SMARCAD1 Contributes to the Regulation of Naive Pluripotency by Interacting with Histone Citrullination.

Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R26Cit peaks and SMARCAD1 peaks. These data suggest a model in which SMARCAD1 regulates naive pluripotency by interacting with H3R26Cit and suppressing heterochromatin formation.

Maintenance, Transgene Delivery, and Pluripotency Measurement of Mouse Embryonic Stem Cells.

This chapter describes standard techniques to (1) maintain mouse embryonic stem cell culture, (2) deliver transgenes into mouse embryonic stem cells mediated by electroporation, nucleofection, lipofection, and retro/lentiviruses, and (3) assess the pluripotency of mouse embryonic stem cells. The last part of this chapter presents induction of random cell differentiation followed by the alkaline phosphatase and embryoid body formation assays, immunofluorescence microscopy, and the teratoma formation assay.

Generation of organized germ layers from a single mouse embryonic stem cell.

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell-cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell-matrix and cell-cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation.

Gene expression profiling reveals the heterogeneous transcriptional activity of Oct3/4 and its possible interaction with Gli2 in mouse embryonic stem cells.

We examined the transcriptional activity of Oct3/4 (Pou5f1) in mouse embryonic stem cells (mESCs) maintained under standard culture conditions to gain a better understanding of self-renewal in mESCs. First, we built an expression vector in which the Oct3/4 promoter drives the monocistronic transcription of Venus and a puromycin-resistant gene via the foot-and-mouth disease virus self-cleaving peptide T2A. Then, a genetically-engineered mESC line with the stable integration of this vector was isolated and cultured in the presence or absence of puromycin. The cultures were subsequently subjected to Illumina expression microarray analysis. We identified approximately 4600 probes with statistically significant differential expression. The genes involved in nucleic acid synthesis were overrepresented in the probe set associated with mESCs maintained in the presence of puromycin. In contrast, the genes involved in cell differentiation were overrepresented in the probe set associated with mESCs maintained in the absence of puromycin. Therefore, it is suggested with these data that the transcriptional activity of Oct3/4 fluctuates in mESCs and that Oct3/4 plays an essential role in sustaining the basal transcriptional activities required for cell duplication in populations with equal differentiation potential. Heterogeneity in the transcriptional activity of Oct3/4 was dynamic. Interestingly, we found that genes involved in the hedgehog signaling pathway showed unique expression profiles in mESCs and validated this observation by RT-PCR analysis. The expression of Gli2, Ptch1 and Smo was consistently detected in other types of pluripotent stem cells examined in this study. Furthermore, the Gli2 protein was heterogeneously detected in mESC nuclei by immunofluorescence microscopy and this result correlated with the detection of the Oct3/4 protein. Finally, forced activation of Gli2 in mESCs increased their proliferation rate. Collectively, it is suggested with these results that Gli2 may play a novel role in the self-renewal of pluripotent stem cells.

Force via integrins but not E-cadherin decreases Oct3/4 expression in embryonic stem cells.

Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. Recently we have demonstrated that a local force applied via Arg-Gly-Asp (RGD) peptides coated magnetic beads to mouse embryonic stem (ES) cells increases cell spreading and cell stiffness and decreases Oct3/4 (Pou5f1) gene expression. However, it is not clear whether the effects of the applied stress on these functions of ES cells can be extended to natural extracellular matrix proteins or cell-cell adhesion molecules. Here we show that a local cyclic shear force applied via fibronectin or laminin to integrin receptors increased cell spreading and stiffness, downregulated Oct3/4 gene expression, and decreased cell proliferation rate. In contrast, the same cyclic force applied via cell-cell adhesion molecule E-cadherin (Cdh1) had no effects on cell spreading, Oct3/4 gene expression, and the self-renewal of mouse ES cells, but induced significant cell stiffening. Our findings demonstrate that biological responses of ES cells to force applied via integrins are different from those to force via E-cadherin, suggesting that mechanical forces might play different roles in different force transduction pathways to shape early embryogenesis.

Short-term serum-free culture reveals that inhibition of Gsk3ß induces the tumor-like growth of mouse embryonic stem cells.

Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3ß. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3ß, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3ß in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3ß governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.

Functional analysis of CTRP3/cartducin in Meckel's cartilage and developing condylar cartilage in the fetal mouse mandible.

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage.

A possible role of Reproductive Homeobox 6 in primordial germ cell differentiation.

