Cultured Mouse Embryonic Stem Cells Research Articles

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.

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

Cross-activation of FGF, NODAL, and WNT pathways constrains BMP-signaling-mediated induction of the totipotent state in mouse embryonic stem cells

Embryonic stem cells (ESCs) are an attractive model to study the relationship between signaling and cell fates. Cultured mouse ESCs can exist in multiple states resembling distinct stages of early embryogenesis, such as totipotent, pluripotent, primed, and primitive endoderm. The signaling mechanisms regulating the totipotent state and coexistence of these states are poorly understood. Here we identify bone morphogenetic protein (BMP) signaling as an inducer of the totipotent state. However, we discover that BMP's role is constrained by the cross-activation of FGF, NODAL, and WNT pathways. We exploit this finding to enhance the proportion of totipotent cells by rationally inhibiting the cross-activated pathways. Single-cell mRNA sequencing reveals that induction of the totipotent state is accompanied by suppression of primed and primitive endoderm states. Furthermore, reprogrammed totipotent cells we generate in culture resemble totipotent cells of preimplantation embryo. Our findings reveal a BMP signaling mechanism regulating both the totipotent state and heterogeneity of ESCs.

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

Succinate as a New Actor in Pluripotency and Early Development?

Pluripotent cells have been stabilized from pre- and post-implantation blastocysts, representing respectively naïve and primed stages of embryonic stem cells (ESCs) with distinct epigenetic, metabolic and transcriptomic features. Beside these two well characterized pluripotent stages, several intermediate states have been reported, as well as a small subpopulation of cells that have reacquired features of the 2C-embryo (2C-like cells) in naïve mouse ESC culture. Altogether, these represent a continuum of distinct pluripotency stages, characterized by metabolic transitions, for which we propose a new role for a long-known metabolite: succinate. Mostly seen as the metabolite of the TCA, succinate is also at the crossroad of several mitochondrial biochemical pathways. Its role also extends far beyond the mitochondrion, as it can be secreted, modify proteins by lysine succinylation and inhibit the activity of alpha-ketoglutarate-dependent dioxygenases, such as prolyl hydroxylase (PHDs) or histone and DNA demethylases. When released in the extracellular compartment, succinate can trigger several key transduction pathways after binding to SUCNR1, a G-Protein Coupled Receptor. In this review, we highlight the different intra- and extracellular roles that succinate might play in the fields of early pluripotency and embryo development.

Recapitulating early human development with 8C-like cells.

In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An invitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.

Zfp281 Inhibits the Pluripotent-to-Totipotent State Transition in Mouse Embryonic Stem Cells.

The cell-fate transition between pluripotent and totipotent states determines embryonic development and the first cell-lineage segregation. However, limited by the scarcity of totipotent embryos, regulators on this transition remain largely elusive. A novel model to study the transition has been recently established, named the 2-cell-like (2C-like) model. The 2C-like cells are rare totipotent-like cells in the mouse embryonic stem cell (mESC) culture. Pluripotent mESCs can spontaneously transit into and out of the 2C-like state. We previously dissected the transcriptional roadmap of the transition. In this study, we revealed that Zfp281 is a novel regulator for the pluripotent-to-totipotent transition in mESCs. Zfp281 is a transcriptional factor involved in the cell-fate transition. Our study shows that Zfp281 represses transcripts upregulated during the 2C-like transition via Tet1 and consequentially inhibits mESCs from transiting into the 2C-like state. Interestingly, we found that the inhibitory effect of Zfp281 on the 2C-like transition leads to an impaired 2C-like-transition ability in primed-state mESCs. Altogether, our study reveals a novel mediator for the pluripotent-to-totipotent state transition in mESCs and provides insights into the dynamic transcriptional control of the transition.

