Yunhua Xie

Epigenetic Marks in Cloned Rhesus Monkey Embryos: Comparison with Counterparts Produced In Vitro

Abstract Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy. During preimplantation development, 67% of two-cell- and 50% of eight-cell-cloned embryos showed higher DNA methylation levels than their in vitro fertilization (IVF) counterparts, which undergo gradual demethylation until the early morula stage. Moreover, whereas an asymmetric distribution of DNA methylation was established in an IVF blastocysts with a lower methylation level in the inner cell mass (ICM) than in the trophectoderm, in most cloned blastocysts, ICM cells maintained a high degree of methylation. Finally, two donor cell lines (S11 and S1–04) that showed a higher level of H3K9 acetylation supported more blastocyst formation after nuclear transfer than the other cell line (S1–03), with a relatively low level of acetylation staining. In conclusion, we propose that abnormal DNA methylation patterns contribute to the poor quality of cloned preimplantation embryos and may be one of the obstacles to successful cloning in primates.

Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monkey Embryonic Stem Cells

In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast‐like (MFG) cells, monkey follicular granulosa epithelium‐like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor β1, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage‐specific embryonic antigen (SSEA)‐3, SSEA‐4, tumor‐related antigen (TRA)‐1‐60, and TRA‐1‐81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.

Transplantable Neural Progenitor Populations Derived from Rhesus Monkey Embryonic Stem Cells

Cell‐based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3% ± 8.1%) populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65%–80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP‐based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.

Folic acid deficiency inhibits neural rosette formation and neuronal differentiation from rhesus monkey embryonic stem cells

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.

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.

Feeder layer- and serum-free culture of rhesus monkey embryonic stem cells

The common culture system of rhesus monkey embryonic stem (rES) cells depends largely on feeder cells and serum, which limits the research and application of rES cells. This study reports a feeder layer-free and serum-free system for culture of rES cells. rES cells could be cultured through at least 22 passages on laminin in medium supplemented with serum replacement (SR), basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGFbeta1), and maintained stable proliferation rates and normal karyotypes, while displaying all the embryonic stem cell characteristics including morphology, alkaline phosphatase (AKP), Oct-4, cell surface markers SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and formed cystic embryoid bodies in vitro. In addition, the studies showed that TGFbeta1, bFGF and laminin are necessary for maintaining the undifferentiated growth of rES cells in long-term culture. Moreover, withdrawal of TGFbeta1 increased the differentiation rate by decreasing the expression of integrins. Therefore, this system would provide a well-defined culture system for rES cells, and would facilitate research into self-renewal and differentiation mechanisms of rES cells.

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.

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.