Rhox6 is one of the Reproductive Homeobox genes on the X chromosome (Rhox) that is expressed in the placenta and the post-migratory primordial germ cells (PGCs) in the nascent gonad. Despite its novel expression pattern, the significance of Rhox6 expression in the differentiation of these cell types remains unknown. To investigate the role that Rhox6 plays in PGCs, cDNA encoding Rhox6 and short-hairpin (sh) RNA directed against Rhox6 transcripts were introduced by unique expression vectors into a genetically engineered mouse embryonic stem cell (ESC) line. This ESC line expresses enhanced green fluorescent protein (EGFP) under the Oct3/4 promoter, thereby allowing us to monitor the presence of undifferentiated ESCs and PGCs in culture in real time. This ESC line was used to isolate clones that stably expressed Rhox6 cDNA, shRNA against Rhox6 transcripts, or controls. Quantitative RT-PCR results validated that overexpression had been achieved, as well as knockdown of Rhox6 transcripts in these ESC clones. However, these clones exhibited a normal appearance of undifferentiated ESCs and expressed EGFP. Next, these ESC clones were induced to differentiate into PGCs by generating embryoid bodies (EBs) in culture medium without leukemia inhibitory factor. Detection of EGFP expression by fluorescence microscopy and germ cell markers by RT-PCR validated the differentiation of PGCs in EBs. The Rhox6 transgene had little, if any, effect on EGFP expression in EBs, whereas Rhox6 knockdown significantly decreased EGFP expression in EBs. Thus, it is suggested with these results that Rhox6 is necessary for determination of the germ cell lineage.

Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions.

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.

Integrated biochemical and mechanical signals regulate multifaceted human embryonic stem cell functions.

Human embryonic stem cells (ESCs [hESCs]) proliferate as colonies wherein individual cells are strongly adhered to one another. This architecture is linked to hESC self-renewal, pluripotency, and survival and depends on epithelial cadherin (E-cadherin), NMMIIA (nonmuscle myosin IIA), and p120-catenin. E-cadherin and p120-catenin work within a positive feedback loop that promotes localized accumulation of E-cadherin at intercellular junctions. NMMIIA stabilizes p120-catenin protein and controls E-cadherin-mediated intercellular adhesion. Perturbations of this signaling network disrupt colony formation, destabilize the transcriptional regulatory circuitry for pluripotency, and impair long-term survival of hESCs. Furthermore, depletion of E-cadherin markedly reduces the efficiency of reprogramming of human somatic cells to an ESC-like state. The feedback regulation and mechanical-biochemical integration provide mechanistic insights for the regulation of intercellular adhesion and cellular architecture in hESCs during long-term self-renewal. Our findings also contribute to the understanding of microenvironmental regulation of hESC identity and somatic reprogramming.

Characterization of the 38 kDa protein lacking in gastrula-arrested mutant Xenopus embryos.

We have reported elsewhere that offspring from the No. 65 female of Xenopus laevis cleaved normally, but their development was arrested at the onset of gastrulation, like the Ambystoma ova-deficient (o) mutant, irrespective of mating with different wild-type males, and that an acidic, 38 kDa protein present in wild-type eggs was lacking in eggs of the female. In the current study, we first determined the partial amino acid sequence (VANLE) of one of the well-separated tryptic peptides from the protein, which was found in elongation factor 1 delta (Ef1delta) in Xenopus, and finally identified the protein as one of the Ef1delta isoforms, Ef1delta2, by peptide mass spectrometry. RT-PCR analyses for Ef1delta2 and its close homolog Ef1delta1 in wild-type oocytes and embryos demonstrated that both transcripts are maternal and Ef1delta1 is present more abundantly than Ef1delta2 throughout the stages examined. Importantly, the amount of the Ef1delta2 transcript per embryo decreased gradually after gastrulation, in accordance with the gradual decrease of the 38 kDa protein per embryo reported in our earlier study. Because pharmacological inhibition of translation induces gastrulation arrest in wild-type embryos, it is reasonable to conclude that the mutant embryos arrest in development due to the lack of Ef1delta2 that is indispensable for translation. Thus, the present study provides the first molecular information on the cause of the gastrulation-defective mutation in Amphibia.

Embryonic stem cells do not stiffen on rigid substrates.

