Early Embryonic Stem Cell Differentiation Research Articles

Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells.

Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood. To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2, Pim-2, and Met-1, respectively. mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays. Our findings showed that the upregulation of Bcl-2, Pim-2, and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression. Our findings revealed that the upregulation of Bcl-2, Pim-2, and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.

Role of Histamine H3 Receptor Antagonist Pitolisant in Early Neural Differentiation of Mouse Embryonic Stem Cells.

The histamine H3 receptor, prominently expressed in neurons with a minor presence in glial cells, acts as both an autoreceptor and an alloreceptor, controlling the release of histamine and other neurotransmitters. The receptor impacts various essential physiological processes. Our team's initial investigations had demonstrated that the histamine H3 receptor antagonists could facilitate nerve regeneration by promoting the histamine H1 receptors on primary neural stem cells (NSCs) in the traumatic brain injury mouse, which suggested the potential of histamine H3 receptor as a promising target for treating neurological disorders and promoting nerve regeneration. Pitolisant (PITO) is the only histamine H3 receptor antagonist approved by the Food and Drug Administration (FDA) for treating narcolepsy. However, there is no report on Pitolisant in neural development or regeneration, and it is urgent to be further studied in strong biological activity models in vitro. The embryonic stem (ES) cells were differentiated into neural cells in vitro, which replicated the neurodevelopmental processes that occur in vivo. It also provided an alternative model for studying neurodevelopmental processes and testing drugs for neurological conditions. Therefore, we aimed to elucidate the regulatory role of Pitolisant in the early differentiation of ES cells into neural cells. Our results demonstrated that Pitolisant could promote the differentiation of ES cells toward NSCs and stimulated the formation of growth cones. Furthermore, Pitolisant was capable of inducing the polarization of NSCs through the cAMP-LKB1-SAD/MARK2 pathway, but had no significant effect on later neuronal maturation. Pitolisant altered mitochondrial morphology and upregulated the levels of mitochondrion-related proteins TOM20, Drp1, and p-Drp1, and reversed the inhibitory effect of Mdivi-1 on mitochondrial fission during the early neural differentiation of ES cells. In addition, Pitolisant induced the increase in cytosolic Ca2+. Our study provided an experimental foundation for the potential application of histamine H3 receptor-targeted modulators in the field of neuroregeneration.

RNA degradation eliminates developmental transcripts during murine embryonic stem cell differentiation via CAPRIN1-XRN2.

Embryonic stem cells (ESCs) are self-renewing and pluripotent. In recent years, factors that control pluripotency, mostly nuclear, have been identified. To identify non-nuclear regulators of ESCs, we screened an endogenously labeled fluorescent fusion-protein library in mouse ESCs. One of the more compelling hits was the cell-cycle-associated protein 1 (CAPRIN1). CAPRIN1 knockout had little effect in ESCs, but it significantly altered differentiation and gene expression programs. Using RIP-seq and SLAM-seq, we found that CAPRIN1 associates with, and promotes the degradation of, thousands of RNA transcripts. CAPRIN1 interactome identified XRN2 as the likely ribonuclease. Upon early ESC differentiation, XRN2 is located in the nucleus and colocalizes with CAPRIN1 in small RNA granules in a CAPRIN1-dependent manner. We propose that CAPRIN1 regulates an RNA degradation pathway operating during early ESC differentiation, thus eliminating undesired spuriously transcribed transcripts in ESCs.

Two Enhancers Regulate HoxB Genes Expression During Retinoic Acid-Induced Early Embryonic Stem Cells Differentiation Through Long-Range Chromatin Interactions.

Homeobox B cluster (HoxB) genes play important roles in retinoic acid (RA)-induced early embryonic stem cells (ESCs) differentiation. Knowledge of regulation network of HoxB is important to further unveil the mechanism of ESCs differentiation. In this study, we identified two enhancers that were activated by RA treatment and 4C data showed long-range interactions between HoxB genes and the two enhancers. CRISPR/Cas9-mediated individual or compound deletion of the two enhancers significantly inhibits HoxB gene expression, and transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in the enhancer KO cells. We propose new mechanism by which two enhancers regulate HoxB gene expression by different regulation modes during RA-induced early ESCs differentiation through long-range chromatin interactions.

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA

Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer–enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.

Retinoic acid (RA) induces rapid differentiation of embryonic stem cells (ESCs), partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ∼40% of genes whose expression changed following enhancer deletion, and ∼18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target genes expression and then orchestrates RA-induced early differentiation of ESCs. This discovery reveals mechanisms of a novel enhancer regulating RA-induced Hoxa genes expression and early ESCs differentiation.

