Inhibition of MEK1/2 and GSK3 (2i system) affects blastocyst quality and early differentiation of porcine parthenotes

Inhibition of both MEK1/2 and glycogen synthase kinase-3 (GSK3; 2i system) facilitates the maintenance of naïve stemness for embryonic stem cells in various mammalian species. However, the effect of the inhibition of the 2i system on porcine early embryogenesis is unknown. We investigated the effect of the 2i system on early embryo development, expression of pluripotency-related genes, and epigenetic modifications. Inhibition of MEK1/2 (by PD0325901) and/or GSK3 (by CHIR99021) did not alter the developmental potential of porcine parthenogenetic embryos, but improved blastocyst quality, as judged by the blastocyst cell number, diameter, and reduction in the number of apoptotic cells. The expression levels of octamer-binding transcription factor 4 and SOX2, the primary transcription factors that maintain embryonic pluripotency, were significantly increased by 2i treatments. Epigenetic modification-related gene expression was altered upon 2i treatment. The collective results indicate that the 2i system in porcine embryos improved embryo developmental potential and blastocyst quality by regulating epigenetic modifications and pluripotency-related gene expression

Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based Parkinson\u27s disease modeling

Recent studies have demonstrated the generation of midbrain-like organoids (MOs) from human pluripotent stem cells. However, the low efficiency of MO generation and the relatively immature and heterogeneous structures of the MOs hinder the translation of these organoids from the bench to the clinic. Here we describe the robust generation of MOs with homogeneous distribution of midbrain dopaminergic (mDA) neurons. Our MOs contain not only mDA neurons but also other neuronal subtypes as well as functional glial cells including astrocytes and oligodendrocytes. Furthermore, our MOs exhibit mDA neuron-specific cell death upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, indicating that MOs could be a proper human model system for studying the in vivo pathology of Parkinson\u27s disease (PD). Our optimized conditions for producing homogeneous and mature MOs might provide an advanced patient-specific platform for in vitro disease modeling as well as for drug screening for PD

ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development

In vitro modeling of urological diseases by three-dimensional printed miniature bladder

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CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

<div><p>The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine <i>OCT4</i> function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against <i>OCT4</i> decreased the percentages of <i>OCT4</i>-positive embryos to 37–50% of total embryos, while ~100% of control embryos exhibited clear <i>OCT4</i> immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. <i>OCT4</i> mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including <i>NANOG</i>, and <i>CDX2</i> were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (<i>eGFP</i>) vector targeting the <i>OCT4</i> exon with Cas9 and sgRNA to create a knockin. We confirmed <i>eGFP</i> fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had <i>eGFP</i> sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig.</p></div

CRISPR/Cas9 mediated <i>OCT4</i> targeting in porcine embryos.

<p>(A) Design of sgRNAs for targeting exon 2 or exon 5. Guide sequences correspond to sgRNAs are marked as underlined and the protospacer adjacent motif(PAM) sites for each guide sequences are marked. (B) Sequencing of PCR amplification product confirmed the introduction of indel in exon 2 or exon 5. Locations of guide sequences are marked as blue and bases deleted in Cas9/sgRNAs injected embryos are marked in red box. (C-D) Various deletion/insertions induced by Cas9/sgRNA injections in exon 2 (C) or exon 5(D). Positions of PAM sites are marked as underlined and deleted base was marked as red. Note that insertion can be induced in some case(marked in blue). (E-F) Detections of <i>OCT4</i> and <i>CDX2</i> protein using immunostaining in targeted porcine embryos. Embryos were injected with 5–10pl of 100ng/μl of Cas9 mRNA mixed with 0, 10, 100ng/μl of sgRNA1 or sgRNA2, respectively. Location of nucleus was stained with Hoechst 33342 (blue). <i>OCT4</i>(left panel) and CDX2(right panel) are presented as green. (G) mRNA expression levels of <i>OCT4</i>, <i>CDX2</i>, and <i>NANOG</i> measured by qRT-PCR. Expression levels are presented as relative expression levels to those in control embryo at 1 cell stages. Control: Cas9 mRNA injection only; CRISPR: Cas9 mRNA and sgRNA 2 injected. In each developmental stages, 20 embryos were collected for RNA extraction.</p

Generation of in-frame fusion of <i>eGFP</i> locus in Porcine <i>OCT4</i> using CRISPR/Cas9 mediated homology dependent repair (HDR).

<p>(A) Scheme for the HDR-mediated integration of <i>eGFP</i> into <i>OCT4</i> locus. Donor vector for HDR was consisted with the left homology arm (1kb) spanning exon 3—exon 5, <i>eGFP</i> fused after exon 5 as in-frame fusion and the right homology arm (1kb). Guide sequence for sgRNA was designed near stop codon of <i>OCT4</i> located at exon 5. Location of PAM are underlined and stop codon of <i>OCT4</i> is marked. Note that <i>eGFP</i> was inserted between stop codon and last codon of <i>OCT4</i> and PAM of sgRNA was located just after stop codon. Therefore donor vector cannot recognize and digested with Cas9/sgRNA. (B) Confirmation of insertion of <i>eGFP</i> locus in genomic DNA of porcine embryo. PCR amplification spanning exon 5 confirmed the presence of in-frame fusion of <i>eGFP</i> at the end of <i>OCT4</i> coding region. (C) Expression of <i>eGFP</i> fused <i>Oct4</i> in HDR mediated <i>eGFP</i> knockin porcine embryos. <i>eGFP</i> expression was detected byConfocal microscopy. Control (Cas9 injected) and Targeted (Cas9/sgRNA/Donor Plasmid) have been compared. Note that localization of <i>eGFP</i> signal in nucleus.</p

Developmental competence and <i>eGFP</i> knockin efficiency in Porcine <i>OCT4</i> locus.

<p>Different combination of Cas9, sgRNA and donor plasmid were injected as shown. Cleavage and blastocyst formation rates of each groups are presented. <i>OCT4</i> targeting efficiency was measured by PCR amplification and sequencing of single blastocysts. Statistical significance was tested using chi-square test.</p><p>Developmental competence and <i>eGFP</i> knockin efficiency in Porcine <i>OCT4</i> locus.</p

Targeted deletion of region between exon 2 and exon 5 of porcine <i>OCT4</i> locus using sgRNA pairs.

<p>(A) Location of sgRNA pair in <i>OCT4</i> locus and scheme for resulting deletion (B) Sequencing of PCR amplification product confimed the deletion of 1.8kb region between exon 2 and exon 5 of <i>OCT4</i> locus. Locations of guide sequences in exon 2 and exon 5 are marked as blue(exon 2) or red (exon 5) respectively, and resulting exon 2—exon 5 fusions are shown. (C, D) Deletion of <i>OCT4</i> and <i>CDX2</i> protein using immunostaining in targeted porcine embryos. Embryos were injected with 5–10pl of 100ng/μl of Cas9 mRNA mixed with 0, 10, 100ng/μl of sgRNA 1 and sgRNA 2, respectively. Location of nucleus was stained with Hoechst 33342 (blue). <i>OCT4</i>(C) and <i>CDX2</i> (D) are presented as green.</p