells with >100 M l-proline and some l-proline-containing peptides resulted in changes in colony morphology, cell proliferation, gene expression, and differentiation kinetics consistent with differentiation toward a primitive ectoderm-like cell. This activity appeared to be associated with l-proline since other amino acids and analogs of proline did not exhibit an equivalent activity. Activation of the mammalian target of rapamycin (mTOR) signaling pathway was found to be necessary but not sufficient for l-proline activity; addition of other activators of the mTOR signaling pathway failed to alter the ES cell phenotype. This is the first report describing a role for amino acids in the regulation of pluripotency and cell differentiation and identifies a novel role for the imino acid l-proline.

Transcriptional control of pluripotency: decisions in early development.

The pathways controlling the maintenance and loss of pluripotency in cells of the early embryo regulate the formation of the tissues that will support development. Several transcription factors have been identified as being integral to the establishment and/or maintenance of pluripotency, coordinately regulating the expression of genes within pluripotent cells and acting as gene targets of these same processes. Recent advances in understanding the transcriptional regulation of these factors have revealed differences in the transcriptional complexes present within sub-populations of the pluripotent lineage and in the mechanisms regulating the loss of pluripotency on differentiation.

A novel role for gamma-secretase in the formation of primitive streak-like intermediates from ES cells in culture.

gamma-Secretase is a membrane-associated protease with multiple intracellular targets, a number of which have been shown to influence embryonic development and embryonic stem (ES) cell differentiation. This paper describes the use of the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) to evaluate the role of gamma-secretase in the differentiation of pluripotent stem cells to the germ lineages. The addition of DAPT did not prevent the formation of primitive ectoderm-like cells from ES cells in culture. In contrast, the addition of DAPT during primitive ectoderm-like cell differentiation interfered with the ability of both serum and BMP4 to induce a primitive streak-like intermediate and resulted in the preferential formation of neurectoderm. Similarly, DAPT reduced the formation of primitive streak-like intermediates from differentiating human ES cells; the culture conditions used resulted in a population enriched in human surface ectoderm. These data suggest that gamma-secretase may form part of the general pathway by which mesoderm is specified within the primitive streak. The addition of an E-cadherin neutralizing antibody was able to partially reverse the effect of DAPT, suggesting that DAPT may be preventing the formation of primitive streak-like intermediates and promoting neurectoderm differentiation by stabilizing E-cadherin and preventing its proteolysis.

Physiological oxygen culture reveals retention of metabolic memory in human induced pluripotent stem cells.

Reprogramming somatic cells to a pluripotent cell state (induced Pluripotent Stem (iPS) cells) requires reprogramming of metabolism to support cell proliferation and pluripotency, most notably changes in carbohydrate turnover that reflect a shift from oxidative to glycolytic metabolism. Some aspects of iPS cell metabolism differ from embryonic stem (ES) cells, which may reflect a parental cell memory, or be a consequence of the reprogramming process. In this study, we compared the metabolism of 3 human iPS cell lines to assess the fidelity of metabolic reprogramming. When challenged with reduced oxygen concentration, ES cells have been shown to modulate carbohydrate use in a predictably way. In the same model, 2 of 3 iPS cell lines failed to regulate carbohydrate metabolism. Oxygen is a well-characterized regulator of cell function and embryo viability, and an inability of iPS cells to modulate metabolism in response to oxygen may indicate poor metabolic fidelity. As metabolism is linked to the regulation of the epigenome, assessment of metabolic responses of iPS cells to physiological stimuli during characterization is warranted to ensure complete cell reprogramming and as a measure of cell quality.

Ethics and Governance of Stem Cell Banks.

This chapter examines the ethical principles and governance frameworks for stem cell banks. Good governance of stem cell banks should balance facilitation of the clinical use of stem cells with the proper respect and protection of stem cell sample providers and stem cell recipients and ensure compliance with national regulatory requirements to foster public trust in the use of stem cell technology. Stem cell banks must develop with regard to the science, the needs of scientists, and the requirements of the public, which will benefit from this science. Given the international reach of this promising research and its clinical application, it is necessary for stem cell bank governance frameworks to be harmonized across jurisdictions.

Metaboloepigenetic Regulation of Pluripotent Stem Cells.

The differentiation of pluripotent stem cells is associated with extensive changes in metabolism, as well as widespread remodeling of the epigenetic landscape. Epigenetic regulation is essential for the modulation of differentiation, being responsible for cell type specific gene expression patterns through the modification of DNA and histones, thereby establishing cell identity. Each cell type has its own idiosyncratic pattern regarding the use of specific metabolic pathways. Rather than simply being perceived as a means of generating ATP and building blocks for cell growth and division, cellular metabolism can directly influence cellular regulation and the epigenome. Consequently, the significance of nutrients and metabolites as regulators of differentiation is central to understanding how cells interact with their immediate environment. This review serves to integrate studies on pluripotent stem cell metabolism, and the regulation of DNA methylation and acetylation and identifies areas in which current knowledge is limited.

