Dynamic conversion of cell sorting patterns in aggregates of embryonic stem cells with differential adhesive affinity.

BACKGROUND: Mammalian early development comprises the proliferation, differentiation, and self-assembly of the embryonic cells. The classic experiment undertaken by Townes and Holtfreter demonstrated the ability of dissociated embryonic cells to sort and self-organize spontaneously into the original tissue patterns. Here, we further explored the principles and mechanisms underlying the phenomenon of spontaneous tissue organization by studying aggregation and sorting of mouse embryonic stem (ES) cells with differential adhesive affinity in culture. RESULTS: As observed previously, in aggregates of wild-type and E-cadherin-deficient ES cells, the cell assemblies exhibited an initial sorting pattern showing wild-type cells engulfed by less adhesive E-cadherin-deficient ES cells, which fits the pattern predicted by the differential adhesive hypothesis proposed by Malcom Steinberg. However, in further study of more mature cell aggregates, the initial sorting pattern reversed, with the highly adhesive wild-type ES cells forming an outer shell enveloping the less adhesive E-cadherin-deficient cells, contradicting Steinberg's sorting principle. The outer wild-type cells of the more mature aggregates did not differentiate into endoderm, which is known to be able to sort to the exterior from previous studies. In contrast to the naive aggregates, the mature aggregates presented polarized, highly adhesive cells at the outer layer. The surface polarity was observed as an actin cap contiguously spanning across the apical surface of multiple adjacent cells, though independent of the formation of tight junctions. CONCLUSIONS: Our experimental findings suggest that the force of differential adhesive affinity can be overcome by even subtle polarity generated from strong bilateral ligation of highly adhesive cells in determining cell sorting patterns.

Establishment of porcine nuclear transfer-derived embryonic stem cells using induced pluripotent stem cells as donor nuclei.

We investigated whether sequential reprogramming via porcine induced pluripotent stem cells (piPSCs) or exposure to oocyte cytoplasm following nuclear transfer could generate nuclear transfer-derived ESCs (piPSCs-ntESCs). Nuclear transfer embryos were reconstructed with piPSCs possessing a ZsGreen fluorescent marker for expression of exogenous Nanog and Lin28. Reconstructed oocytes developed to morphologically normal 8-cell/morulae (35/93, 37.6%) and blastocysts (12/93, 12.9%). Although most green fluorescent protein-positive blastocysts showed efficient outgrowth (8/10, 80%), none formed primary colonies and all cultures degenerated. Conversely, 15% of fluorescent positive 8-cell/morula stage embryos showed outgrowth (6/40), with three forming primary colonies (7.5%). All three were expanded and maintained as piPSC-ntESC lines. These cell lines expressed stem cell marker genes and proteins. Despite inactivation of one X chromosome, all piPSC-ntESC lines formed teratomas comprising derivatives from all three embryonic germ layers. Strong SSEA1, 3, and 4 expression was detected at the 8-cell/morula stage in embryos reconstructed from both piPSCs and porcine embryonic fibroblasts (PEFs). SSEA3 was notably absent from IVF controls at pre-implantation embryo stages. Finally, we attempted to establish ntESCs from 8-cell/morulae reconstructed with PEFs using the same culture conditions as those for piPSC-ntESC derivation. Although eight primary colonies arose from 107 embryos (7.5%), they all degenerated after the first passage culture. Early and sustained expression of key reprogramming regulatory factors may be critical for pluripotent stem cell derivation to derive piPSC-ntESCs from 8-cell/morula stages, while the expression of SSEAs may be involved in the initial stem cell colony formation phases.

The frequency and consequences of multipolar mitoses in undifferentiated embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability.

SC1 Promotes MiR124-3p Expression to Maintain the Self-Renewal of Mouse Embryonic Stem Cells by Inhibiting the MEK/ERK Pathway.

BACKGROUND/AIMS: Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. METHODS: In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. RESULTS: SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. CONCLUSION: SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1.

