Establishing a 3D culture system for early organogenesis of monkey embryos ex vivo and single-cell transcriptome analysis of cultured embryos.

Creating in vitro culture platforms for monkey embryos is crucial for understanding the initial 4 weeks of early primate embryogenesis. Here, we present a protocol to culture cynomolgus monkey embryos in vitro for 25 days post-fertilization and to delineate the key developmental events of gastrulation and early organogenesis. We describe steps for culturing with a 3D system, immunofluorescence analysis, single-cell RNA sequencing, and bioinformatic analysis. For complete details on the use and execution of this protocol, please refer to Gong et al. (2023).1.

Ex utero embryogenesis of non-human primate embryos and beyond.

Understanding cellular and molecular processes underlying the human early post-implantation development represents one of the most fundamental questions in development and stem cell biology. As embryos implant into the uterus a week after fertilization, human development beyond the blastocyst stage is extremely difficult to study due to the inaccessibility of embryos and ethical concerns. The advents in the human embryo in vitro culture system provide an easily accessible, tractable, and perturbable platform to dissect key developmental events of human early embryonic development. However, these studies stopped around gastrulation to technical and ethical limitations, and our understanding of human gastrulation and early organogenesis remains poor. As closely related species to humans, non-human primates (NHPs) are suitable surrogate species to interrogate mechanisms underpinning human embryonic development. Here, we review the most recent advances in embryo in vitro culture systems of NHP and discuss their potential optimization strategies and applications.

Ex utero monkey embryogenesis from blastocyst to early organogenesis.

The third and fourth weeks of gestation in primates are marked by several developmental milestones, including gastrulation and the formation of organ primordia. However, our understanding of this period is limited due to restricted access to in vivo embryos. To address this gap, we developed an embedded 3D culture system that allows for the extended ex utero culture of cynomolgus monkey embryos for up to 25 days post-fertilization. Morphological, histological, and single-cell RNA-sequencing analyses demonstrate that ex utero cultured monkey embryos largely recapitulated key events of in vivo development. With this platform, we were able to delineate lineage trajectories and genetic programs involved in neural induction, lateral plate mesoderm differentiation, yolk sac hematopoiesis, primitive gut, and primordial germ-cell-like cell development in monkeys. Our embedded 3D culture system provides a robust and reproducible platform for growing monkey embryos from blastocysts to early organogenesis and studying primate embryogenesis ex utero.

Mapping developmental paths of monkey primordial germ-like cells differentiation from pluripotent stem cells by single cell ribonucleic acid sequencing analysis†.

The induction of primordial germ-like cells (PGCLCs) from pluripotent stem cells (PSCs) provides a powerful system to study the cellular and molecular mechanisms underlying germline specification, which are difficult to study in vivo. The studies reveal the existence of a species-specific mechanism underlying PGCLCs between humans and mice, highlighting the necessity to study regulatory networks in more species, especially in primates. Harnessing the power of single-cell RNA sequencing (scRNA-seq) analysis, the detailed trajectory of human PGCLCs specification in vitro has been achieved. However, the study of nonhuman primates is still needed. Here, we applied an embryoid body (EB) differentiation system to induce PGCLCs specification from cynomolgus monkey male and female PSCs, and then performed high throughput scRNA-seq analysis of approximately 40 000 PSCs and cells within EBs. We found that EBs provided a niche for PGCLCs differentiation by secreting growth factors critical for PGCLC specification, such as bone morphogenetic protein 2 (BMP2), BMP4, and Wnt Family Member 3. Moreover, the developmental trajectory of PGCLCs was reconstituted, and gene expression dynamics were revealed. Our study outlines the roadmap of PGCLC specification from PSCs and provides insights that will improve the differentiation efficiency of PGCLCs from PSCs.

Developmental dynamics of chromatin accessibility during post-implantation development of monkey embryos.

BACKGROUND: Early post-implantation development, especially gastrulation in primates, is accompanied by extensive drastic chromatin reorganization, which remains largely elusive. RESULTS: To delineate the global chromatin landscape and understand the molecular dynamics during this period, a single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) was applied to in vitro cultured cynomolgus monkey (Macaca fascicularis, hereafter referred to as monkey) embryos to investigate the chromatin status. First, we delineated the cis-regulatory interactions and identified the regulatory networks and critical transcription factors involved in the epiblast (EPI), hypoblast, and trophectoderm/trophoblast (TE) lineage specification. Second, we observed that the chromatin opening of some genome regions preceded the gene expression during EPI and trophoblast specification. Third, we identified the opposing roles of FGF and BMP signaling in pluripotency regulation during EPI specification. Finally, we revealed the similarity between EPI and TE in gene expression profiles and demonstrated that PATZ1 and NR2F2 were involved in EPI and trophoblast specification during monkey post-implantation development. CONCLUSIONS: Our findings provide a useful resource and insights into dissecting the transcriptional regulatory machinery during primate post-implantation development.

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

Long noncoding RNA HOTTIP cooperates with CCCTC-binding factor to coordinate HOXA gene expression.

The spatiotemporal control of HOX gene expression is dependent on positional identity and often correlated to their genomic location within each loci. Maintenance of HOX expression patterns is under complex transcriptional and epigenetic regulation, which is not well understood. Here we demonstrate that HOTTIP, a lincRNA transcribed from the 5' edge of the HOXA locus, physically associates with the CCCTC-binding factor (CTCF) that serves as an insulator by organizing HOXA cluster into disjoint domains, to cooperatively maintain the chromatin modifications of HOXA genes and thus coordinate the transcriptional activation of distal HOXA genes in human foreskin fibroblasts. Our results reveal the functional connection of HOTTIP and CTCF, and shed light on lincRNAs in gene activation and CTCF mediated chromatin organization.