Decoupling the impact of microRNAs on translational repression versus RNA degradation in embryonic stem cells.

Translation and mRNA degradation are intimately connected, yet the mechanisms that link them are not fully understood. Here, we studied these mechanisms in embryonic stem cells (ESCs). Transcripts showed a wide range of stabilities, which correlated with their relative translation levels and that did not change during early ESC differentiation. The protein DHH1 links translation to mRNA stability in yeast; however, loss of the mammalian homolog, DDX6, in ESCs did not disrupt the correlation across transcripts. Instead, the loss of DDX6 led to upregulated translation of microRNA targets, without concurrent changes in mRNA stability. The Ddx6 knockout cells were phenotypically and molecularly similar to cells lacking all microRNAs (Dgcr8 knockout ESCs). These data show that the loss of DDX6 can separate the two canonical functions of microRNAs: translational repression and transcript destabilization. Furthermore, these data uncover a central role for translational repression independent of transcript destabilization in defining the downstream consequences of microRNA loss.

Isolation and Comparative Characteristics of Mesenchymal Stem-Cell Lines Derived from Foreskin of Two Donors of Similar Age

Two new nonimmortalized human cell lines FRSN-1 and FRSN-2 were established from foreskin of two similarly aged donors (2.5 years). Growth characteristics and differentiation potential of these cell lines studied on the sixth passage confirmed their status as mesenchymal stem cells (MSCs). A number of characteristics have been analyzed during long-term cultivation up to the 26th passage. The dynamics of the process of replicative senescence defined by the activity of β-galactosidase differed between these lines. However, at the 26th passage, the process of replicative senescence was equally enhanced in both lines. The plating efficiency markedly differed between the lines on the sixth passage. In FRSN-1, it was higher than in FRSN-2. The plating efficiency substantially dropped to the 26th passage in FRSN-1 and was lost in FRSN-2 line. Growth curves showed active proliferation of these lines at the 6th passage. The average doubling time did not differ between the lines and was 36.9 and 39.0 h, respectively. Analysis of growth curves on the 26th passage revealed a decline in proliferative activity and increase in average doubling time of cell populations in both lines, more in FRSN-2 than in FRSN-1 lines. The patterns of growth curves differed in these lines. Morphological analysis revealed increased cell size and spreading typical for the phase of the replicative senescence. Numerical and structural karyotypic analysis at the sixth passage showed that both lines have normal karyotype 46, XY. We did not discover interline differences in the frequency of chromosomal aberrations. To determine the status of these cell lines, comparative analysis of the surface markers was performed using flow cytometry. It was revealed that cells of both lines expressed surface antigens characteristic of human MSCs: CD44, CD73, CD90, CD105, and HLA-ABC and did not express CD34, CD45 and HLADR. Cells of both lines displayed SSEA-4 and SOX2, markers of human embryonic stem cells (ESCs). Expression of SSEA-4 was also detected at the 26th passage in both lines. FRSN-1 and FRSN-2 cells expressed the markers of early ESC differentiation into three germ layers. The ability of these cell lines to differentiate into osteogenic, chondrogenic, and adipogenic lineages was shown on the sixth passage. Both lines exhibited substantially reduced adipogenic potential on the 20th passage. These data indicate that in contrast to growth characteristics the adipogenic differentiation potential changes even with an average degree of replicative senescence. It appears that the cell replicative senescence contributed to the change in MSC differentiation potential. Overall, the results demonstrate that cell lines derived from different donors are distinguished in growth characteristics and pattern of replicative senescence. The disparity is due to a direct genetic influence and indirectly by different microenvironment in their donor organisms before cell isolation.

RBM14 is indispensable for pluripotency maintenance and mesoderm development of mouse embryonic stem cells

The pluripotency of embryonic stem cells (ESCs) is maintained by core pluripotency transcription factors, cofactors and several signaling pathways. RBM14 is a component of the para-speckle complex, which has been implicated in multiple important biological processes. The role of RBM14 in ESCs and lineage differentiation remains to be elucidated. In the present study, we provided evidence that RBM14 plays important roles in maintaining pluripotency and in the early differentiation of ESCs. RBM14 was demonstrated to be expressed in mouse embryonic stem cells (mESCs) and localized in the nucleus. RBM14 expression was depleted in mESCs using clustered regularly interspaced short palindromic repeats (CRISPR) technology. Our results also showed that RBM14 depletion altered the gene expression profiles of mESCs. In particular, pluripotency-associated genes and genes involved in the Wnt and TGF-β signaling pathways were downregulated in RBM14 knockout mESCs. Furthermore, RBM14 was found to be essential for mesoderm development in vitro and in vivo. The specific effects of RBM14 depletion were verified by conducting a rescue experiment. Our findings demonstrated that RBM14 not only plays an important role in maintaining the pluripotency of mESCs but is also indispensable for mesoderm development.

