Shaohui Ji

Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

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.

Transgenic rhesus monkeys produced by gene transfer into early-cleavage-stage embryos using a simian immunodeficiency virus-based vector

The development of transgenic technologies in monkeys is important for creating valuable animal models of human physiology so that the etiology of diseases can be studied and potential therapies for their amelioration may be developed. However, the efficiency of producing transgenic primate animals is presently very low, and there are few reports of success. We have developed an improved methodology for the production of transgenic rhesus monkeys, making use of a simian immunodeficiency virus (SIV)-based vector that encodes EGFP and a protocol for infection of early-cleavage–stage embryos. We show that infection does not alter embryo development. Moreover, the timing of infection, either before or during embryonic genome activation, has no observable effect on the level and stability of transgene expression. Of 70 embryos injected with concentrated virus at the one- to two-cell stage or the four- to eight-cell stage and showing fluorescence, 30 were transferred to surrogate mothers. One transgenic fetus was obtained from a fraternal triple pregnancy. Four infant monkeys were produced from four singleton pregnancies, of which two expressed EGFP throughout the whole body. These results demonstrate the usefulness of SIV-based lentiviral vectors for the generation of transgenic monkeys and improve the efficiency of transgenic technology in nonhuman primates.

Directional freezing as an alternative method for cryopreserving rhesus macaque (Macaca mulatta) sperm.

The objective was to develop a freezing protocol using a directional freezing (DF) technique for cryopreservation of rhesus macaque sperm and achieve a survival rate comparable to that achieved with a conventional freezing (CF) technique. Rhesus macaque sperm frozen with a DF technique, with cooling rates of 12 or 16 °C/min, had higher post-thaw motility (P < 0.05) than those cooled at 7 °C/min (59.3, 61.1, and 50.3%, respectively). Furthermore, sperm cryopreserved with 5% glycerol and a DF technique had similar frozen-thawed sperm motility to those cryopreserved by a CF technique (63.7 vs. 53.9%, P > 0.05). The function of sperm cryopreserved at the optimized cooling rate using a DF technique was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated rhesus macaques. Of the 38 mature oocytes collected, 78.9% were fertilized and 71.1, 47.4, and 42.1% of the oocytes developed to the 2-cell, morulae, and blastocyst stages, respectively. In conclusion, rhesus macaque sperm was effectively cryopreserved using a DF technique, providing a new and effective method for genetic preservation in this important species.

The Available Time Window for Embryo Transfer in the Rhesus Monkey (Macaca mulatta)

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.

Isolation and characterization of liver epithelial progenitor cells from normal adult rhesus monkeys (Macaca mulatta)

Isolation and characterization of liver epithelial progenitor cells from normal adult rhesus monkeys ( Macaca mulatta )

Impairments in embryonic genome activation in rhesus monkey somatic cell nuclear transfer embryos

Somatic cell nuclear transfer (SCNT) is a remarkable process in which a somatic cell nucleus is acted upon by the ooplasm via mechanisms that today remain unknown. Here we show the developmental competence (% blastocyst) of embryos derived from SCNT (21%) was markedly (p < 0.05) impaired compared with those derived from in vitro fertilization (IVF) (42.1%) in rhesus monkey. Also, SCNT embryos were abnormal in their time course of embryonic development. SCNT produced embryos reached the eight-cell stage faster than did IVF produced embryos. We compare the transcription patterns of five nucleolar-related proteins-nucleolin, nucleophosmin, fibrillarin, PAF53, and UBF-in single IVF and SCNT blastocysts by RT-PCR. The SCNT embryos showed abnormal gene transcription. Immunolocalization of fibrillarin was undetectable in 8-cell and 16-cell SCNT embryos, indicating embryonic genomic activation was delayed in monkey embryos produced by SCNT compared to their IVF-derived counterparts. Some of SCNT embryos appeared to relative higher developmental potential and fibrillarin expression by prolonged exposure of incoming nuclei to a cytoplasm. Thus, our data show that SCNT embryos are characterized by abnormal cleavage and the timely onset of embryonic genome transcription, deficits that may explain their reduced pre- and postimplantation developmental capacity.

Notch signaling dependent differentiation of cholangiocyte-like cells from rhesus monkey embryonic stem cells.

Rhesus monkey embryonic stem (rES) cells have similar characteristics to human ES cells, and might be useful as a substitute model for preclinical research. Notch signaling is involved in the formation of bile ducts, which are composed of cholangiocytes. However, little is known about the role of Notch signaling in cholangiocytic commitment of ES cells. We analyzed the effect of Notch signaling on the induction of cholangiocyte-like cells from rES cells. About 80% of definitive endoderm (DE) cells were generated from rES cells after treatment with activin A. After treatment with BMP4 and FGF1 on matrigel coated wells in serum-free medium, rES-derived DE gave rise to cholangiocyte-like cells by expression of cholangiocytic specific proteins (CK7, CK18, CK19, CK20, and OV-6) and genes (GSTPi, IB4, and HNF1β). At the same time, expression of Notch 1 and Notch 2 mRNA were detected during cell differentiation, as well as their downstream target genes such as Hes 1 and Hes 5. Inhibition of the Notch signal pathway by L-685458 resulted in decreased expression of Notch and their downstream genes. In addition, the proportion of cholangiocyte-like cells declined from approximately 90% to approximately 20%. These results suggest that Notch signaling may play a critical role in cholangiocytic development from ES cells.

Hepatocytic differentiation of rhesus monkey embryonic stem cells promoted by collagen gels and growth factors

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.

Neural progenitors derived from monkey embryonic stem cells in a simple monoculture system.

A simple monoculture system, combined with a chemically defined medium containing hepatocyte growth factor (HGF) and G5 supplement, was used to induce rhesus monkey embryonic stem cells (rESC) directly into neuroepithelial (NE) cells. Under these conditions, the generation of NE cells did not require the formation of embryoid bodies or co-culture with other cell types. The NE cells could further develop to generate neurons, astrocytes and oligodendrocytes. These results demonstrate a simple approach to obtain enriched and expandable populations of neural progenitors. Importantly, unlike other systems, the neural progenitors obtained using this approach may possess the potential to differentiate into various regional neural cells. Finally, the results suggest that the time-dependent shift in the differentiation potential of the rESC-derived neural progenitors in vitro reflects the developmental events that occur during neurogenesis in vivo. Thus, this system can be used to study the mechanisms of cell fate specification during non-human primate neurogenesis.