Suppression of AcMNPV replication by adf and thymosin protein up-regulation in a new testis cell line, Ha-shl-t.

Host cytoskeletons facilitate the entry, replication, and egress of viruses because cytoskeletons are essential for viral survival. One mechanism of resisting viral infections involves regulating cytoskeletal polymerization/depolymerization. However, the molecular mechanisms of regulating these changes in cytoskeleton to suppress viral replication remain unclear. We established a cell line (named Ha-shl-t) from the pupal testis of Helicoverpa armigera (Lepidoptera: Noctuidae). The new testis cell line suppresses Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) replication via disassembly of cytoskeleton. Up-regulation of thymosin (actin disassembling factor) and adf (actin depolymerizing factor) reduces F-actin. Silencing thymosin or adf or treating cells with the F-actin stabilizer phalloidin led to increased AcMNPV replication, while treating cells with an F-actin assembly inhibitor cytochalasin B decreased viral replication. We infer that Ha-shl-t cells utilize F-actin depolymerization to suppress AcMNPV replication by up-regulating thymosin and adf. We propose Ha-shl-t as a model system for investigating cytoskeletal regulation in antiviral action and testicular biology generally.

Adenylate kinase 2 (AK2) promotes cell proliferation in insect development.

BACKGROUND: Adenylate kinase 2 (AK2) is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP) ↔ ATP(GTP) + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms. RESULTS: This study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown. CONCLUSION: These results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development.

Molecular cloning and expression analysis of signal transducer and activator of transcription (STAT) from the Chinese white shrimp Fenneropenaeus chinensis.

Innate immunity is the first line of defense by a host against invading pathogens. Several signaling pathways participate in the immune response, one of which is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Various evidences have been provided to suggest that the JAK/STAT pathway is involved in both antibacterial and antiviral immunities. In this study, the full-length cDNA and gene sequence of STAT (designated as FcSTAT) was cloned from the Chinese white shrimp, Fenneropenaeus chinensis. Phylogenetic analysis reveals that the FcSTAT is clustered with STAT5s and STAT6s from vertebrates and STATs from invertebrates. Quantitative real-time PCR exhibited that the FcSTAT had a wide distribution in all detected tissues and developmental stages. Time course analysis of the transcription level after WSSV challenge showed a noticeably early up-regulation of FcSTAT in hemocytes, hepatopancreas, and intestines. The expression levels of FcSTAT increased corresponding to Vibrio anguillarum stimulation in both hemocytes and hepatopancreas as well. All these imply that the JAK/STAT pathway participates in the immune response against bacteria and virus in F. chinensis.

Establishment of a new cell line from lepidopteran epidermis and hormonal regulation on the genes.

When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes.

Purification and characterisation of an inhibitor of a cathepsin B-like proteinase from sunflower seed.

Cathepsin B is a vitally important enzyme in various physiological processes and in tumor invasion and metastasis. A cathepsin B inhibitor, HCB-SunI, was identified and purified from sunflower seeds, Helianthus annuus, using ammonium sulfate precipitation and two steps of conventional chromatography. The molecular mass of HCB-SunI was estimated to be 12 kDa by SDS-PAGE and 12.32 kDa by MALDI TOF MS. Its N-terminal amino acid sequence was determined to be: PYGGGGTESG. HCB-SunI not only inhibited Helicoverpa cathepsin B (HCB) but also decreased the growth of HeLa and glioma cells by 7-27% and 6-22%, respectively, when the cells were grown in a final concentration of 0.002-0.008 microM inhibitor.

Cathepsin B-like proteinase is involved in the decomposition of the adult fat body of Helicoverpa armigera.

Cathepsin B-like proteinase (HCB, EC 3.4.22.1) is expressed in Helicoverpa armigera oocytes and adult fat bodies. Previous work has revealed that HCB plays a key role in the degradation of yolk proteins during embryogenesis. This study investigated the function and regulatory activation of HCB in adult fat bodies during aging and oogenesis. The HCB transcript was detected at all stages from larval to adult fat bodies with Northern blot analysis. Pro-HCB was also detected in fat bodies at these stages with an immunoblot assay using a monoclonal antibody against HCB. However, mature HCB and its activity were only detected in fat bodies of pre-adults and adults. This evidence suggested that HCB is regulated post-translationally by activation of the pro-enzyme during the pupa-adult metamorphosis. The activation of HCB was coupled with the expression of hormone receptor 3 (HHR3), and was up-regulated by the ecdysteroid agonist, RH-2485, suggesting that HCB activation is related to the ecdysone regulatory system. The decomposition of the adult fat bodies during aging and oogenesis was found to occur via programmed cell death, in which HCB took part.

Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis.

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal sequence (1-19) followed by a mature peptide (20-71). The sequence identity with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The mature peptide, with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp, and that Ch-penaeidin was constitutively expressed mainly in haemocytes.

Determination of glutathione in single human hepatocarcinoma cells by capillary electrophoresis with electrochemical detection.

A method for determination of glutathione (GSH) in single human hepatocarcinoma (HH) cells was described by capillary zone electrophoresis with electrochemical detection at a gold/mercury amalgam micro-disk electrode. When HH cells were washed with the running buffer instead of physiological buffer saline, only one electrophoretic peak for GSH is depicted on the electropherograms of single HH cells. When electroosmotic injection of 0.01 mol/l NaOH for lysing the cell introduced into the capillary, the lysis time can be shorten to 5 s. The whole cell injection and no need of derivatization reaction lead more accurate and precise results. The average amount of GSH in an individual HH cell is 22.3+/-5.8 fmol (mean+/-standard deviation), which is consistent with that of its homogenate.