Regulation of amino acid transporters in pluripotent cell populations in the embryo and in culture; novel roles for sodium-coupled neutral amino acid transporters.

The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment, and inappropriate concentrations of amino acids, or the loss of amino acid-sensing mechanisms, can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells, a cell population derived from the blastocyst, has been shown in culture. l-proline acts as a signalling molecule, exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2, Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo, reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore, SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation, an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development.

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture.

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3ß are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3ß are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation.

Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities.

Pluripotent cells of embryonic origin proliferate at unusually rapid rates and have a characteristic cell cycle structure with truncated gap phases. To define the molecular basis for this we have characterized the cell cycle control of murine embryonic stem cells and early primitive ectoderm-like cells. These cells display precocious Cdk2, cyclin A and cyclin E kinase activities that are conspicuously cell cycle independent. Suppression of Cdk2 activity significantly decreased cycling times of pluripotent cells, indicating it to be rate-limiting for rapid cell division, although this had no impact on cell cycle structure and the establishment of extended gap phases. Cdc2-cyclin B was the only Cdk activity that was identified to be cell cycle regulated in pluripotent cells. Cell cycle regulation of cyclin B levels and Y(15) regulation of Cdc2 contribute to the temporal changes in Cdc2-cyclin B activity. E2F target genes are constitutively active throughout the cell cycle, reflecting the low activity of pocket proteins such as p107 and pRb and constitutive activity of pRb-kinases. These results show that rapid cell division cycles in primitive cells of embryonic origin are driven by extreme levels of Cdk activity that lack normal cell cycle periodicity.

Embryonic stem cell differentiation and the analysis of mammalian development.

Molecular and cellular analysis of early mammalian development is compromised by the experimental inaccessibility of the embryo. Pluripotent embryonic stem (ES) cells are derived from and retain many properties of the pluripotent founder population of the embryo, the inner cell mass. Experimental manipulation of these cells and their environment in vitro provides an opportunity for the development of differentiation systems which can be used for analysis of the molecular and cellular basis of embryogenesis. In this review we discuss strengths and weaknesses of the available ES cell differentiation methodologies and their relationship to events in vivo. Exploitation of these systems is providing novel insight into embryonic processes as diverse as cell lineage establishment, cell progression during differentiation, patterning, morphogenesis and the molecular basis for cell properties in the early mammalian embryo.

Formation of neural precursor cell populations by differentiation of embryonic stem cells in vitro.

Recent interest in the generation of neural lineages by differentiation of embryonic stem cells arises from the opportunities represented by a developmentally normal, unlimited source of material that can be manipulated genetically with precision. Several experimental approaches, which differ conceptually, in the route of differentiation and the characteristics of the resulting cell population have been reported. In this review we undertake a comparative analysis of these approaches and their suitability for experimental investigation or implantation.

Endoderm complexity in the mouse gastrula is revealed through the expression of spink3.

Endoderm formation in the mammalian embryo occurs first in the blastocyst, when the primitive endoderm and pluripotent cells resolve into separate lineages, and again during gastrulation, when the definitive endoderm progenitor population emerges from the primitive streak. The formation of the definitive endoderm can be modeled using pluripotent cell differentiation in culture. The differentiation of early primitive ectoderm-like (EPL) cells, a pluripotent cell population formed from embryonic stem (ES) cells, was used to identify and characterize definitive endoderm formation. Expression of serine peptidase inhibitor, Kazal type 3 (Spink3) was detected in EPL cell-derived endoderm, and in a band of endoderm immediately distal to the embryonic-extra-embryonic boundary in pregastrula and gastrulating embryos. Later expression marked a region of endoderm separating the yolk sac from the developing gut. In the embryo, Spink3 expression marked a region of endoderm comprising the distal visceral endoderm, as determined by an endocytosis assay, and the proximal region of the definitive endoderm. This region was distinct from the more distal definitive endoderm population, marked by thyrotropin-releasing hormone (Trh). Endoderm expressing either Spink3 or Trh could be formed during EPL cell differentiation, and the prevalence of these populations could be influenced by culture medium and growth factor addition. Moreover, further differentiation suggested that the potential of these populations differed. These approaches have revealed an unexpected complexity in the definitive endoderm lineage, a complexity that will need to be accommodated in differentiation protocols to ensure the formation of the appropriate definitive endoderm progenitor in the future.