Revisiting the role of GCNF in embryonic development.

GCNF (NR6A1) is essential for embryonic development. GCNF belongs to the nuclear receptor (NR) gene family, it is distantly related to other NRs and is the only member of subfamily 6. As the ligand for GCNF has not been identified, GCNF is designated an orphan nuclear receptor. GCNF has been found to be a transcriptional repressor, through specific binding to DR0 response elements, which is found in the Oct4 proximal promoter for example. GCNF is expressed widely in early mouse embryos, and later in the developing nervous system. GCNF knockout mouse embryos die around E10.5. GCNF is required for the restriction of Oct4 expression to primordial germ cells after gastrulation. GCNF is expressed in ES/EC cells and during their differentiation, and has been reported to be required for pluripotency gene repression during retinoic acid (RA)-induced mES cell differentiation. GCNF can interact with DNA methylation proteins, and is suggested to recruit DNA methylation complexes to repress and silence Oct4 expression. Nuclear receptor regulation in embryonic development is a complex process, as different nuclear receptors have overlapping and distinct functions. In-depth exploration of GCNF function and mechanism of action will help to comprehensively understand the nuclear receptor regulation in embryonic development.

EphB4 forward-signaling regulates cardiac progenitor development in mouse ES cells.

Eph receptor (Eph)-ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4-ephrinB2 regulates cardiovascular development. To assess the role of EphB4-ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5(+) cardiac progenitor cells and in α-MHC(+) cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5-GFP(+) cardiac progenitor cells, but not in α-MHC-GFP(+) cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL-RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4-ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full-length EphB4 or intracellular domain-truncated EphB4 in EphB4-null ES cells was established to investigate the role of EphB4-forward signaling in ES cells. Interestingly, while full-length EphB4 was able to restore the cardiac lineage development in EphB4-null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor-kinase-dependent EphB4-forward signaling plays a crucial role in the development of cardiac progenitor cells.

Efficient gene-targeting in rat embryonic stem cells by CRISPR/Cas and generation of human kynurenine aminotransferase II (KAT II) knock-in rat.

The relative proportion of kynurenine aminotransferase (KAT) I-IV activities in the brain is similar between humans and rats. Moreover, KAT II is considered to be the main enzyme for kynurenic acid production in the brain. Taken together, human KAT II knock-in (hKAT II KI) rats will become a valuable tool for the evaluation of KAT II targeted drugs as a human mimetic model. Although we initially tried the approach by conventional gene-targeting via embryonic stem cells (ESCs) to generate them, we had to give up the production because of no recombinant ESCs. Accordingly, we developed a method to improve the efficiency of homologous recombination (HR) in ESCs by the combination with the CRISPR/Cas system. Co-electroporation of Cas9 plasmid, single guide RNA plasmid and hKAT II KI vector increased the number of drug-resistant colonies and greatly enhanced the HR efficiency from 0 to 36 %. All the clones which we obtained showed the same sequence as designed. These recombinant clones resulted in chimeras that transmitted the hKAT II KI allele to their offspring. hKAT II KI rats showed no reduction of KATs mRNA expression and the amount of kynurenic acid was similar between the hKAT II KI rats and the wild type in their brains. These results indicate that the methodology presented in this report can overcome the problem encountered in conventional gene-targeting that prevented production of humanized rats.

Inter-subspecies mouse F1 hybrid embryonic stem cell lines newly established for studies of allelic imbalance in gene expression.