BMP4 preserves the developmental potential of mESCs through Ube2s- and Chmp4b-mediated chromosomal stability safeguarding

Chemically defined medium is widely used for culturing mouse embryonic stem cells (mESCs), in which N2B27 works as a substitution for serum, and GSK3β and MEK inhibitors (2i) help to promote ground-state pluripotency. However, recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs. Here, we demonstrated the deficient bone morphogenetic protein (BMP) signal in the chemically defined condition is one of the main causes for the impaired pluripotency. Mechanistically, activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream targets Ube2s and Chmp4b. More importantly, BMP4 promotes a distinct in vivo developmental potential and a long-term pluripotency preservation. Besides, the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression. Taken together, our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system.

Cell-cell communication through FGF4 generates and maintains robust proportions of differentiated cell types in embryonic stem cells.

ABSTRACTDuring embryonic development and tissue homeostasis, reproducible proportions of differentiated cell types are specified from populations of multipotent precursor cells. Molecular mechanisms that enable both robust cell-type proportioning despite variable initial conditions in the precursor cells, and the re-establishment of these proportions upon perturbations in a developing tissue remain to be characterized. Here, we report that the differentiation of robust proportions of epiblast-like and primitive endoderm-like cells in mouse embryonic stem cell cultures emerges at the population level through cell-cell communication via a short-range fibroblast growth factor 4 (FGF4) signal. We characterize the molecular and dynamical properties of the communication mechanism and show how it controls both robust cell-type proportioning from a wide range of experimentally controlled initial conditions, as well as the autonomous re-establishment of these proportions following the isolation of one cell type. The generation and maintenance of reproducible proportions of discrete cell types is a new function for FGF signaling that might operate in a range of developing tissues.

Generation of mouse-human chimeric embryos.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3-4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1-4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals.

Retinoic acid signaling is critical during the totipotency window in early mammalian development

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

MgFe-LDH Nanoparticles: A Promising Leukemia Inhibitory Factor Replacement for Self-Renewal and Pluripotency Maintenance in Cultured Mouse Embryonic Stem Cells.

Leukemia inhibitory factor (LIF), an indispensable bioactive protein that sustains self‐renewal and pluripotency in stem cells, is vital for mouse embryonic stem cell (mESC) culture. Extensive research is conducted on reliable alternatives for LIF as its clinical application in stable culture and large‐scale expansion of ESCs is limited by its instability and high cost. However, few studies have sought to replace LIF with nanoparticles to provide a xeno‐free culture condition. MgAl‐LDH (layered double hydroxide) nanoparticles can partially replace LIF in maintaining pluripotency of mESCs; however, the requirement and tolerance for aluminum ions in mice are far lesser than those of iron ions. Hence, MgFe‐LDH nanoparticles are selected for this study. MgFe‐LDH is superior to MgAl‐LDH in maintaining self‐renewal and pluripotency of mESCs, in the absence of LIF and mouse embryonic fibroblast. Furthermore, combined transcriptomic and proteomic analysis confirms that MgFe‐LDH can activate the LIF receptor (LIFR)/phosphatidylinositol 3‐kinase (PI3K)/protein kinase B(AKT), LIFR/JAK/janus kinase (JAK)/signal transducer and activator of transcription 3(STAT3), and phospho‐signal transducer and activator of transcription 3(p‐STAT3)/ten‐eleven translocation (TET) signaling pathways, while the extra Fe2+ provided by MgFe‐LDH would also enhance TET1/2 abundance thus affecting the TET1/2 regulated pluripotency related marker expression and TET1/2 meditated DNA demethylation. These results suggest that MgFe‐LDH nanoparticles can thus be used as an affordable and efficient replacement for LIF in mESC cultivation.

Mouse Embryonic Stem Cell Culture in Serum-Containing or 2i Conditions.