RAS Regulates the Transition from Naive to Primed Pluripotent Stem Cells.

SummaryThe transition from naive to primed state of pluripotent stem cells is hallmarked by epithelial-mesenchymal transition, metabolic switch from oxidative phosphorylation to aerobic glycolysis, and changes in the epigenetic landscape. Since these changes are also seen as putative hallmarks of neoplastic cell transformation, we hypothesized that oncogenic pathways may be involved in this process. We report that the activity of RAS is repressed in the naive state of mouse embryonic stem cells (ESCs) and that all three RAS isoforms are significantly activated upon early differentiation induced by LIF withdrawal, embryoid body formation, or transition to the primed state. Forced expression of active RAS and RAS inhibition have shown that RAS regulates glycolysis, CADHERIN expression, and the expression of repressive epigenetic marks in pluripotent stem cells. Altogether, this study indicates that RAS is located at a key junction of early ESC differentiation controlling key processes in priming of naive cells.

Transcriptomics analysis of early embryonic stem cell differentiation under osteoblast culture conditions: Applications for detection of developmental toxicity

The mouse embryonic stem cell test (mEST) is a promising in vitro assay for predicting developmental toxicity. In the current study, early differentiation of D3 mouse embryonic stem cells (mESCs) under osteoblast culture conditions and embryotoxicity of cadmium sulfate were examined. D3 mESCs were exposed to cadmium sulfate for 24, 48 or 72h, and whole genome transcriptional profiles were determined. The results indicate a track of differentiation was identified as mESCs differentiate. Biological processes that were associated with differentiation related genes included embryonic development and, specifically, skeletal system development. Cadmium sulfate inhibited mESC differentiation at all three time points. Functional pathway analysis indicated biological pathways affected included those related to skeletal development, renal and reproductive function. In summary, our results suggest that transcriptional profiles are a sensitive indicator of early mESC differentiation. Transcriptomics may improve the predictivity of the mEST by suggesting possible modes of action for tested chemicals.

MiR-29 regulates Tet1 expression and contributes to early differentiation of mouse ESCs.

The ten-eleven translocation-1 (Tet1), which converts 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC), plays important roles in many important biological processes, such as mouse embryonic stem cells (ESCs) maintenance. However, the mechanisms for Tet-1 regulation remain largely unknown. Here we showed that miR-29 family (miR-29a, miR-29b and miR-29c) can directly repress Tet1 expression. We found that Tet1 was highly expressed and 5hmC was presented at relatively high levels in mouse ESCs, but the levels of both Tet1 and 5hmC were reduced during the early differentiation of ESCs. On the contrary, miR-29 level was increased in this process. ESCs stably transfecting with miR-29 precursors showed lower levels of Tet1 protein and 5hmC. Furthermore, we demonstrated that miR-29 overexpression selectively affected cell lineage markers and skewed ESC differentiation, which was similar in Tet1 knockdown ESCs. Our results indicate that miR-29 is a direct regulator of Tet1 in mouse ESCs.

Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1.

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8(-/Y) ) model, we found that deletion of phf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8(+/Y) ) and ph8(-/Y) ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown of pmaip1 mimicked the phenotype of ph8(-/Y) by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8(-/Y) ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells 2016;34:1527-1540.

Dynamic changes in energy metabolism upon embryonic stem cell differentiation support developmental toxicant identification.

Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST.

Rotary suspension culture enhances mesendoderm differentiation of embryonic stem cells through modulation of Wnt/β-catenin pathway.

Recently, physical factors in the local cellular microenvironment have been confirmed with strong influences on regulating stem cell fate. Despite the recent identification of the rotary cell culture system (RCCS) as a bioreactor for culturing stem cells, the underlying biological role provided by RCCS in the lineage differentiation of embryonic stem cells (ESCs) remains largely undefined. Here, we explored the embryoid body (EB) formation and subsequent differentiation of mouse ESCs in RCCS. We demonstrated that EBs formed in RCCS were more homogeneous and bigger in size compared with those in the static condition. Further, we determined that mesendoderm differentiation was prominently enhanced, while neuroectodermal differentiation was significantly suppressed in RCCS. Surprisingly, we found that Wnt/β-catenin signaling was greatly enhanced mainly due to the increased expression of Wnt3 during ESC differentiation in RCCS. Inhibition of Wnt/β-catenin signaling by DKK1 decreased the expression of Brachyury and attenuated mesendoderm differentiation in RCCS. Intriguingly, Wnt3a markedly increased Brachyury expression under static condition rather than in RCCS. Taken together, our findings uncover a new role of rotary suspension culture in initializing the early differentiation of ESCs.