It has been previously established that living cells, including mesenchymal stem cells, stiffen in response to elevation of substrate stiffness. This stiffening is largely attributed to the elevation of the tractions at the cell base that is associated with increases in cell spreading on more-rigid substrates. We show here, surprisingly, that mouse embryonic stem cells (ESCs) do not stiffen when substrate stiffness increases. As shown recently, these cells do not increase spreading on more-rigid substrates either. However, these ESCs do increase their basal tractions as substrate stiffness increases. We conclude that these ESCs exhibit mechanical behaviors distinct from those of mesenchymal stem cells and of terminally differentiated cells, and decouple its apical cell stiffness from its basal tractional stresses during the substrate rigidity response.

Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells.

Growing evidence suggests that physical microenvironments and mechanical stresses, in addition to soluble factors, help direct mesenchymal-stem-cell fate. However, biological responses to a local force in embryonic stem cells remain elusive. Here we show that a local cyclic stress through focal adhesions induced spreading in mouse embryonic stem cells but not in mouse embryonic stem-cell-differentiated cells, which were ten times stiffer. This response was dictated by the cell material property (cell softness), suggesting that a threshold cell deformation is the key setpoint for triggering spreading responses. Traction quantification and pharmacological or shRNA intervention revealed that myosin II contractility, F-actin, Src or cdc42 were essential in the spreading response. The applied stress led to oct3/4 gene downregulation in mES cells. Our findings demonstrate that cell softness dictates cellular sensitivity to force, suggesting that local small forces might have far more important roles in early development of soft embryos than previously appreciated.

Transcriptional heterogeneity in mouse embryonic stem cells.

The embryonic stem (ES) cell is a stem cell derived from early embryos that can indefinitely repeat self-renewing cell division cycles as an undifferentiated cell in vitro and give rise to all specialised cell types in the body. However, manipulating ES cell differentiation in vitro is a challenge due to, at least in part, heterogeneous gene induction. Recent experimental evidence has demonstrated that undifferentiated mouse ES cells maintained in culture exhibit heterogeneous expression of Dppa3, Nanog, Rex1, Pecam1 and Zscan4 as well as genes (Brachyury/T, Rhox6/9 and Twist2) normally expressed in specialised cell types. The Nanog-negative, Rex1-negative or T-positive ES cell subpopulation has a unique differentiation potential. Thus, studying the mechanism that generates ES cell subpopulations will improve manipulation of ES cell fate and help our understanding of the nature of embryonic development.

Development of a gene-trap vector with a highly sensitive fluorescent protein reporter system for expression profiling.

SUMMARY: Combining high-content screening (HCS) with random gene-trap mutagenesis could be a powerful tool to investigate transcriptional networks, cell signaling, chemical genetics, and developmental processes. However, a critical limitation has been poor quantification of reporter expression per cell. To overcome this hurdle, we generated a variety of Gtx-based expression cassettes and re-evaluated translational enhancement of arrayed Gtx segments in tandem by HCS. We then modified the cassette into a new polyA trap vector, which consists of a variant of yellow fluorescent protein, Venus, in combination with the Gtx segments. Expression of Venus was detected in about 60% of trapped genes assayed in embryonic stem cell (ESC) cultures, comparable to expression screening of LacZ-based vectors. Furthermore, tetraploid aggregations using a clone encoding a gene-trap insertion into Twist2 demonstrated identical spatiotemporal expression between Venus and Twist2. This highly sensitive reporter system is amenable to high-throughput expression-based real-time HCS including single cell analyses.

Prediction and testing of novel transcriptional networks regulating embryonic stem cell self-renewal and commitment.

Stem cell fate is governed by the integration of intrinsic and extrinsic positive and negative signals upon inherent transcriptional networks. To identify novel embryonic stem cell (ESC) regulators and assemble transcriptional networks controlling ESC fate, we performed temporal expression microarray analyses of ESCs after the initiation of commitment and integrated these data with known genome-wide transcription factor binding. Effects of forced under- or overexpression of predicted novel regulators, defined as differentially expressed genes with potential binding sites for known regulators of pluripotency, demonstrated greater than 90% correspondence with predicted function, as assessed by functional and high-content assays of self-renewal. We next assembled 43 theoretical transcriptional networks in ESCs, 82% (23 out of 28 tested) of which were supported by analysis of genome-wide expression in Oct4 knockdown cells. By using this integrative approach, we have formulated novel networks describing gene repression of key developmental regulators in undifferentiated ESCs and successfully predicted the outcomes of genetic manipulation of these networks.

