The researchers next showed that this approach could be coupled with CRISPR–Cas9 editing of donor ES cells as a way of interrogating the function of genes of interest in the forebrain. As a proof of principle, they focused on the gene doublecortin (Dcx), which encodes a protein involved in neuronal migration and causes malformations of the cerebral cortex when mutated in patients. Chimaeras produced using Dcx-deficient donor ES cells exhibited hippocampal defects that mimicked those observed8 in transgenic mice lacking Dcx.
Chang and colleagues’ NBC approach could facilitate the study of brain development and disease in several ways. First, generating transgenic animals in which both copies of a gene are mutated requires multiple breeding steps: even if both copies of the gene are mutated in the injected ES cells, the chimaeric mice must be crossed to wild-type partners to produce offspring with one normal copy, and an extra step is needed to produce animals in which both gene copies are mutated. The need for these breeding steps is eliminated with NBC. Similarly, numerous modifications can be introduced into donor ES cells at the same time using CRISPR–Cas9, rather than being brought together through complex breeding strategies. This could accelerate studies into disorders involving more than one gene, such as autism spectrum disorders, and improve our understanding of gene–gene interactions during development.
Second, although Chang et al. chose to ablate forebrain progenitors, the same principle can be applied to other regions or cell types in the CNS. Moreover, alternative approaches for targeted genetic ablation could also be used, such as the removal of genes essential for development of a specific brain region, or the forced expression of proteins that induce programmed cell death. However, because most of the proteins that regulate development have roles in various cell types and at a range of embryonic stages, caution is warranted when designing targeted ablation strategies, to avoid undesired side effects.
Third, this study raises the possibility of generating interspecies chimaeras of the CNS. In particular, the generation of human–animal chimaeras, in which human cells are integrated into an animal’s neural circuits, would allow some human-specific brain features to be studied in a more physiological environment than can be provided by current in vitro systems9. However, the ethical implications of this work need to be closely considered10. Indeed, organizations such as the International Society for Stem Cell Research recommend restrictions on experiments that incorporate human cells into animals early in development, together with specialized oversight and review of the research11.
There are technical difficulties, too. Although human cells such as neural-precursor cells have successfully been transplanted into mouse embryos to generate chimaeric tissues12, it has proved harder to efficiently generate whole-organism chimaeras from blastocyst injections. This barrier could be overcome by strategies that confer a selective advantage on or enhance the survival of donor human cells, or by the development of techniques to better monitor the state of human pluripotent stem cells. For instance, human pluripotent stem cells that are in a ground state known as naive can be engrafted into pig and cattle blastocysts, but show little chimaera-forming abilities; by contrast, pluripotent stem cells in a different cell state known as intermediate can be engrafted and generate differentiated progeny13.