With their unique capabilities of self-renewal and differentiation into three germ layers, mouse embryonic stem cells (mESCs) are widely used as an in vitro cellular model for early mammalian developmental studies. mESCs are traditionally cultured in high-serum and LIF-containing medium on a growth-deficient mouse embryonic fibroblast layer. A more recent culturing system with two inhibitors (for GSK3β (CHIR99021) and MEK1/2 (PD0325901)) and LIF enables the derivation of mESC lines from various mouse strains. Here we describe methods for the mESC growth and maintenance in each medium composition as well as their adaptation to either condition.

Induction of Functional Hypothalamus and Pituitary Tissues From Pluripotent Stem Cells for Regenerative Medicine.

The hypothalamus and pituitary have been identified to play essential roles in maintaining homeostasis. Various diseases can disrupt the functions of these systems, which can often result in serious lifelong symptoms. The current treatment for hypopituitarism involves hormone replacement therapy. However, exogenous drug administration cannot mimic the physiological changes that are a result of hormone requirements. Therefore, patients are at a high risk of severe hormone deficiency, including adrenal crisis.Pluripotent stem cells (PSCs) self-proliferate and differentiate into all types of cells. The generation of endocrine tissues from PSCs has been considered as another new treatment for hypopituitarism. Our colleagues established a 3-dimensional (3D) culture method for embryonic stem cells (ESCs). In this culture, the ESC-derived aggregates exhibit self-organization and spontaneous formation of highly ordered patterning. Recent results have shown that strict removal of exogenous patterning factors during early differentiation efficiently induces rostral hypothalamic progenitors from mouse ESCs. These hypothalamic progenitors generate vasopressinergic neurons, which release neuropeptides upon exogenous stimulation.Subsequently, we reported adenohypophysis tissue self-formation in 3D cultures of mouse ESCs. The ESCs were found to differentiate into both nonneural oral ectoderm and hypothalamic neuroectoderm in adjacent layers. Interactions between the 2 tissues appear to be critically important for in vitro induction of a Rathke’s pouch-like developing embryo. Various endocrine cells were differentiated from nonneural ectoderm. The induced corticotrophs efficiently secreted adrenocorticotropic hormone when engrafted in vivo, which rescued hypopituitary hosts.For future regenerative medicine, generation of hypothalamic and pituitary tissues from human PSCs is necessary. We and other groups succeeded in establishing a differentiation method with the use of human PSCs. Researchers could use these methods for models of human diseases to elucidate disease pathology or screen potential therapeutics.

InVitro Meiosis of Male Germline Stem Cells.

SummaryIn vitro spermatogenesis has been achieved by culturing mouse embryonic stem cells (ESCs) together with a cell suspension of male juvenile gonad. However, for human fertility treatment or preservation, patient-specific ESCs or juvenile gonad is not available. We therefore aim to achieve in vitro spermatogenesis using male germline stem cells (GSCs) without the use of juvenile gonad. GSCs, when cultured on immortalized Sertoli cells, were able to enter meiosis, reach the meiotic metaphase stages, and sporadically form spermatid-like cells. However, the in vitro-formed pachytene-like spermatocytes did not display full chromosome synapsis and did not form meiotic crossovers. Despite this, the meiotic checkpoints that usually eliminate such cells to prevent genomic instabilities from being transmitted to the offspring were not activated, allowing the cells to proceed to the meiotic metaphase stages. In vitro-generated spermatid-like cells should thus be thoroughly investigated before being considered for clinical use.

Effects of DMSO on the Pluripotency of Cultured Mouse Embryonic Stem Cells (mESCs).

