Generation of functional organs and tissues using organism interspecific blastocyst complementation

项目概要： 培养的多能干细胞（PSC）的体外分化为产生用于细胞替代疗法的特定细胞类型提供了有希望的工具。尽管在过去十年中取得了快速进展，但在全面的临床应用成为现实之前，包括细胞生产数量，质量和安全性在内的众多挑战仍有待解决。此外，从培养的PSC中产生功能性器官，这是满足对器官供体日益增长的需求所急需的突破，仍然是不可能的。这在很大程度上是由于我们对体内组织/器官生成的分子和遗传过程的理解有限，而体内组织/器官生成已经通过数百万年的进化而完善。自然界已经进化出一个复杂而强大的系统，在胚胎发育的正常过程中产生功能性组织和器官。内在遗传程序以完美的时空方式与外在发育小生境无缝合作，使胚胎细胞能够致力于特定的细胞谱系，并组织成更高级的组织结构。胚泡互补是基于清空这些通过敲除对特定组织/器官发育至关重要的基因而产生的发育小生境，并使用供体嵌合能力PSC来定殖空的小生境并产生供体来源的所需组织/器官。随着CRISPR/CAS9介导的基因组工程的蓬勃发展，可以通过将Cas9 mRNA和gRNA直接共注射到受精卵中来高效地产生基因敲除动物。此外，嵌合能力的灵长类PSC最近已经看到了几个有希望的发展。通过结合合子基因组编辑和嵌合能力灵长类PSC的优势，我们提出建立一种新的种间囊胚互补系统，用于在大型动物宿主猪中产生功能器官。对于这第一项研究，我们将专注于使用来自非人灵长类物种的PSC，以评估我们提出的策略的有效性和安全性。我们的研究将为未来应用于人类提供关键信息。如果成功，这种方法将导致再生医学的范式转变，并将有助于克服器官捐献者短缺的问题。

PROJECT SUMMARY: In vitro differentiation of cultured pluripotent stem cells (PSCs) provides a promising tool for the generation of specific cell types useful for cell replacement therapies. Despite rapid progress over the last decade, numerous challenges encompassing cell-production quantity, quality and safety remain to be addressed before full-fledged clinical application becomes a reality. Moreover, generation of functional organs from cultured PSCs, a much needed breakthrough to meet the rising demands for organ donors, remains improbable. This is largely due to our limited understanding of molecular and genetic processes underlying in vivo tissue/organ generation, which has been perfected through millions of years’ evolution. Nature has evolved a sophisticated and robust system to generate functional tissues and organs during normal course of embryo development. The intrinsic genetic program works seamlessly with extrinsic developmental niches in a perfect spatiotemporal manner to enable embryonic cells to commit to specific cell lineages and be organized into higher-order tissue architectures. Blastocyst complementation is based on emptying these developmental niches created by knocking out gene(s) critical for the specific tissue/organ development and use donor chimeric-competent PSCs to colonize the vacant niche and generate desired tissues/organs of donor origin. With the booming of CRISPR/CAS9-mediated genome engineering gene knockout animals can be generated with high efficiency by co-injection of Cas9 mRNA and gRNAs directly into zygotes. Also, chimeric-competent primate PSCs have seen several promising developments very recently. By combining the strength of zygote genome editing and chimeric-competent primate PSCs, we propose to establish a novel interspecific blastocyst complementation system for the generation of functional organs in a large animal host, the pig. For this first study, we will focus on using PSCs derived from non-human primate species, to evaluate the efficacy and safety of our proposed strategy. Our study will provide critical information for future application into humans. If successful, this approach will lead to a paradigm shift in regenerative medicine and will help to overcome the shortage of organ donors.