Blastocyst complementation: current progress and future directions in xenogeneic organogenesis

Abstract

The generation of organs derived from pluripotent stem cells can be achieved in vivo through the blastocyst complementation technique. This method is based on the introduction of pluripotent stem cells into organogenesis-disabled pre-implantation embryos, where environmental signals instruct donor cells to colonize the vacant niche and to develop into the missing organ. When applied interspecies, this approach has the potential to produce human organs in genetically engineered livestock, offering a promising solution to the global transplants' shortage crisis. In this review, we summarize the current progress in blastocyst complementation research and highlight the key challenges that must be addressed to advance this field.

Keywords: Blastocyst complementation; Chimera; Interspecies organogenesis; Organ generation; Organ transplantation; Pluripotent stem cells.

Fig. 1

Blastocyst complementation technique overview. Patient-derived PSCs are microinjected into genetically engineered pig embryos that are unable of developing the desired organ (heart in this example). After that, microinjected embryos are transferred to surrogate sows to let them develop to term. In the end, hPSC-derived organs from the resulting pig could be used for human transplantation

Fig. 2

Strategies to obtain organ-deficient embryos. A Gene disruption methods to obtain genetic KOs include I) Crossbreeding heterozygous mice carrying a mutation in a master regulator gene of organ development results in an organ-deficient phenotype in homozygous mutants, which accounts for 25% of the progeny. II) Nuclear transfer of a previously gene edited somatic cell into an enucleated oocyte followed by proper stimulation, allows the generation of a genetically modified homozygous cloned organism. III) Microinjection of the CRISPR/Cas9 system (Cas9 mRNA or protein along with guide RNAs) into 1-cell stage embryos results in the direct production of KO embryos. IV) Conditional KO by using the Cre-lox system (used if systemic deletion of the target gene interferes with normal development of multiple tissues). Mice expressing Cre recombinase under a tissue-specific promoter are crossbred with mice carrying the target gene flanked by LoxP sites. To obtain 50% organ-deficient progeny, mice homozygous for the tissue-specific Cre and heterozygous for the LoxP flanked target gene should be crossed with mice homozygous for the LoxP flanked target gene. B DTA-mediated cell ablation: engineered mice expressing the Cre-recombinase under a promoter specific for the tissue to be ablated are crossed with mice carrying the DTA gene preceded by a LoxP flanked STOP cassette. When homozygous mice are used, all resulting progeny will express the DTA in the target tissue, leading to an organ-deficient phenotype

Fig. 3

The intricate balance to achieve interspecies organ complementation. Low chimerism levels may not be sufficient to complement an empty organ niche, resulting in embryonic lethality. However, if chimerism levels are too high, embryonic development is impaired and complemented embryos ultimately die