Culture Of Embryonic Stem Cells Research Articles

Early neural specification of stem cells is mediated by a set of SOX2-dependent neural-associated enhancers

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells.

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.

Chronic hypoxia remodels the tumor microenvironment to support glioma stem cell growth

Cerebral organoids co-cultured with patient derived glioma stem cells (GLICOs) are an experimentally tractable research tool useful for investigating the role of the human brain tumor microenvironment in glioblastoma. Here we describe long-term GLICOs, a novel model in which COs are grown from embryonic stem cell cultures containing low levels of GSCs and tumor development is monitored over extended durations (ltGLICOs). Single-cell profiling of ltGLICOs revealed an unexpectedly long latency period prior to GSC expansion, and that normal organoid development was unimpaired by the presence of low numbers of GSCs. However, as organoids age they experience chronic hypoxia and oxidative stress which remodels the tumor microenvironment to promote GSC expansion. Receptor-ligand modelling identified astrocytes, which secreted various pro-tumorigenic ligands including FGF1, as the primary cell type for GSC crosstalk and single-cell multi-omic analysis revealed these astrocytes were under the control of ischemic regulatory networks. Functional validation confirmed hypoxia as a driver of pro-tumorigenic astrocytic ligand secretion and that GSC expansion was accelerated by pharmacological induction of oxidative stress. When controlled for genotype, the close association between glioma aggressiveness and patient age has very few proposed biological explanations. Our findings indicate that age-associated increases in cerebral vascular insufficiency and associated regional chronic cerebral hypoxia may contribute to this phenomenon.

A Political and ethical landscape of brain organoid research

Human cerebral organoids (hCOs), produced in labs through directed cell culture of embryonic or induced pluripotent stem cells, closely replicate the 3-dimensional architecture of the human brain on a micro scale. This technology has been used to model neurological disease and shows promise to complement or supplant animal subjects in preclinical therapeutic investigation. However, attention must be paid by researchers and institutions to the various ethical concerns associated with hCO development. Human-animal chimeras produced through the grafting of hCOs have shown integration of neurological function, calling into question the moral status of both the animal chimeras and the organoid itself. Sensationalist reporting on such acts may also prompt public backlash, potentially jeopardizing hCO research and the promised benefits thereof. Moreover, concerns arise over privacy and consent for past and prospective donors of stem cells used to produce organoids. Genetic information may be considered privileged to the public domain and disrupted trust can reduce the supply of willing donors. Though hCOs are believed thus far to lack the capacity for consciousness and cognitive function, consideration must be given to their potential status as moral agents with further development or enhancement. Boundaries concerning organoids adhered to by researchers have been largely voluntary and informal to this point. By edict or by the power of the purse, governmental regulatory agencies ought to formalize necessary guidelines to ensure compliance with ethical principles and the adequate representation of all affected stakeholders in future decisions.

A Modular Microfluidic Organoid Platform Using LEGO-Like Bricks.

The convergence of organoid and organ-on-a-chip (OoC) technologies is urgently needed to overcome limitations of current 3D in vitro models. However, integrating organoids in standard OoCs faces several technical challenges, as it is typically laborious, lacks flexibility, and often results in even more complex and less-efficient cell culture protocols. Therefore, specifically adapted and more flexible microfluidic platforms need to be developed to facilitate the incorporation of complex 3D in vitro models. Here, a modular, tubeless fluidic circuit board (FCB) coupled with reversibly sealed cell culture bricks for dynamic culture of embryonic stem cell-derived thyroid follicles is developed. The FCB is fabricated by milling channels in a polycarbonate (PC) plate followed by thermal bonding against another PC plate. LEGO-like fluidic interconnectors allow plug-and-play connection between a variety of cell culture bricks and the FCB. Lock-and-play clamps are integrated in the organoid brick to enable easy (un)loading of organoids. A multiplexed perfusion experiment is conducted with six FCBs, where thyroid organoids are transferred on-chip within minutes and cultured up to 10 d without losing their structure and functionality, thus validating this system as a flexible, easy-to-use platform, capable of synergistically combining organoids with advanced microfluidic platforms.

