[Show abstract][Hide abstract] ABSTRACT: To develop a whole-genome methylation sequencing method that fulfills the needs for studies using ultra-low-input DNA.
The tagmentation-based whole-genome bisulfite sequencing (T-WGBS) technology is modified, enabling stable library construction with complexity from minimally 0.5 ng of initial genomic DNA, which equals less than 100 mammalian cells.
We thoroughly assessed the performance of this T-WGBS method by sequencing the methylomes of a rice strain and pre-implantation embryos of rhesus monkey and compare to traditional WGBS approach, thereby demonstrating the efficacy of this new approach.
This new approach is highly attractive for the complete methylome analysis of very few cells, for example, mammalian pre-implantation embryos, or tiny human biopsy specimens.
[Show abstract][Hide abstract] ABSTRACT: Spermatogonial stem cells (SSCs) play fundamental roles in spermatogenesis. Although a handful of genes have been discovered as key regulators of SSC self-renewal and differentiation, the regulatory network responsible for SSC function remains unclear. In particular, small RNA signatures during mouse spermatogenesis are not yet systematically investigated. Here, using next generation sequencing, we compared small RNA signatures of in vitro expanded SSCs, testis-derived somatic cells (Sertoli cells), developing germ cells, mouse embryonic stem cells (ESCs), and mouse mesenchymal stem cells among mouse embryonic stem cells (ESCs) to address small RNA transition during mouse spermatogenesis. The results manifest that small RNA transition during mouse spermatogenesis displays overall declined expression profiles of miRNAs and endo-siRNAs, in parallel with elevated expression profiles of piRNAs, resulting in the normal biogenesis of sperms. Meanwhile, several novel miRNAs were preferentially expressed in mouse SSCs, and further investigation of their functional annotation will allow insights into the mechanisms involved in the regulation of SSC activities. We also demonstrated the similarity of miRNA signatures between SSCs and ESCs, thereby providing a new clue to understanding the molecular basis underlying the easy conversion of SSCs to ESCs.
BioMed Research International 07/2014; 2014. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent advances in gene editing technology have introduced the potential for application of mutagenesis approaches in nonhuman primates to model human development and disease. Here we report successful TALEN-mediated mutagenesis of an X-linked, Rett syndrome (RTT) gene, methyl-CpG binding protein 2 (MECP2), in both rhesus and cynomolgus monkeys. Microinjection of MECP2-targeting TALEN plasmids into rhesus and cynomolgus zygotes leads to effective gene editing of MECP2 with no detected off-target mutagenesis. Male rhesus (2) and cynomolgous (1) fetuses carrying MECP2 mutations in various tissues including testes were miscarried during midgestation, consistent with RTT-linked male embryonic lethality in humans. One live delivery of a female cynomolgus monkey occurred after 162 days of gestation, with abundant MECP2 mutations in peripheral tissues. We conclude that TALEN-mediated mutagenesis can be an effective tool for genetic modeling of human disease in nonhuman primates.
[Show abstract][Hide abstract] ABSTRACT: Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.
[Show abstract][Hide abstract] ABSTRACT: Serum microRNAs (miRNAs) have recently emerged as promising biomarkers for a variety of diseases including cancer and metabolic disorders. However, despite the increasing category of small RNAs, the existence and diagnostic value of other serum small RNAs are surprisingly few. In present study, by using RNA-Seq and quantitative PCR, we discovered an ancient class of tRNA-derived small RNAs (tsRNAs) abundantly, conservatively existed in the serum of a wide range of vertebrate species (from fish to human). tsRNAs are stably existed in the serum, free from serum microvesicles (exosomes). The stabilization mechanism of serum tsRNAs in an RNase-rich blood environment involves at least two layers of protection: through binding and co-existed with serum protein complexes, as well as by nucleotide modifications inheriting from their tRNA predecessors. Most importantly, serum tsRNAs showed surge upregulation during LPS-induced acute inflammation in mouse and monkey, as well as in human patients under virus infection (HBV replication phase), suggesting their active involvements in infection-induced defensive response. Overall, our data unveiled another hidden layer of serum small RNAs linking with disease condition, opening future avenues for the development of novel biomarker approaches based on analyzing serum tsRNAs.
