Single-cell RNA sequencing reveals regulation of fetal ovary development in the monkey (Macaca fascicularis).

Germ cells are vital for reproduction and heredity. However, the mechanisms underlying female germ cell development in primates, especially in late embryonic stages, remain elusive. Here, we performed single-cell RNA sequencing of 12,471 cells from whole fetal ovaries, and explored the communications between germ cells and niche cells. We depicted the two waves of oogenesis at single-cell resolution and demonstrated that progenitor theca cells exhibit similar characteristics to Leydig cells in fetal monkey ovaries. Notably, we found that ZGLP1 displays differentially expressed patterns between mouse and monkey, which is not overlapped with NANOG in monkey germ cells, suggesting its role in meiosis entry but not in activating oogenic program in primates. Furthermore, the majority of germ cell clusters that sharply express PRDM9 and SPO11 might undergo apoptosis after cyst breakdown, leading to germ cell attrition. Overall, our work provides new insights into the molecular and cellular basis of primate fetal ovary development at single-cell resolution.

Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species.

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Heterogeneous nuclear ribonucleoprotein A3 controls mitotic progression of neural progenitors via interaction with cohesin.

Cortex development is controlled by temporal patterning of neural progenitor (NP) competence with sequential generation of deep and superficial layer neurons, but underlying mechanisms remain elusive. Here, we report a role for heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) in regulating the division of early cortical NPs that mainly give rise to deep-layer neurons via direct neurogenesis. HNRNPA3 is expressed at high levels in NPs of mouse and human cortex at early stages, with a unique peri-chromosome pattern. Intriguingly, downregulation of HNRNPA3 caused chromosome disarrangement, which hindered normal separation of chromosomes during NP division, leading to mitotic delay. Furthermore, HNRNPA3 is associated with the cohesin-core subunit SMC1A and controls its association with chromosomes, implicating a mechanism for the role of HNRNPA3 in regulating chromosome segregation in dividing NPs. Hnrnpa3-deficient mice exhibited reduced cortical thickness, especially of deep layers. Moreover, downregulation of HNRNPA3 in cultured human cerebral organoids led to marked reduction in NPs and deep-layer neurons. Thus, this study has identified a crucial role for HNRNPA3 in NP division and highlighted the relationship between mitosis progression and early neurogenesis.

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs.

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2.

Methyl-CpG binding protein 2 (MeCP2) has crucial roles in transcriptional regulation and microRNA processing. Mutations in the MECP2 gene are found in 90% of patients with Rett syndrome, a severe developmental disorder with autistic phenotypes. Duplications of MECP2-containing genomic segments cause the MECP2 duplication syndrome, which shares core symptoms with autism spectrum disorders. Although Mecp2-null mice recapitulate most developmental and behavioural defects seen in patients with Rett syndrome, it has been difficult to identify autism-like behaviours in the mouse model of MeCP2 overexpression. Here we report that lentivirus-based transgenic cynomolgus monkeys (Macaca fascicularis) expressing human MeCP2 in the brain exhibit autism-like behaviours and show germline transmission of the transgene. Expression of the MECP2 transgene was confirmed by western blotting and immunostaining of brain tissues of transgenic monkeys. Genomic integration sites of the transgenes were characterized by a deep-sequencing-based method. As compared to wild-type monkeys, MECP2 transgenic monkeys exhibited a higher frequency of repetitive circular locomotion and increased stress responses, as measured by the threat-related anxiety and defensive test. The transgenic monkeys showed less interaction with wild-type monkeys within the same group, and also a reduced interaction time when paired with other transgenic monkeys in social interaction tests. The cognitive functions of the transgenic monkeys were largely normal in the Wisconsin general test apparatus, although some showed signs of stereotypic cognitive behaviours. Notably, we succeeded in generating five F1 offspring of MECP2 transgenic monkeys by intracytoplasmic sperm injection with sperm from one F0 transgenic monkey, showing germline transmission and Mendelian segregation of several MECP2 transgenes in the F1 progeny. Moreover, F1 transgenic monkeys also showed reduced social interactions when tested in pairs, as compared to wild-type monkeys of similar age. Together, these results indicate the feasibility and reliability of using genetically engineered non-human primates to study brain disorders.

Leukemia propagating cells rebuild an evolving niche in response to therapy.