Nuclear structure and function

The Minisymposium on Nuclear Structure and Function featured new strategies and approaches for understanding how the vast amount of information in the nucleus is parsed out in individual cells. The field faces the problem of deducing the structure of a dynamic polymer (chromatin) in a living cell. Classical structural biology approaches have not been as forthcoming for dissecting the structure of this highly flexible and dynamic polymer. From a statistical mechanics perspective, the number of states that chromatin can adopt provides a rich source of conformational energy. Extrapolating to nuclear domains, we appreciate these are statistical definitions for regions within the nucleus that are on average differentiated from adjacent regions. Nuclear domains are not confined by membranes; rather, they are more like eddies in a highly viscous environment that provide chemistry favoring one chromatin state over another. The question is how we probe subtle features in such a fragile environment as the cell. Domain targeting Jason Brickner (Northwestern University) has cracked the code for clustering of active genes at the nuclear periphery in yeast. Gene promoters contain “DNA zip codes” that target genes to nuclear pores as one mechanism to enhance expression. The positional information is epigenetically propagated through several cell cycles, providing a mechanism of transcriptional memory. Aspects of this mechanism are conserved to humans, suggesting that the interaction of genes with nuclear pore proteins plays an important, conserved role in regulating transcription and chromatin structure. Gatekeeping function of the nuclear pore Weidong Yang (Temple University) is interested in the mechanism of RNA transport through the nuclear pore and has developed a new method called SPEED (single-point edge excitation subdiffusion) microscopy. SPEED microscopy enabled him to beat the loss of spatial resolution in z and visualize single RNA molecules as they traverse a nuclear pore with an unprecedented spatiotemporal superaccuracy of 8 nm and 2 ms. The three-dimensional mapping of export routes indicates that mRNAs primarily interact with the periphery on the nucleoplasmic side and in the center of the NPC (nuclear pore complex) without entering the central conduit utilized for passive diffusion of small molecules. These are important findings that enable discrimination of various models in the field. Gene and chromosome pairing Megan Bodnar (David Spector's laboratory, Cold Spring Harbor Laboratory) investigated the organization of pluripotency genes during early embryonic stem cell differentiation. She reported that a DNA element mediates allelic pairing at the Oct4 gene locus during the onset of Oct4 gene repression. Thus, just as active genes may be corralled for maximum expression, inactive genes may also rely on positional cues. On a larger scale, Ofer Rog (Abby Dernberg's laboratory, University of California–Berkeley) showed us chromosome synapsis in live meiosis. These remarkable images revealed a rapid and processive event that initiates from previously identified pairing sites and elongates the length of the chromosome. This study opens up a new understanding of the intimate contacts and exchange that occur between homologous chromosomes. Finally, we heard two talks on mechanisms of chromosome segregation. Ajit Joglekar (University of Michigan) updated his nanometer localization mapping of kinetochore protein position in live cells to address mechanisms of force transduction. Using a sensitive fluorescence resonance energy transfer (FRET) assay and statistical analysis of the data, Joglekar demonstrated that the microtubule (mt)-binding complex (Ndc80) is responsible for attachment to shortening mt-plus ends, while the homologue of XMAP215 (Stu2) seems to be required for corralling growing mt-plus ends. Kerry Bloom (University of North Carolina–Chapel Hill) discussed the structure of the chromatin spring that is required for tension sensing and distribution of forces between sister kinetochores. The spring is most likely a cross-linked polymer network. The cluster of 16 kinetochores in budding yeast is analogous in structure to one mammalian kinetochore that makes multiple microtubule attachments in mitosis.