DMSO is a commonly used solvent in biological studies, as it is an amphipathic molecule soluble in both aqueous and organic media. For that reason, it is the vehicle of choice for several water-insoluble substances used in research. At the molecular and cellular level, DMSO is a hydrogen-bound disrupter, an intercellular electrical uncoupler, and a cryoprotectant, among other properties. Importantly, DMSO often has overlooked side effects. In stem cell research, the literature is scarce, but there are reports on the effect of DMSO in human embryoid body differentiation and on human pluripotent stem cell priming towards differentiation, via modulation of cell cycle. However, in mouse embryonic stem cell (mESC) culture, there is almost no available information. Taking into consideration the almost ubiquitous use of DMSO in experiments involving mESCs, we aimed to understand the effect of very low doses of DMSO (0.0001%-0.2%), usually used to introduce pharmacological inhibitors/modulators, in mESCs cultured in two different media (2i and FBS-based media). Our results show that in the E14Tg2a mESC line used in this study, even the smallest concentration of DMSO had minor effects on the total number of cells in serum-cultured mESCs. However, these effects could not be explained by alterations in cell cycle or apoptosis. Furthermore, DMSO did not affect pluripotency or differentiation potential. All things considered, and although control experiments should be carried out in each cell line that is used, it is reasonable to conclude that DMSO at the concentrations used here has a minimal effect on this particular mESC line.

Nicotinamide restricts neural precursor proliferation to enhance catecholaminergic neuronal subtype differentiation from mouse embryonic stem cells.

Emerging evidence indicates that a strong relationship exists between brain regenerative therapies and nutrition. Early life nutrition plays an important role during embryonic brain development, and there are clear consequences to an imbalance in nutritional factors on both the production and survival of mature neuronal populations and the infant’s risk of diseases in later life. Our research and that of others suggest that vitamins play a fundamental role in the formation of neurons and their survival. There is a growing body of evidence that nicotinamide, the water-soluble amide form of vitamin B3, is implicated in the conversion of pluripotent stem cells to clinically relevant cells for regenerative therapies. This study investigated the ability of nicotinamide to promote the development of mature catecholaminergic neuronal populations (associated with Parkinson’s disease) from mouse embryonic stem cells, as well as investigating the underlying mechanisms of nicotinamide’s action. Nicotinamide selectively enhanced the production of tyrosine hydroxylase-expressing neurons and serotonergic neurons from mouse embryonic stem cell cultures (Sox1GFP knock-in 46C cell line). A 5-Ethynyl-2´-deoxyuridine (EdU) assay ascertained that nicotinamide, when added in the initial phase, reduced cell proliferation. Nicotinamide drove tyrosine hydroxylase-expressing neuron differentiation as effectively as an established cocktail of signalling factors, reducing the proliferation of neural progenitors and accelerating neuronal maturation, neurite outgrowth and neurotransmitter expression. These novel findings show that nicotinamide enhanced and enriched catecholaminergic differentiation and inhibited cell proliferation by directing cell cycle arrest in mouse embryonic stem cell cultures, thus driving a critical neural proliferation-to-differentiation switch from neural progenitors to neurons. Further research into the role of vitamin metabolites in embryogenesis will significantly advance cell-based regenerative medicine, and help realize their role as crucial developmental signalling molecules in brain development.

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos

CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F0 generation.

Enhanced Induction of Definitive Endoderm Differentiation of Mouse Embryonic Stem Cells in Simulated Microgravity.

Directed in vitro differentiation of pluripotent stem cells toward definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two-dimensional monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2, Sox17, CxCr4), and mesodermal (Vimentin, Meox1) lineages. Culture in the form of EBs in maintenance media in the presence of leukemia inhibitory factor, in static or SMG conditions, induced expression of some of the differentiation markers, suggesting heterogeneity of the cells. This is in line with previous studies showing that differentiation is initiated as cells are aggregated into EBs even without supplementing differentiation factors to the media. Culturing EBs in static conditions in differentiation media (DM) in the presence of activin A reduced Oct3/4 expression and significantly increased Brachyury T and CxCr4 expression, but downregulated FoxA2 and Sox17. However, culturing in SMG BRs in DM upregulated Brachyury T and all of the DE markers and reduced Oct3/4 expression, indicating the advantage of dynamic cultures in BRs to specifically enhance directed DE differentiation. Given the potential discrepancies between the SMG conditions on earth and actual microgravity conditions, as observed in other studies, future experiments in space flight are required to validate the effects of reduced gravity on mESC differentiation.