ISRIB facilitates the co-culture of human trophoblast stem cells and embryonic stem cells.

The embryo-like structures (embryoids) constructed by aggregating embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have provided revolutionary tools for studying the intricate interaction between embryonic and extra-embryonic tissues during early embryonic development, which has been achieved in mice. However, due to the opposite dependence on some signalling pathways for in vitro culture of human ESCs (hESCs) and TSCs (hTSCs), particularly WNT and TGFβ signalling pathways, which limits the construction of human post-implantation embryoids by aggregating hESCs and hTSCs. To overcome this challenge, here, by screening 1639 chemicals, we found that an inhibitor of integrated stress response, ISRIB, can replace WNT agonists and TGFβ inhibitors to maintain the stemness and differentiation capacity of hTSCs. Thus, we developed an ISRIB-dependent in vitro culture medium for hTSCs, namely nTSM. Furthermore, we demonstrated that ISRIB could also maintain the hESC stemness. Using a 3D co-culture system (hESCs and hTSCs aggregate, ETA), we demonstrated that a 1:1 mixture of hESC culture medium (ESM) and nTSM improved the cell proliferation and organisation of both hESC- and hTSC-compartments and the lumenogenesis of hESC-compartment in ETAs. Overall, our study provided an ISRIB-dependent system for co-culturing hESCs and hTSCs, which facilitated the construction of human embryoids by aggregating hESCs and hTSCs.

Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts.

Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. MEFs are therefore widely used as a standard model for functional characterisation of gene knockouts, and can also be used in co-cultures, commonly to support embryonic stem cell cultures. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wild-type primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were abundant in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70S6K, and MTOR. Cell surface markers and matrisome proteins were also annotated. Data are available via ProteomeXchange with identifier PXD043244. This provides a comprehensive catalogue of the wild-type MEF proteome and phosphoproteome which can be utilised by the field to guide future work.

Dissecting the Role of the Ccr4-Not Deadenylase Complex in Pluripotency and Differentiation.

In this chapter, we describe methods and assays to examine the role of the Ccr4-Not mRNA deadenylase complex in regulating mouse embryonic stem cell (ESC) pluripotency and differentiation. We present the following procedures: sgRNA design and cloning, the culture of mouse ESCs, transfection and colony picking, genotyping, generation of Cnot3 conditional deletion ESCs, assessment of Cnot3 deletion efficiency, and examination of the impact on ESC maintenance and differentiation. The above approach and protocols can also be readily applied to study other Ccr4-Not subunits in ESC fate regulation, thereby facilitating the systematic dissection of Ccr4-Not function in stem cells and development.

Development of Reproducible and Scalable Culture Conditions for In Vitro Maintenance of Pig Embryonic Stem Cells Using the Sandoz Inbred Swiss Mouse Thioguanine-Resistant Ouabain-Resistant Cell Line as a Feeder Layer.

Feeder cells play a crucial role in maintaining the pluripotency of embryonic stem cells (ESCs) by secreting various extrinsic regulators, such as extracellular matrix (ECM) proteins and growth factors. Although primary mouse embryonic fibroblasts (MEFs) are the most widely used feeder cell type for the culture of ESCs, they have inevitable disadvantages such as batch-to-batch variation and labor-intensive isolation processes. Here, we revealed that the Sandoz inbred Swiss Mouse (SIM) thioguanine-resistant ouabain-resistant (STO) cell line, an immortalized cell line established from mouse SIM embryonic fibroblasts, can be used as a feeder layer for in vitro culture of authentic pig ESCs instead of primary MEFs. First, the expression of genes encoding ECM proteins and growth factors was analyzed to compare their secretory functions as feeder cells. Quantitative real-time polymerase chain reaction (qPCR) showed that the gene expression of these pluripotency-associated factors was downregulated in STO cells compared to primary MEFs of similar density. Therefore, subsequent optimization of the culture conditions was attempted using higher STO cell densities. Notably, pig ESCs cultured on STO cell density of 3 × (187,500 cells/cm2) exhibited the most similar pluripotent state to pig ESCs cultured on primary MEF density of 1 × (62,500 cells/cm2), as determined by alkaline phosphatase staining, qPCR, and immunocytochemistry. In addition, pig ESCs cultured on STO cell density of 3 × formed complex teratoma containing multiple types of tissues derived from all three germ layers. Our culture conditions using optimal STO cell density can be applied to fields requiring reproducible and scalable production of pig ESCs, such as preclinical research and cellular agriculture.

Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carry out comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. Notably, we find a fundamental difference between optimal DIA methods for high- and low-load samples. We further improve our low-input DIA method by relying on high-resolution MS1 quantification, thus enhancing sensitivity by more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we are able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we demonstrate the capability of our approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes and highlighting distinct differences in key metabolic enzyme expression in distinct cell subclusters.

Efficient generation of functional neurons from mouse embryonic stem cells via neurogenin-2 expression.

The production of induced neuronal (iN) cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells by the forced expression of proneural transcription factors is rapid, efficient and reproducible. The ability to generate large numbers of human neurons in such a robust manner enables large-scale studies of human neural differentiation and neuropsychiatric diseases. Surprisingly, similar transcription factor-based approaches for converting mouse ESCs into iN cells have been challenging, primarily because of low cell survival. Here, we provide a detailed approach for the efficient and reproducible generation of functional iN cells from mouse ESC cultures by the genetically induced expression of neurogenin-2. The resulting iN cells display mature pre- and postsynaptic specializations and form synaptic networks. Our method provides the basis for studying neuronal development and enables the direct comparison of cellular phenotypes in mouse and human neurons generated in an equivalent way. The procedure requires 14 d and can be carried out by users with expertise in stem cell culture.

A genome-wide screen reveals new regulators of the 2-cell-like cell state.

In mammals, only the zygote and blastomeres of the early embryo are totipotent. This totipotency is mirrored in vitro by mouse '2-cell-like cells' (2CLCs), which appear at low frequency in cultures of embryonic stem cells (ESCs). Because totipotency is not completely understood, we carried out a genome-wide CRISPR knockout screen in mouse ESCs, searching for mutants that reactivate the expression of Dazl, a gene expressed in 2CLCs. Here we report the identification of four mutants that reactivate Dazl and a broader 2-cell-like signature: the E3 ubiquitin ligase adaptor SPOP, the Zinc-Finger transcription factor ZBTB14, MCM3AP, a component of the RNA processing complex TREX-2, and the lysine demethylase KDM5C. All four factors function upstream of DPPA2 and DUX, but not via p53. In addition, SPOP binds DPPA2, and KDM5C interacts with ncPRC1.6 and inhibits 2CLC gene expression in a catalytic-independent manner. These results extend our knowledge of totipotency, a key phase of organismal life.

IN VITRO PROPAGATION OF PLATELETS FROM EMBRYONIC STEM CELLS WITH C. PAPAYA LEAF EXTRACT

Objective: The depletion of platelets in patients under different clinical conditions like thrombocytopenia is often associated with a significant mortality factor. Alternative modes of platelet propagation from other resources, including stem cells, are evaluated under in vitro conditions. Embryonic stem (ES) cells can proliferate and differentiate into various cell types. This study evaluated the generation of functional platelets from embryonic stem cells with C. papaya leaf extract through a controlled sequential pathway to meet the clinical demand.
 Methods: In vitro culture of embryonic stem cells was carried out with the help of growth factors and a suitable medium. The differentiated megakaryocytes and platelets were analyzed by staining, flow cytometer and functional studies. The normal megakaryocytes were compared with the megakaryocytes differentiated from Embryonic Stem cells.
 Results: The platelets derived from embryonic stem cells are morphologically identical to normal platelets isolated from peripheral blood, and the number of viable cells was analyzed by flow cytometer. The distinctive two waves of platelet production represented normal primitive and definitive hematopoiesis.
 Conclusion: This study is a preliminary milestone in platelet propagation from embryonic stem cells with C. papaya leaf extract. The differentiation of ESCs into megakaryocyte-mediated platelets with C. papaya leaf extract has shown valuable information on the positive effects that embryonic stem cells can be used for large-scale platelet production.

The people behind the papers - Nicole Repina and David Schaffer.