Journal of Molecular Cell Biology 12/2013; · 8.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian mesenchymal stem cells (MSCs) have been shown to be strongly immunosuppressive in both animal disease models and human clinical trials. We have reported that the key molecule mediating immunosuppression by MSCs is species dependent: indoleamine 2,3-dioxygenase (IDO) in human and inducible nitric oxide synthase (iNOS) in mouse. In the present study, we isolated MSCs from several mammalian species, each of a different genus, and investigated the involvement of IDO and iNOS during MSC-mediated immunosuppression. The characterization of MSCs from different species was by adherence to tissue culture plastic, morphology, specific marker expression, and differentiation potential. On the basis of the inducibility of IDO and iNOS by inflammatory cytokines in MSCs, the tested mammalian species fall into two distinct groups: IDO utilizers and iNOS utilizers. MSCs from monkey, pig, and human employ IDO to suppress immune responses, whereas MSCs from mouse, rat, rabbit, and hamster utilize iNOS. Interestingly, based on the limited number of species tested, the iNOS-utilizing species all belong to the phylogenetic clade, Glires. Although the evolutionary significance of this divergence is not known, we believe that this study provides critical guidance for choosing appropriate animal models for preclinical studies of MSCs.Cell Death and Differentiation advance online publication, 25 October 2013; doi:10.1038/cdd.2013.149.
Cell Death and Differentiation 10/2013; · 8.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Defects in multiple coagulation factor deficiency protein 2 (MCFD2) are a cause of factor V and factor VIII combined deficiency type 2 (F5F8D). MCFD2 was also suggested to play an important role as an autocrine/paracrine factor in maintaining neural stem cell potential. The current work provided direct evidence that both amphibian and human MCFD2 can maintain stem cell pluripotency or stemness of rhesus monkey embryonic stem cells (rESCs) as basic fibroblast growth factor 2 (FGF-2) does. In most cases, MCFD2 had identical effects on stem cells as FGF-2. We investigated the possible mechanism of MCFD2 to support stem cell pluripotency by highlighting the effects of MCFD2 and FGF-2 on several signaling pathways in rESCs, namely MAPK, TGF-β, Wnt, and Akt, and 3 core transcriptional factors (Oct4, Nanog, and Sox2). In addition, some features of signaling pathways (MAPK and Akt), which are different from human embryonic stem cells (hESCs) and mouse embryonic stem cells (mESCs), are found in rESCs, indicating that primate ESCs have unique signaling mechanisms. These results may shed light on the biological roles of MCFD2, the conserved protein family distributed in both vertebrates and invertebrates. The ability to support stem cell self-renewal may be the general function of the conserved protein family.-Liu, H., Zhao, B., Chen, Y., You, D., Liu, R., Rong, M., Ji, W., Zheng, P., Lai, R. Multiple coagulation factor deficiency protein 2 contains the ability to support stem cell self-renewal.
[Show abstract][Hide abstract] ABSTRACT: Nonhuman primates (NHPs) provide powerful experimental models to study human development, cognitive functions and disturbances as well as complex behavior, because of their genetic and physiological similarities to humans. Therefore, NHPs are appropriate models for the study of human diseases, such as neurodegenerative diseases including Parkinson's, Alzheimer's and Huntington's diseases, which occur as a result of genetic mutations. However, such diseases afflicting humans do not occur naturally in NHPs. So transgenic NHPs need to be established to understand the etiology of disease pathology and pathogenesis. Compared to rodent genetic models, the generation of transgenic NHPs for human diseases is inefficient, and only a transgenic monkey model for Huntington's disease has been reported. This review focuses on potential approaches and contributing factors for generating transgenic NHPs to study human diseases.
Journal of Genetics and Genomics 06/2012; 39(6):247-51. · 2.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Evidence from epidemiological studies has proved that periconceptional use of folic acid (FA) can significantly reduce the risk of neural tube defects (NTDs). However, it is hard to explore when and how FA plays roles in neurogenesis and brain development in vivo, especially in human or other nonhuman primate systems. Primate embryonic stem cell (ESC) lines are ideal models for studying cell differentiation and organogenesis in vitro. In the present study, the roles of FA in neural differentiation were assessed in a rhesus monkey ESC system in vitro. The results showed no significant difference in the expression of neural precursor markers, such as nestin, Sox-1, or Pax-6, among neural progenitors obtained from different FA concentrations or with the FA antagonist methotrexate (MTX). However, FA depletion decreased cell proliferation and affected embryoid body (EB) and neural rosette formation, as well as neuronal but not neuroglia differentiation. Our data imply that the ESC system is a suitable model for further exploring the mechanism of how FA works in prevention of NTDs in primates.
Journal of Neuroscience Research 03/2012; 90(7):1382-91. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Much effort has been focused on improving assisted reproductive technology procedures in humans and nonhuman primates (NHPs). However, the pregnancy rate after embryo transfer (ET) has not been satisfactory, indicating that some barriers still need to be overcome in this important procedure. One of the key factors is embryo–uterine synchronicity, which is little known in NHPs. The objective of this study was to investigate the available ET time window in rhesus monkey (Macaca mulatta). Eighty-two adult female rhesus monkeys were superovulated with recombinant human FSH. Ovarian phases were identified according to estrogen (E2) and progesterone (P4) levels as well as ovarian examination by ultrasonography and laparoscopy. A total of 259 embryos were transferred by the laparoscopic approach into the oviducts of 63 adult female monkeys. Ovarian phases were divided into late follicular and early luteal phases. Similar pregnancy rates (30–36.4%) were obtained from recipients receiving ET either in their late follicular or early luteal phases, regardless of embryo developmental stages. This study indicates that the available time window for ET in rhesus monkeys is from the late follicular to early luteal phases.
