Institute of Primate Translational Medicine, KMUST

The research team led by Prof. Tianqing Li and Weizhi Ji uncovered the molecular and morphogenetic developmental landscape of human embryos pre-gastrulation that occurs during human blastocyst stage to the primitive streak anlage stage with the help of a three-dimensional (3D) blastocyst-culture system developed by them (Nature 2020 Jan; 577(7791):537-542).

At Day 7 post-fertilization (7 d.p.f), human embryo implants into the mother's uterus for further development. Clinically, a large number of embryos arrest growth due to failure of implantation in the second week. Human embryo development as well as the key cellular and molecular events in the uterus after 7 d.p.f are locked in a "black box" due to ethical limits and technological barriers.

Human embryogenesis has been modeled in 2D cultures up to days 12–13. However, these 2D-cultured embryos only recapitulate some developmental landmarks and self-organize into some simple structures. Structures or processes such as amniotic epithelium (AME)–epiblast separation, basement membrane, secondary yolk sac, anterior visceral endoderm, anterior–posterior (AP) polarity initiation and primitive streak anlage (PSA) are not present in human 2D-embryos, making it difficult to study spatial self-organization and cell lineages. Thus, better models for studying human pre-gastrulation embryogenesis are needed.

To address this limitation, Li and their teams first developed a 3D-culture system that enables human blastocyst at 5–6 d.p.f development up to 14 d.p.f in culture. The researchers could observe that epiblast, primitive endoderm and trophoblast fates were well defined after 1–2 days of culture. Then the epiblast started to polarize and the primary yolk sac developed, followed by the secondary yolk sac. Moreover, a clear difference between the AME and the epiblast arose. Additional features that had previously not been observed were the appearance of AP polarity and the formation of the PSA in the embryo. More excitingly, the researchers revealed some key developmental features and mechanisms of human embryogenesis based on the system. First, the AME displays unique and characteristic phenotypes distinct from the epiblast. Second, after implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Third, epiblasts undergo a transition to pluripotency upon implantation, and their developmental processes are driven by different pluripotency factors. Finally, key differences between human and macaque embryos in the epiblast indicate monkey embryos cannot completely replace human embryos in uncovering human development. In summary, these findings revealed by the Nature paper provide crucial insights into the pluripotency of human pluripotent stem cells and uncover stem-cell self-renewal and differentiation processes, and will inform future strategies to improve in vitro fertilization success rates.

Figure Model of human pre-gastrulation embryo development landmarks. AC, amniotic cavity; PYS, primary yolk sac; SYS, secondary yolk sac

Early embryonic development is the key stage in primate species that will influence their long-term development and health conditions. Due to ethical concerns and technological limits, little was known about the post-implantation embryo development. Until recently, the researchers of Profs. Tao Tan, Yuyu Niu and Weizhi Ji from Yunnan Key Laboratory of Primate Biomedical Research / Institute of Primate Translational Medicine, Kunming University of Science and Technology have grown the nonhuman primate embryos in a dish longer than before till Day 20 covering pre-implantation to gastrulation. The in vitro cultured embryos exhibited highly consistent morphology and gene expression characteristics with in vivo embryos. The study has been published on Science (Science. 2019; 366 (6467): eaaw5754)

The research reveals the dynamic regulation of pluripotency during post-implantation development and provides new insights into the development of primate trophoblasts and primordial germ cells. This can shed light on previously unknown aspects of human post-implantation development and possible causes for birth defect and developmental disorder. Meanwhile, the study has important guiding significance for cell replacement therapy and organ regeneration research.

The published results have received extensive attentions and feedbacks from peer researchers and positively commented by top tier journals including Nature and Science.