During embryogenesis, cells differentiate and organise into spatially defined regions in response to varying patterns of signalling. A new paper in Development uses an optogenetic system (optoWnt) to investigate how cells organise into distinct domains in response to Wnt signalling in two-dimensional human embryonic stem cell cultures. To hear more about the story behind the paper, we caught up with the first author Nicole Repina and her PhD supervisor David Schaffer, Professor at University of California, Berkeley.

Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells

Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

Hyperosmotic stress induces 2-cell-like cells through ROS and ATR signaling.

Mouse embryonic stem cells (ESCs) display pluripotency features characteristic of the inner cell mass of the blastocyst. Mouse embryonic stem cell cultures are highly heterogeneous and include a rare population of cells, which recapitulate characteristics of the 2-cell embryo, referred to as 2-cell-like cells (2CLCs). Whether and how ESC and 2CLC respond to environmental cues has not been fully elucidated. Here, we investigate the impact of mechanical stress on the reprogramming of ESC to 2CLC. We show that hyperosmotic stress induces 2CLC and that this induction can occur even after a recovery time from hyperosmotic stress, suggesting a memory response. Hyperosmotic stress in ESCs leads to accumulation of reactive-oxygen species (ROS) and ATR checkpoint activation. Importantly, preventing either elevated ROS levels or ATR activation impairs hyperosmotic-mediated 2CLC induction. We further show that ROS generation and the ATR checkpoint act within the same molecular pathway in response to hyperosmotic stress to induce 2CLCs. Altogether, these results shed light on the response of ESC to mechanical stress and on our understanding of 2CLC reprogramming.

P-799 Morphine causes global hypomethylation through active and passive demethylation mechanisms in mESCs hindering optimal embryonic development

Abstract Study question Can morphine create changes in the mESC methylation pattern that are potentially related to the mismatches observed during embryonic development? Summary answer Morphine creates an even more hypomethylated state of the mESC genome, which results in a delay in mESC differentiation and, consequently, an increase in pluripotency. What is known already Epigenetic changes are essential for normal development. However, some environmental factors can cause epigenetic changes that leads to health problems or diseases. Morphine is known to pass through the placental barrier and impact normal embryo development by affecting the neural tube, frontal cortex and spinal cord development, and, as a consequence, delaying nervous system development. In fact, in-utero morphine exposure has shown alterations in anxiety-like behaviours, analgesic tolerance, synaptic plasticity and the neuronal structure of offspring. Nonetheless, how morphine leads to abnormal neurogenesis and other physiological consequences during embryo development is still unknown. Study design, size, duration Considering that DNA methylation is a key epigenetic factor crucial for embryo development, our aim is to elucidate the suitability of using this opioid in pregnant women, analyzing the role of methylation in response to morphine. To study morphine effects on embryo development, we used mouse embryonic stem cells (mESCs) culture, one of the most widely used models for studying embryo development in vitro. Participants/materials, setting, methods mESCs was treated with a chronic morphine treatment (24 h, 10 μM). DNA extraction was done using a classic phenol-chloroform/isoamyl methodology, and RNA extraction with commercial Nucleozol reagent according to the manufacturer’s instructions. Liquid chromatography–mass spectrometry (LC-MS/MS) measured global genome methylation/hydroxymethylation levels, Whole Genome Bisulfite Sequencing (WGBSeq) measured cytosines methylation, and Real Time quantitative Polymerase Chain Reaction (RT-qPCR) measured gene expression. The statistical analysis (LC-MS/MS + RT-qPCR) was made by t-student. Main results and the role of chance By MS/MS approaches, we observed a global methylation decrease and a global hydroxymethylation increase in mESCs, changes that occurred after a chronic morphine treatment (24 h, 10 μM). WGBSeq identified 13329 sensitive cytosines to morphine that are involved in embryo development, signalling pathways, metabolism and/or gene expression. This suggests that morphine might impact methylation levels at developmental genes. Integrative analyses between WGBSeq and RNASeq identified Tet Methylcytosine Dioxygenase 1 (Tet1) as sensitive morphine gene. Morphine increased the gene expression of Tet1, modifying the methylation levels at the promoter. Otherwise, RNASeq and qRT-PCR analyses revealed that DNA Methyltransferase 1 (Dnmt1) gene expression decreased after morphine treatment. In conclusion, morphine induces a global hypomethylation in mESC through different mechanisms that involves active and passive demethylation, and the first one has a self-regulating mechanism that significantly enhances the demethylation process. Limitations, reasons for caution Taking into account all the ethical problems caused by the use of hESCs, this study has been carried out with mESCs. Although the physiology of mice and humans is somewhat different, this is the most recommended model for this type of pilot study. Wider implications of the findings This hypomethylation can potentially lead to a significant delay in optimal embryonic development; The global hypomethylated state of the genome is one of the most significant characteristics of stem cells. Consequently, the possible use of this and other opioids in patients who are pregnant should be strongly discouraged. Trial registration number Not applicable