Esg1, expressed exclusively in preimplantation embryos, germline, and embryonic stem cells, is a putative RNA-binding protein with broad RNA targets.

In our earlier attempt to identify genes involved in the maintenance of cellular pluripotency, we found that KH-domain protein Embryonal stem cell-specific gene 1 (Esg1) showed similar expression patterns to those of Oct3/4 (Pou5f1), whereas the forced repression of Oct3/4 in mouse embryonic stem cells immediately downregulated the expression of Esg1. Here we further confirm this overlap by in situ hybridization and immunohistochemical analyses. Both Esg1 transcript and protein exist in the egg and preimplantation embryos. At embryonic day 3.5, blastocyst stage, however, ESG1 protein was more abundant in the inner cell mass (ICM) than in trophectoderm (TE), whereas Esg1 transcript was detected in both the ICM and the TE, particularly in the polar trophectoderm. The presence of an RNA-binding KH-domain in ESG1 led us to search for and identify 902 target transcripts by microarray analysis of immunoprecipitated ESG1 complex. Interaction of 20 target mRNA with ESG1, including Cdc25a, Cdc42, Ezh2, Nfyc and Nr5a2, was further validated by reverse transcriptase-polymerase chain reaction of the immunoprecipitation material, supporting the notion that ESG1 is an RNA-binding protein which associates with specific target transcripts.

Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages.

Interferon regulatory factor-8 (IRF-8)/interferon consensus sequence-binding protein (ICSBP) is a transcription factor that controls myeloid-cell development. Microarray gene expression analysis of Irf-8-/- myeloid progenitor cells expressing an IRF-8/estrogen receptor chimera (which differentiate into macrophages after addition of estradiol) was used to identify 69 genes altered by IRF-8 during early differentiation (62 up-regulated and 7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes (cystatin C, cathepsin C, lysozyme, and prosaposin) did not require de novo protein synthesis for induction, suggesting that they were direct targets of IRF-8. We developed a reporter assay system employing a self-inactivating retrovirus and analyzed the cystatin C and cathepsin C promoters. We found that a unique cis element mediates IRF-8-induced activation of both promoters. Similar elements were also found in other IRF-8 target genes with a consensus sequence (GAAANN[N]GGAA) comprising a core IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8 and the PU.1 Ets transcription factor bind to this element in vivo. Collectively, these data indicate that IRF-8 stimulates transcription of target genes through a novel cis element to specify macrophage differentiation.

Thrombin mediated migration of osteogenic cells.

Given that thrombin is ubiquitously expressed at sites of vascular injury, and that osteogenic cells express receptors for thrombin, we questioned whether thrombin could attract osteogenic cells to a bony wound. Using a scratch wound assay, thrombin stimulated a significant increase in migration of osteogenic cultures of primary marrow cells. This effect was dependent on thrombin proteolytic activity; however, thrombin was unable to stimulate the migration of a more differentiated marrow-derived osteogenic cell line. To better understand the role of thrombin in osteoprogenitor migration, we developed an osteoprogenitor migration assay that combines a modified Boyden chamber with a bone nodule assay. Primary cells that migrated through the transwell filter in the presence of thrombin formed significantly more bone nodules compared to the condition without thrombin. This was not due to proliferation or differentiation effects of thrombin. In contrast, thrombin was unable to stimulate an increase in the number of nodules for the more differentiated osteogenic cell line. Thus, our results suggest that thrombin exhibits differential motogenic effects on osteogenic cells depending on their differentiation state. The cell migration/bone nodule assay described here is the first assay that can be specifically used to examine the effects of factors on the migration of osteoprogenitor cells, particularly those derived from primary populations.

A global view of gene expression in the preimplantation mouse embryo: morula versus blastocyst.

As a first step to understand preimplantation development, we performed global gene-expression profiling of morula and blastocyst using the NIA 15k mouse cDNA microarray. Gene expression levels were measured four times for blastocyst and five times for morula. Student's t-test at the 5% significance level identified 428 genes upregulated and 748 downregulated in blastocyst compared to morula. This trend was consistent with semi-quantative RT-PCR analysis of sample genes. The upregulated genes known to be involved in critical regulatory processes, included Mist1, Id2, Hd1, and Requiem; the downregulated genes included CREB-binding protein, Per3, zinc finger protein 217, Krox-25, and miwi1. Such well-characterized genes and many novel genes provide markers for early stages in development and starting materials for further functional studies.