On May 18th, 2017, the world's top journal "Cell" published the Kunming University of Science and Technology, Yunnan Key Laboratory of Primate Biomedical Research (LPBR), Ji Weizhi, Chen Yongchang team and Sun Yi team of Tongji University, using TALEN technology to carry out Rettsyndrome(RTT) Crab Monkey model.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder that mostly manifests in girls with a morbidity rate of 1:10,000–1:15,000. Almost 95% of RTT is believed to be caused by mutations of an X-linked gene methyl-CpG-binding protein 2 (MECP2) . MECP2 mutations are most often embryonic lethal for boys, except for very few, who are born with severe encephalopathy leading to death before 2 years of age. RTT girls seem to have normal development for up to 6–18 months but manifest a series of symptoms associated with intellectual disability, loss of acquired language, and compromised cognitive, social, and motor skills, etc. As RTT is a monogenic disorder, genetic modification technologies have made it possible to develop animal models for further study. RTT animal models were first generated in mice and recently in rats.Gene-edited nonhuman primates (NHPs),such as rhesus and cynomolgus monkeys,share high levels of genetic, physiological, social behavioral, and CNS developmental similarities with humans, making them suitable for the study of neurodevelopmental disorders. In order to establish the RTT monkey model, researchers from Kunming University of Science and Technology, Yunnan Key Laboratory of Primate Biomedical Research (LPBR) and Tongji University, after more than one year of research, first reportedthat they successfully developed a monkey model of the MECP2 gene using TALEN, a targeted gene editing method , published in the "Cell stem cell" magazine.

This report in "Cell" is different from the rodent model,Mecp2 knockout rodents show "limb-clasping" phenotype, but this phenotype is shared by neurodegeneration conditions in mice, which is not characteristic for RTT. In contrast, monkey models showed complicated behavioral features, such as fragmented sleep, increased stereotypy, reduced active avoidance of noisy or heat stimuli, and reduced environmental exploration, all of which resemble symptoms of patients. These activities are not widely set up for detection in mouse models. This study showed that a valid monkey model might offer robust phenotype or endophenotype, which could be implemented as outcome measures in human clinical trials in the future to facilitate drug development.

By improving the culture conditions of stem cells, Yunnan Key Laboratory of Primate Biomedical Research (LPBR) first isolated and established Cynomolgus monkey ESCs which capable of producing chimeric monkeys in the world. 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 naivelike cell state. The results of this study are published in Cell Stem Cell (Cell stem cell. 2014; 14(3): 323-328).

The generation of monkey ESCs with the ability to produce same-species chimeras has important implications for stem cell research, clinical regenerative medicine, and non-human primate models of human disease. First, this approach can be an important platform upon which to test the pluripotency of primate ESCs or iPSCs. Second, this type of approach could pave the way for the use of chimerism to generate functional organs, such as pancreas and kidney, from PSCs by injection of monkey PSCs into pancreatogenesis- or nephrogenesis-disabled monkey early embryos as demonstrated by previous reports . Third, monkey chimeras generated by ESCs could be used to produce human disease models in non-human primates for the study of mechanisms of diseases by gene editing.

In 2014, Yunnan Key Laboratory of Primate Biomedical Research (LPBR) cooperated with domestic research groups,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, successfully achieve precise gene targeting in cynomolgus monkeys,and successfully obtained the first Cas9-mediated gene knockout cynomolgus monkeys.

The results of the study were published in the first unit in Cell (Cell. 2014; 156(4): 836-843), which attracted widespread attention. More than a dozen foreign media have reported this, and Nature has referred to this result as "Milestone for targeted gene-editing technology promises better models for human diseases." At the end of 2014, the study was selected by Nature as one of the successful research events in 2014.

(http://www.nature.com/news/365-days-2014-in-science-1.16573). The article was named one of the 10 best research papers from Cell in 2014 (http://onlinedigeditions.com/publication/frame.php?i=236801&p=&pn=&ver=flex). In the subsequent studies, we further confirmed that CRISPR/Cas9-mediated genetic modification was performed on the germ cells of the experimental animals, and the results were published in Cell Research (Cell research. 2015; 25(2): 262-265).