Residence of cancer-propagating cells (CPCs) within preferential microenvironmental niches has a major part in evading therapy. However, the nature of niches involved and the mechanisms protecting CPCs remain largely unknown. We addressed these issues in mouse transplantation models of acute lymphoblastic leukemia (ALL). When the engrafted leukemic cells substantially damaged adjacent microenvironment in the bone marrow (BM), after chemotherapy small foci of CPCs were retained, surrounded by sheaths of supporting cells that comprise a protective niche. We investigated patients' BM biopsies and found evidence of a similar process in patients receiving induction therapy. The efficacy of chemotherapy was enhanced by interfering with the niche formation or function. We therefore identified a therapy-induced niche that protects CPCs.

Generation of a monkey with MECP2 mutations by TALEN-based gene targeting.

Gene editing in model organisms has provided critical insights into brain development and diseases. Here, we report the generation of a cynomolgus monkey (Macaca fascicularis) carrying MECP2 mutations using transcription activator-like effector nucleases (TALENs)-mediated gene targeting. After injecting TALENs mRNA into monkey zygotes achieved by in vitro fertilization and embryo transplantation into surrogate monkeys, we obtained one male newborn monkey with an MECP2 deletion caused by frameshifting mutation in various tissues. The monkey carrying the MECP2 mutation failed to survive after birth, due to either the toxicity of TALENs or the critical requirement of MECP2 for neural development. The level of MeCP2 protein was essentially depleted in the monkey's brain. This study demonstrates the feasibility of introducing genetic mutations in non-human primates by site-specific gene-editing methods.

A study of the immune properties of human umbilical cord lining epithelial cells.

BACKGROUND AIMS: Our previous study has demonstrated the stem cell-like properties of human umbilical cord lining epithelial cells (CLECs) and their capability for epidermal reconstitution in organotypic skin culture; however, the immunogenicity of these cells has not been clearly defined. We assessed several aspects of the immune properties of CLECs in vitro. METHODS: We examined CLECs for their immunoregulatory function in a mixed lymphocyte culture experiment. We characterized the expression patterns of the major histocompatibility complex (MHC), co-stimulatory molecules and the pro-/anti-inflammatory cytokines and growth factors in CLECs by means of reverse transcription-polymerase chain reaction, Western blotting, flow cytometry and FlowCytomix multiple analyte detection assays. RESULTS: CLECs were found not to induce but to suppress the proliferation response of the peripheral blood mononuclear cells in a mixed lymphocyte culture assay. They did not express the MHC class II antigen HLA-DR but the non-classic MHC class I antigens HLA-G and HLA-E and lacked the expression of the co-stimulatory molecules CD40, CD80 and CD86. In addition, they produced less interleukin-1ß and transforming growth factor-ß1 but more interleukin-4 and hepatocyte growth factor than did adult keratinocytes, a pattern in favor of wound healing with less inflammation response. CONCLUSIONS: Our data suggest that CLECs have an immunosuppressive function in addition to their low immunogenicity. This could be at least partially explained by their expression of HLA-G and HLA-E molecules associated with immune tolerance and absence of HLA-DR and co-stimulatory molecules. The demonstration that CLECs produce a favorable pattern of cytokines and growth factors for wound healing provides further support for their potential clinical application in allogeneic cell therapy.

Stem cell-like properties of human umbilical cord lining epithelial cells and the potential for epidermal reconstitution.

BACKGROUND AIMS: Stem cells are particularly attractive for many cell-based therapeutic interventions because of their ability to self-renew and their capacity to differentiate into site-specific differentiating cells. Restoration of the integrity of epithelial continuity is an essential aspect of wound repair and tissue regeneration. We are currently looking at the potential of human umbilical cord lining cells as a source of epithelial stem cells with appropriate differentiation capacity for potential epidermal reconstitution. METHODS: We isolated human umbilical cord lining epithelial cells (CLEC) and characterized their phenotype from the perspective of proliferative potential, telomere length, expression of epidermal differentiation markers, as well as stem cell-specific markers, and clonogenicity. Their potential for epidermal reconstitution was investigated in an organotypic culture model. RESULTS: The results demonstrated that CLEC present a long telomere length and have a relatively high proliferative potential and passaging ability in culture. CLEC display some of the stem cell-specific markers for epithelial as well as pluripotent stem cells, including CK19, p63, OCT-4, SSEA-4, TRA-1-60, SOX2 and Nanog. CLEC are capable of generating a fully stratified epithelium in organotypic culture. CONCLUSIONS: The potential of CLEC to be used in clinical applications for specialized epithelial reconstruction is still unexplored. The demonstration that CLEC have stem cell-like properties and are capable of generating fully stratified epithelium provides support for their potential clinical application in epidermal reconstitution.