American Journal of Primatology 02/2012; 74(2):165-73. · 2.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Parthenogenetic embryonic stem cells are considered as a promising resource for regeneration medicine and powerful tools for developmental biology. A lot of studies have revealed that embryonic stem cells have distinct microRNA expression pattern and these microRNAs play important roles in self-renewal and pluripotency of embryonic stem cells. However, few studies concern about microRNA expression pattern in parthenogenetic embryonic stem cells, especially in non-human primate--the ideal model species for human, largely due to the limited rhesus monkey parthenogenetic embryonic stem cells (rpESCs) available and lack of systematic analysis of the basics of rpESCs. Here, we derived two novel rpESCs lines and characterized their microRNA signature by Solexa deep sequencing. These two novel rpESCs shared many properties with other primate ESCs, including expression of pluripotent markers, capacity to generate derivatives representative of all three germ layers in vivo and in vitro, maintaining of euploid karyotype even after long culture. Additionally, lack of some paternally expressed imprinted genes and identity of Single-nucleotide Polymorphism (SNP) compare to their oocyte donors support their parthenogenesis origin. By characterizing their microRNA signature, we identified 91 novel microRNAs, except those are also detected in other primate ESCs. Moreover, these two novel rpESCs display a unique microRNA signature, comparing to their biparental counterpart ESCs. Then we analyzed X chromosome status in these two novel rpESCs; results suggested that one of them possesses two active X chromosomes, the other possesses only one active X chromosome liking biparental female embryonic stem cells. Taken together, our novel rpESCs provide a new alternative to existing rhesus monkey embryonic stem cells, microRNA information expands rhesus monkey microRNA data and may help understanding microRNA roles in pluripotency and parthenogenesis.
PLoS ONE 09/2011; 6(9):e25052. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective was to examine the effect of seminal plasma on cryopreservation of sperm from rhesus macaques. Sperm cryosurvival was evaluated by sperm motility and acrosomal integrity. Compared with slow cooling (-0.4 C/min) from 37 C (body temperature) to 4 C, rapid cooling (-16 C/min) caused cold shock in rhesus macaque sperm. The cryosurvival of sperm was decreased regardless of the presence or absence of seminal plasma (P<0.05). However, the presence of seminal plasma during cold shock at a rapid cooling rate improved sperm motility and acrosomal integrity in individual monkeys. Male-to-male variation in sperm cryosurvival was observed after cryopreservation (P<0.05), and the presence of seminal plasma during sperm cryopreservation improved sperm motility and acrosomal integrity in individual monkeys (P<0.05). Furthermore, by adding seminal plasma from monkeys with good sperm cryosurvival to sperm freezing extender, the frozen-thawed motility and acrosomal integrity of sperm from monkey with poor cryosurvival were improved (P<0.05). The present study indicated that seminal fluid is beneficial to sperm undergoing cold shock or cryopreservation in individual monkeys. The cryosurvival of sperm from rhesus macaques with poor sperm freezability could be improved by the presence of seminal plasma from males with good sperm cryosurvival. This finding provides a useful method for genetic preservation in this important species.
Journal of Reproduction and Development 09/2011; 57(6):737-43. · 1.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: rES (rhesus monkey embryonic stem) cells have similar characteristics to human ES (embryonic stem) cells, and might be useful as a substitute model for preclinical research. Before their clinical application, it is critical to understand the roles of factors that control the differentiation of ES cells into hepatocytes. Here, we analysed the effect of collagen gels on rES cells differentiation into hepatocytes by stepwise protocols. About 80% of DE (definitive endoderm) cells were generated from rES cells after being treated with activin A. The DE cells were then plated on to collagen gels or type I collagen-coated wells with growth factors to induce hepatocyte differentiation. In type I collagen systems, characteristics of immature hepatocytes were observed, including the expression of immature hepatic genes and the generation of 15±3% AFP (alpha fetoprotein)/CK (cytokeratin)18 double-positive cells. In collagen gel culture, differentiated cells exhibited typical hepatocyte morphology and expressed adult liver-specific genes. The mRNA expression of AFP (immature hepatic gene) was detected at day 11 but decreased at day 18. In contrast, mRNA expression of albumin (mature hepatic gene) was detected at day 11 and increased at day 18. Compared with type I collagen systems, significantly higher AFP/CK18 double-positive cells (68±7%) were produced in collagen gel culture. Furthermore, some differentiated cells acquired the hepatocytic function of glycogen storage. However, only immature hepatic genes were observed in collagen gel systems if growth factors were absent. Thus, collagen gels combined with hepatocyte-inducing growth factors efficiently promoted differentiation of hepatocytes from rES.