Allele-specific monoallelic gene expression is a unique phenomenon and a great resource for analyzing gene regulation. To study this phenomenon, we established new embryonic stem (ES) cell lines derived from F1 hybrid blastocysts from crosses between four mouse subspecies (Mus musculus domesticus, C57BL/6; M. musculus molossinus, MSM/Ms; M. musculus musculus, PWK; M. musculus castaneus, HMI/Ms) and analyzed the expression levels of undifferentiated pluripotent stem cell markers and karyotypes of each line. To demonstrate the utility of our cell lines, we analyzed the allele-specific expression pattern of the Inpp5d gene as an example. The allelic expression depended on the parental alleles; this dependence could be a consequence of differences in compatibility between cis- and trans-elements of the Inpp5d gene from different subspecies. The use of parental mice from four subspecies greatly enhanced genetic polymorphism. The F1 hybrid ES cells retained this polymorphism not only in the Inpp5d gene, but also at a genome-wide level. As we demonstrated for the Inpp5d gene, the established cell lines can contribute to the analysis of allelic expression imbalance based on the incompatibility between cis- and trans-elements and of phenotypes related to this incompatibility.

RBM47 is a Critical Regulator of Mouse Embryonic Stem Cell Differentiation.

RNA-binding proteins (RBPs) are pivotal for regulating gene expression as they are involved in each step of RNA metabolism. Several RBPs are essential for viable growth and development in mammals. RNA-binding motif 47 (RBM47) is an RRM-containing RBP whose role in mammalian embryonic development is poorly understood yet deemed to be essential since its loss in mouse embryos leads to perinatal lethality. In this study, we attempted to elucidate the significance of RBM47 in cell-fate decisions of mouse embryonic stem cells (mESCs). Downregulation of Rbm47 did not affect mESC maintenance and the cell cycle but perturbed the expression of primitive endoderm (PrE) markers and increased GATA4 + PrE-like cells. However, the PrE misregulation could be reversed by either overexpressing Rbm47 or treating the knockdown mESCs with the inhibitors of FGFR or MEK, suggesting an implication of RBM47 in regulating FGF-ERK signaling. Rbm47 knockdown affected the multi-lineage differentiation potential of mESCs as it regressed teratoma in NSG mice and led to a skewed expression of differentiation markers in serum-induced monolayer differentiation. Further, lineage-specific differentiation revealed that Rbm47 is essential for proper differentiation of mESCs towards neuroectodermal and endodermal fate. Taken together, we assign a hitherto unknown role(s) to RBM47 in a subtle regulation of mESC differentiation.

Time-Lapse Bioluminescence Imaging of Hes7 Expression In Vitro and Ex Vivo.

Somites are formed sequentially by the segmentation of the anterior parts of the presomitic mesoderm (PSM), and such periodical somite formation is crucial to ensure the proper vertebrae. In the mouse embryo, Hes7, a segmentation clock gene, controls this periodic event with new somites forming every 2 h. Hes7 oscillations are synchronized between neighboring PSM cells and propagate from the posterior to the anterior PSM in the form of traveling waves. However, the exact mechanisms that generate these oscillatory dynamics and control synchronization are still unclear. Given that the half-life of Hes7 is too short to be monitored with most fluorescent proteins, time-lapse bioluminescence imaging (BLI) is a suitable tool to monitor the chronological Hes7 expression dynamics. In this chapter, we introduce a ubiquitinated luciferase reporter which enables the visualization of Hes7 expression dynamics with high temporal and spatial resolution in living cells and tissues.

Targeted Mutations in the Mouse via Embryonic Stem Cells.

Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.

Principles of Genetic Engineering.

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

Design and Analysis of CCN Gene Activity Using CCN Knockout Mice Containing LacZ Reporters.

Two developments have greatly facilitated the construction of CCN mutant mouse strains. The first is the availability of modified embryonic stem (ES) cells and mice developed through several large-scale government-sponsored research programs. The second is the advent of CRISPR/Cas9 technology. In this chapter, we describe the available mouse strains generated by gene targeting techniques and the CCN targeting vectors and genetically modified ES cells that are available for the generation of CCN mutant mice. Many of these mutant mouse lines and ES cells carry a ß-galactosidase reporter that can be used to track CCN expression, facilitating phenotypic analysis and revealing new sites of CCN action. Therefore, we also describe a method for ß-galactosidase staining.

Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7.

Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes.

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.