RNA Interference inhibits hepatitis B virus of different genotypes in vitro and in vivo.

BACKGROUND: Hepatitis B virus (HBV) infection increases the risk of liver disease and hepatocellular carcinoma. Small interfering RNA (siRNA) can be a potential new tool for HBV therapy. Given the high heterogeneity of HBV strains and the sensitivity towards sequences changes of siRNA, finding a potent siRNA inhibitor against the conservative site on the HBV genome is essential to ensure a therapeutic application. RESULTS: Forty short hairpin RNA (shRNA) expression plasmids were constructed to target conserved regions among nine HBV genotypes. HBV 1.3-fold genome plasmids carrying various genotypes were co-transfected with shRNA plasmids into either Huh7 cells or mice. The levels of various viral markers were examined to assess the anti-HBV efficacy of siRNA. Four (B245, B376, B1581 and B1789) were found with the ability to potently inhibit HBV RNA, DNA, surface antigen (HBsAg), e antigen (HBeAg) and core antigen (HBcAg) expression in HBV genotypes A, B, C, D and I (a newly identified genotype) in Huh7 cells and in mice. No unusual cytotoxicity or off-target effects were noted. CONCLUSIONS: Such siRNA suggests an alternate way of inhibiting various HBV genotypes in vitro and in vivo, promising advances in the treatment of HBV.

[Construction of highly effective artifical miRNA targeted to HIV-1 vif and the lentiviral-mediated antiviral research in vitro].

Effective and specific RNA interference (RNAi) elements are essential for the RNAi-based anti-HIV-1 research which has achieved extensive application. vif37 targeted to HIV-1 vi f is a highly effective and conserved RNAi target obtained from the previous study on screening. In this study, we explored the construction of artificial miRNAs to induce RNAi targeted to vif37, which had advantages on inhibition efficiency and flexibility of promoter selection. Three artificial miRNA targeted to vif37 were constructed by walking method using native miR-155 as a backbone and expressed by RNA polymerase II promoter. Then, expression vectors of artificial miRNA were co-transfected with HIV-1 infectious clone pNL4-3 to score its inhibition ability and showed that only miR-vif37 had the significant inhibition efficiency similar to shRNA-vif37. Subsequently, co-transfections with luciferase reporter plasmids into which different target sequences were inserted proved the specificity of miR-vif37 H. The replication of HIV-1 was inhibited in MT-4-miR37 H cells which could express miR-vif37 H stably and were cloned from MT-4 cells transducted with recombinant lentiviral vectors containing the miR-vif37 H expression element. Real-time RT-PCR revealed that miR-vif37 H had much lower expression level than shRNA-vif37. Results also showed that intracellular miR-181 and miR-16 expression levels and stat1 mRNA levels were not effected by the expression of miR-vif37 H in MT-4-miR37 H cells. We conclude miR-vif37 is a specific and highly effective artificial miRNA which will promote the further application of vif37 target.

[Screening of highly effective siRNA sequence targeting to HIV-1 vif and the lentiviral-mediated antiviral research in vitro].

Discovery of the RNA interference (RNAi) pathway has led to exciting new strategies for developing HIV treatment. This study was to find out the highly effective and conserved siRNA target sequences for improving RNAi-based therapy against the HIV-1. We constructed 30 shRNA expression plasmids for expressing different siRNAs targeted to HIV-1 vif and co-transfected them with the pNL4-3 to score for its ability to inhibit the expression of p24 protein of HIV-1. Then, the highly effective siRNAs targeting sequences were selected to align with 625 HIV-1 sequences in database including all HIV-1 subtypes to ana lyze their conserved character. In addition, vif37 the highly effective and most conserved target sequence was confirmed of its sequence-specific inhibition by independent reporter assays. MT-4 cell transduced with lentiviral shRNA-vif37 vector could inhibit HIV-1(NL4.3) replication in vitro. Moreover, MT-4-vif37 cloned from transduced MT-4 cell could stably express shRNA-vif37 and inhibit virus replication more efficiently when challenged with high titer virus. These results showed that RNAi has great potential as an antiviral gene therapy approach and supports the efforts to develop treatment for HIV-1-infected individuals.