Cell Biology International 04/2011; 35(8):775-81. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The origin of the myofibroblast, the primary effector cell of liver fibrosis, is still elusive. Here, we report that fluorescence-activated cell sorting purified E-cad + rhesus monkey liver epithelial progenitor cells (mLEPCs) may serve as a potential source for liver myofibroblasts. Adult mLEPCs colonies were cultured in medium containing 2 ng/ml transforming growth factor β (TGF-β) and 10% fetal bovine serum (FBS) to induce differentiation. Phenotypic changes of cells were analyzed by morphological observation, immunostaining, and reverse transcription-polymerase chain reaction (RT-PCR). After cultured with TGF-β and FBS, some cells in adult mLEPCs colonies converted to fibroblasts-like cells. Immunostaining showed that fibroblasts-like cells had acquired the expression of mesenchymal cell marker vimentin but lost the expression of epithelial cell marker CK8. Fibroblasts-like cells were maintained in culture for up to 40 passages. RT-PCR analysis revealed that fibroblasts-like cells had acquired the expression of mesenchymal genes (snail, PAI-1, and collagen I) and lost the expression of epithelial specific genes (E-cad, ZO-1, CK18, and occludin). In addition, more than 60% of fibroblasts-like cells expressed myofibroblastic-related proteins such as αSMA, vimentin, and N-cad, which were not presented in mLEPCs. Furthermore, increased cell motility was also detected in these fibroblasts-like cells by time-lapse video observation. Our results demonstrate that hepatic epithelial progenitor cells, mLEPCs, transform to myofibroblast-like cells via epithelial-mesenchymal transition. This finding will facilitate understanding of the origin of myofibroblasts in liver fibrosis.
[Show abstract][Hide abstract] ABSTRACT: Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-β receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-β signalings in maintenance of rabbit TS cell self-renewal.
PLoS ONE 02/2011; 6(2):e17124. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ethylene glycol (EG) has been speculated to be the most appropriate penetrating cryoprotectant for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient. The present study aimed to determine the optimal EG concentration, freezing rate and holding time in liquid nitrogen (LN(2)) vapor for rhesus sperm cryopreservation. Among six tested EG concentrations (0, 0.18, 0.35, 0.7, 1.4 and 2.1 M), 0.7 M EG showed the most effective cryoprotection (P<0.05). Sperm frozen with 0.7 M EG at -183°C/min showed higher post-thaw motility than sperm frozen at -10, -67 or -435°C/min (P<0.05). Sperm frozen in LN(2) vapor at -183°C/min with 0.7 M EG and a holding time of 10 min showed higher post-thaw motility compared with a holding time of 5 or 15 min (P<0.05). The function of sperm cryopreserved at the optimized EG concentration, freezing rate and holding time was further evaluated by in vitro fertilization. Of the 36 oocytes collected from gonadotropin-stimulated rhesus macaques, 61.1% were fertilized, and 61.1, 44.4 and 36.1% of the oocytes developed to 2 cells, morulae and blastocysts, respectively. Our findings provide an alternative penetrating cryoprotectant and optimal protocol for genetic preservation purposes in this important species.
Journal of Veterinary Medical Science 01/2011; 73(6):717-23. · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) play important roles in embryonic stem cell (ESC) self-renewal and pluripotency. Numerous studies have revealed human and mouse ESC miRNA profiles. As a model for human-related study, the rhesus macaque is ideal for delineating the regulatory mechanisms of miRNAs in ESCs. However, studies on rhesus macaque (r)ESCs are lacking due to limited rESC availability and a need for systematic analyses of fundamental rESC characteristics.
We established three rESC lines and profiled microRNA using Solexa sequencing resulting in 304 known and 66 novel miRNAs. MiRNA profiles were highly conserved between rESC lines and predicted target genes were significantly enriched in differentiation pathways. Further analysis of the miRNA-target network indicated that gene expression regulated by miRNAs was negatively correlated to their evolutionary rate in rESCs. Moreover, a cross-species comparison revealed an overall conservation of miRNA expression patterns between human, mouse and rhesus macaque ESCs. However, we identified three miRNA clusters (miR-467, the miRNA cluster in the imprinted Dlk1-Dio3 region and C19MC) that showed clear interspecies differences.
rESCs share a unique miRNA set that may play critical roles in self-renewal and pluripotency. MiRNA expression patterns are generally conserved between species. However, species and/or lineage specific miRNA regulation changed during evolution.