Tianqing Li

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.

Generation and Characterization of Rabbit Embryonic Stem Cells

We described the derivation of four stable pluripotent rabbit embryonic stem cell (ESC) lines, one (RF) from blastocysts fertilized in vivo and cultured in vitro and three (RP01, RP02, and RP03) from parthenogenetic blastocysts. These ESC lines have been cultivated for extended periods (RF >1 year, RP01 >8 months, RP02 >8 months, and RP03 >6 months) in vitro while maintaining expression of pluripotent ESC markers and a normal XY or XX karyotype. The ESCs from all lines expressed alkaline phosphatase, transcription factor Oct‐4, stage‐specific embryonic antigens (SSEA‐1, SSEA‐3, and SSEA‐4), and the tumor‐related antigens (TRA‐1‐60 and TRA‐1‐81). Similar to human and mouse ESCs, rabbit ESCs expressed pluripotency (Oct‐4, Nanog, SOX2, and UTF‐1) and signaling pathway genes (fibroblast growth factor, WNT, and transforming growth factor pathway). Morphologically, rabbit ESCs resembled primate ESCs, whereas their proliferation characteristics were more like those seen in mouse ESCs. Rabbit ESCs were induced to differentiate into many cell types in vitro and formed teratomas with derivatives of the three major germ layers in vivo when injected into severe combined immunodeficient mice. Our results showed that pluripotent, stable ESC lines could be derived from fertilized and parthenote‐derived rabbit embryos.

Generation of Cynomolgus Monkey Chimeric Fetuses using Embryonic Stem Cells

Because of their similarity to humans, non-human primates are important models for studying human disease and developing therapeutic strategies. Establishment of chimeric animals using embryonic stem cells (ESCs) could help with these investigations, but has not so far been achieved. Here, we show that cynomolgus monkey ESCs (cESCs) grown in adjusted culture conditions are able to incorporate into host embryos and develop into chimeras with contribution in all three germ layers and in germ cell progenitors. Under the optimized culture conditions, which are based on an approach developed previously for naive human ESCs, the cESCs displayed altered growth properties, gene expression profiles, and self-renewal signaling pathways, suggestive of an altered naive-like cell state. Thus our findings show that it is feasible to generate chimeric monkeys using ESCs and open up new avenues for the use of non-human primate models to study both pluripotency and human disease.

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.

Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells.

Generation of homogeneous oligodendrocytes as donor cells is essential for human embryonic stem cell (hESC)-based cell therapy for demylinating diseases. Herein we present a novel method for efficiently obtaining mature oligodendrocytes from hESCs with high purity (79.7+/-6.9%), using hepatocyte growth factor (HGF) and G5 supplement (containing insulin, transferrin, selenite, biotin, hydrocortisone, basic fibroblast growth factor and epidermal growth factor) in a four-step method. We induced hESCs into neural progenitors (NP) with HGF (5ng/ml) and G5 (1x) supplemented medium in an adherent differentiation system. The purified NPs were amplified in suspension as neurospheres for 1 month, and terminal oligodendrocyte differentiation was then induced by G5 supplement withdrawal and HGF treatment (20ng/ml). The cells generated displayed typical morphologies of mature oligodendrocytes and expressed oligodendrocyte markers O4 and myelin basic protein (MBP). Our result revealed that HGF significantly enhanced the proliferation of hESC-derived NPs and promoted the differentiation as well as the maturation of oligodendrocytes from NPs. Further studies suggest that HGF/c-Met signaling pathway might play an important role in oligodendrocyte differentiation in our system. Our studies provide a means for generating the clinically relevant cell type and a platform for deciphering the molecular mechanisms that control oligodendrocyte differentiation.

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.

A Robust Single Primate Neuroepithelial Cell Clonal Expansion System for Neural Tube Development and Disease Studies

Summary Developing a model of primate neural tube (NT) development is important to promote many NT disorder studies in model organisms. Here, we report a robust and stable system to allow for clonal expansion of single monkey neuroepithelial stem cells (NESCs) to develop into miniature NT-like structures. Single NESCs can produce functional neurons in vitro, survive, and extensively regenerate neuron axons in monkey brain. NT formation and NESC maintenance depend on high metabolism activity and Wnt signaling. NESCs are regionally restricted to a telencephalic fate. Moreover, single NESCs can turn into radial glial progenitors (RGPCs). The transition is accurately regulated by Wnt signaling through regulation of Notch signaling and adhesion molecules. Finally, using the “NESC-TO-NTs” system, we model the functions of folic acid (FA) on NT closure and demonstrate that FA can regulate multiple mechanisms to prevent NT defects. Our system is ideal for studying NT development and diseases.

Neuroprotective Effects of 7, 8-dihydroxyflavone on Midbrain Dopaminergic Neurons in MPP + -treated Monkeys

Parkinson’s disease (PD) is one common neurodegenerative disease caused by a significant loss of midbrain dopaminergic neurons. Previous reports showed that 7, 8- dihydroxyflavone (7, 8-DHF) as a potent TrkB agonist can mimic BDNF and play neuroprotective roles for mouse dopaminergic neurons. Nonetheless, the safety and neuroprotective effects are unclear in monkey models of PD. Here, we find that 7, 8-DHF could be absorbed and metabolized into 7-hydroxy-8-methoxyflavone through oral administration in monkeys. The half-life time of 7, 8-DHF in monkey plasma is about 4–8 hrs. Furthermore, these monkeys maintain health state throughout the course of seven-month treatments of 7, 8-DHF (30 mg/kg/day). Importantly, 7, 8-DHF treatments can prevent the progressive degeneration of midbrain dopaminergic neurons by attenuating neurotoxic effects of MPP+ and display strong neuroprotective effects in monkeys. Our study demonstrates that this promising small molecule may be transited into a clinical useful pharmacological agent.

Conversion of monkey fibroblasts to transplantable telencephalic neuroepithelial stem cells.

Non-human primates provide optimal models for the development of stem cell therapies. Although somatic cells have been converted into neural stem/progenitor cells, it is unclear whether telencephalic neuroepithelial stem cells (NESCs) with stable properties can be generated from fibroblasts in primate. Here we report that a combination of transcription factors (Oct4, Sox2, Klf4) with a new culture medium induces rhesus monkey fibroblasts into NESCs, which can develop into miniature neural tube (NT)-like structures at a cell level. Furthermore, single induced NESCs (iNESCs) can generate later-stage 3D-NTs after grown on matrigel in suspension culture. iNESCs express NT cell markers, have a unique gene expression pattern biasing towards telencephalic patterning, and give rise to cortical neurons. Via transplantation, single iNESCs can extensively survive, regenerate myelinated neuron axons and synapse structures in adult monkey striatum and cortex, and differentiate into cortical neurons. Successful transplantation is closely associated with graft regions and grafted cell identities. The ability to generate defined and transplantable iNESCs from primate fibroblasts under a defined condition with predictable fate choices will facilitate disease modeling and cell therapy.