Shear stress during early embryonic stem cell differentiation promotes hematopoietic and endothelial phenotypes

Pluripotent embryonic stem cells (ESCs) are a potential source for cell-based tissue engineering and regenerative medicine applications, but their translation into clinical use will require efficient and robust methods for promoting differentiation. Fluid shear stress, which can be readily incorporated into scalable bioreactors, may be one solution for promoting endothelial and hematopoietic phenotypes from ESCs. Here we applied laminar shear stress to differentiating ESCs using a 2D adherent parallel plate configuration to systematically investigate the effects of several mechanical parameters. Treatment similarly promoted endothelial and hematopoietic differentiation for shear stress magnitudes ranging from 1.5 to 15 dyne/cm(2) and for cells seeded on collagen-, fibronectin- or laminin-coated surfaces. Extension of the treatment duration consistently induced an endothelial response, but application at later stages of differentiation was less effective at promoting hematopoietic phenotypes. Furthermore, inhibition of the FLK1 protein (a VEGF receptor) neutralized the effects of shear stress, implicating the membrane protein as a critical mediator of both endothelial and hematopoietic differentiation by applied shear. Using a systematic approach, studies such as these help elucidate the mechanisms involved in force-mediated stem cell differentiation and inform scalable bioprocesses for cellular therapies.

Inducing endoderm differentiation by modulating mechanical properties of soft substrates

Early embryonic stem cell (ESC) differentiation is marked by the formation of three germ layers from which all tissues types arise. Conventionally, ESCs are differentiated by altering their chemical microenvironment. Recently however, it was established that a mechanical microenvironment can also contribute towards cellular phenotype commitment. In this study, we report how the cellular mechanical microenvironment of soft substrates affects the differentiation and phenotypic commitment of ESCs. Mouse ESCs were cultured in a fibrin hydrogel matrix in 2D and 3D cultures. The gelation characteristics of the substrates were modulated by systematically altering the fibrinogen concentration and the fibrinogen-thrombin crosslinking ratio. Analysis of the ESCs cultured on different substrate conditions clearly illustrated the strong influence that substrate physical characteristics assert on cellular behaviours. Specifically, it was found that ESCs had a higher proliferation rate in gels of lower stiffness. Early differentiation events were studied by analyzing the gene and protein expression levels of early germ layer markers. Our results revealed that lower substrate stiffness elicited stronger upregulation of endoderm related genes Sox17, Afp and Hnf4 compared to stiffer substrates. While both 2D and 3D cultures showed a similar response, the effects were much stronger in 3D culture. These results suggest that physical cues can be used to modulate ESC differentiation into clinically relevant tissues such as liver and pancreas.

Cited2 Gene Controls Pluripotency and Cardiomyocyte Differentiation of Murine Embryonic Stem Cells through Oct4 Gene

Cited2 (CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail 2) is a transcriptional modulator critical for the development of multiple organs. Although many Cited2-mediated phenotypes and molecular events have been well characterized using in vivo genetic murine models, Cited2-directed cell fate decision in embryonic stem cells (ESCs) remains elusive. In this study, we examined the role of Cited2 in the maintenance of stemness and pluripotency of murine ESCs by a gene-targeting approach. Cited2 knock-out (Cited2(Δ/-), KO) ESCs display defective differentiation. Loss of Cited2 in differentiating ESCs results in delayed silencing of the genes involved in the maintenance of pluripotency and self-renewal of stem cells (Oct4, Klf4, Sox2, and c-Myc) and the disturbance in cardiomyocyte, hematopoietic, and neuronal differentiation. In addition, Cited2 KO ESCs experience a delayed induction of cardiomyocyte differentiation-associated proteins, NFAT3 (along with the reduced expression of NFAT3 target genes, Nkx2.5 and β-MHC), N-cadherin, and smooth muscle actin. CITED2 is recruited to the Oct4 promoter to regulate its expression during early ESC differentiation. This is the first demonstration that Cited2 controls ESC pluripotency and differentiation via direct regulation of Oct4 gene expression.

Histone modifications and lamin A regulate chromatin protein dynamics in early embryonic stem cell differentiation

Embryonic stem cells are characterized by unique epigenetic features including decondensed chromatin and hyperdynamic association of chromatin proteins with chromatin. Here we investigate the potential mechanisms that regulate chromatin plasticity in embryonic stem cells. Using epigenetic drugs and mutant embryonic stem cells lacking various chromatin proteins, we find that histone acetylation, G9a-mediated histone H3 lysine 9 (H3K9) methylation and lamin A expression, all affect chromatin protein dynamics. Histone acetylation controls, almost exclusively, euchromatin protein dynamics; lamin A expression regulates heterochromatin protein dynamics, and G9a regulates both euchromatin and heterochromatin protein dynamics. In contrast, we find that DNA methylation and nucleosome repeat length have little or no effect on chromatin-binding protein dynamics in embryonic stem cells. Altered chromatin dynamics associates with perturbed embryonic stem cell differentiation. Together, these data provide mechanistic insights into the epigenetic pathways that are responsible for chromatin plasticity in embryonic stem cells, and indicate that the genome's epigenetic state modulates chromatin plasticity and differentiation potential of embryonic stem cells.