Controlled generation of human embryoids using optogenetics

项目总结

Project Summary Understanding human embryonic development has tremendous impact on improving assisted reproductive technologies, stem cell-based regenerative therapies, and the prevention of genetic birth defects and teratogenesis. However, our knowledge about human embryonic development is limited due to drastic species divergences between humans and other commonly used mammalian models and limited accessibility to human/non-human primate embryonic tissue samples for research purposes. To overcome these difficulties, stem cell-based in vitro culture methods that allow spontaneous development of multicellular structures while simplifying the experimental system and improving accessibility for high-quality live imaging are of tremendous value. Despite the great promise, the inherent variations of existing culture systems significantly limit their applications for fundamental mechanistic research and clinical applications. Optogenetic approaches utilize genetic engineering methods and optical technologies to control biological functions of cells or tissues modified to express photosensitive proteins. The precise spatiotemporal resolution of optogenetics, coupled with properly engineered illumination systems, can offer a powerful and versatile solution to improve the controllability and reproducibility of stem-cell derived human development models. Our preliminary studies reveal that exogenous BMP4 stimulation efficiently drives human pluripotent stem cells (hPSCs) to differentiate into amniotic ectoderm-like cells (AMLCs); and localized exogenous BMP stimulation to hPSC aggregates generates human embryonic-like structures (or embryoids). In this proposed research, we will pursue an exploratory, high-risk but high-reward study to demonstrate the integration of optogenetics and stem cell-derived human embryo models. Specifically, we will derive optogenetic hPSC lines using an optoBMP system; and examine their intracellular BMP activities and amniotic differentiation potentials under light exposure. Fully characterized optoBMP-hPSCs will then be utilized to generate, the first of its kind, optogenetic human embryoid system. This research, if successful, will demonstrate, for the first time, the successful generation of human embryoids with optogenetic perturbation of developmental signals. This is very significant, as there are crucial issues pervading in existing embryoid culture systems: a lack of reproducibility and controllability and, coupled to this, our lack of understanding of the processes that guide their development and self-organization. Incorporating optogenetic tools into existing embryoid cultures will offer the experimenter precise spatiotemporal control over the key development signals. The improved reproducibility and experimental consistency will greatly facilitate meaningful mechanistic studies to understand mechanisms that underlie human embryonic development in normal and pathological situations, and to use them as targets for genetic manipulation or drug testing.