Generation of pulsatile ERK activity in mouse embryonic stem cells is regulated by Raf activity

The extracellular signal-regulated kinase (ERK) is a serine/threonine kinase that is known to regulate cellular events such as cell proliferation and differentiation. The ERK signaling pathway is activated by fibroblast growth factors, and is considered to be indispensable for the differentiation of primitive endoderm cells, not only in mouse preimplantation embryos, but also in embryonic stem cell (ESC) culture. To monitor ERK activity in living undifferentiated and differentiating ESCs, we established EKAREV-NLS-EB5 ESC lines that stably express EKAREV-NLS, a biosensor based on the principle of fluorescence resonance energy transfer. Using EKAREV-NLS-EB5, we found that ERK activity exhibited pulsatile dynamics. ESCs were classified into two groups: active cells showing high-frequency ERK pulses, and inactive cells demonstrating no detectable ERK pulses during live imaging. Pharmacological inhibition of major components in the ERK signaling pathway revealed that Raf plays an important role in determining the pattern of ERK pulses.

Culture conditions of mouse ESCs impact the tumor appearance in vivo

Various culture conditions by small molecules have been explored to extend pluripotency of stem cells, but their impacts on cell fate invivo remain elusive. We systematically compared the effects of various culture conditions on the pluripotency and cell fate invivo of mouse embryonic stem cells (ESCs) by tetraploid embryo complementation assay. Conventional ESC cultures in serum/LIF-based condition produced complete ESC mice and also the survival to adulthood at the highest rates of all other chemical-based cultures. Moreover, long-term examination of the survived ESC mice demonstrated that conventional ESC cultures did not lead to visible abnormality for up to 1.5-2 years, whereas the prolonged chemical-based cultures developed retroperitoneal atypical teratomas or leiomyomas. The chemical-based cultures exhibited transcriptomes and epigenomes that typically differed from those of conventional ESC cultures. Our results warrant further refinement of culture conditions in promoting the pluripotency and safety of ESCs in future applications.

Political and ethical landscape of brain organoid research

Human cerebral organoids (hCOs), produced in labs through directed cell culture of embryonic or induced pluripotent stem cells, closely replicate the 3-dimensional architecture of the human brain on a micro scale. This technology has been used to model neurological disease and shows promise to complement or supplant animal subjects in preclinical therapeutic investigation. However, attention must be paid by researchers and institutions to the various ethical concerns associated with hCO development. Human-animal chimeras produced through the grafting of hCOs have shown integration of neurological function, calling into question the moral status of both the animal chimeras and the organoid itself. Sensationalist reporting on such acts may also prompt public backlash, potentially jeopardizing hCO research and the promised benefits thereof. Moreover, concerns arise over privacy and consent for past and prospective donors of stem cells used to produce organoids. Genetic information may be considered privileged to the public domain and disrupted trust can reduce the supply of willing donors. Though hCOs are believed thus far to lack the capacity for consciousness and cognitive function, consideration must be given to their potential status as moral agents with further development or enhancement. Boundaries concerning organoids adhered to by researchers have been largely voluntary and informal to this point. By edict or by the power of the purse, governmental regulatory agencies ought to formalize necessary guidelines to ensure compliance with ethical principles and the adequate representation of all affected stakeholders in future decisions.