Generating Human Organs via Interspecies Chimera Formation: Advances and Barriers

Abstract

The shortage of human organs for transplantation is a devastating medical problem. One way to expand organ supply is to derive functional organs from patient-specific stem cells. Due to their capacity to grow indefinitely in the laboratory and differentiate into any cell type of the human body, patient-specific pluripotent stem (PS) cells harbor the potential to provide an inexhaustible supply of donor cells for transplantation. However, current efforts to generate functional organs from PS cells have so far been unsuccessful. An alternative and promising strategy is to generate human organs inside large animal species through a technique called interspecies blastocyst complementation. In this method, animals comprised of cells from human and animal species are generated by injecting donor human PS cells into animal host embryos. Critical genes for organ development are knocked out by genome editing, allowing donor human PS cells to populate the vacated niche. In principle, this experimental approach will produce a desired organ of human origin inside a host animal. In this mini-review, we focus on recent advances that may bring the promise of blastocyst complementation to clinical practice. While CRISPR/Cas9 has accelerated the creation of transgenic large animals such as pigs and sheep, we propose that further advances in the generation of chimera-competent human PS cells are needed to achieve interspecies blastocyst complementation. It will also be necessary to define the constituents of the species barrier, which inhibits efficient colonization of host animal embryos with human cells. Interspecies blastocyst complementation is a promising approach to help overcome the organ shortage facing the practice of clinical medicine today.

Keywords: CRISPR; Cas9; Pdx1; blastocyst complementation; chimeras; human pluripotent stem cells; interspecies blastocyst complementation; interspecies chimeras; mouse pluripotent stem cells; naive pluripotency; naive pluripotent stem cells; organ generation; organ shortage; organ transplantation; pluripotency; pluripotent stem cells; primed pluripotency; primed pluripotent stem cells; reprogramming.

Figure 1

Interspecies blastocyst complementation. Organ generation via interspecies blastocyst complementation could help to solve the severe shortage of organ donors worldwide. The genetic modification of host animals to disable organ development may enable donor human PS cells or progenitors to populate the targeted organ with minimal competition from the host. First, embryos of large animal hosts such as pigs or sheep are edited using CRISPR/Cas9 to disable formation of a target organ. Second, human xenogenic chimera-competent pluripotent stem cells are generated – first by: 1) reprogramming somatic cells to generate conventional human induced pluripotent stem cells (iPSCs) followed by 2) converting conventional human iPSCs to a chimera-competent state. Human xenogenic PS cells are then introduced into host animal embryos by blastocyst injection and the resulting chimeric embryo is transferred into a pseudopregnant foster mother. The chimeric embryo is allowed to develop in utero and if the method is successful, human-pig or human-sheep chimeras are born.

Figure 2

Mouse and human naive and primed pluripotent stem cells. (Top left) Mouse naive embryonic stem (ES) cells; (top right) Mouse primed epiblast stem (EpiS) cells; (bottom left) putative human naive induced pluripotent stem (iPS) cells; (bottom right) human primed iPS cells. Mouse ES cells were grown in N2B27-2i/LIF conditions. Mouse EpiS cells and human iPS cells were grown in FGF-containing medium. Human naive iPS cells were grown in a modified 2i/LIF medium (ADLA, data unpublished).

Figure 3

Species barrier that impedes interspecies chimerism. A. Understanding the species barrier: synchronizing developmental speed. It is unclear why the efficiency of interspecies chimerism between humans and large animal species is low. The undefined parameters that impede interspecies chimerism are referred to as the species barrier. One possible component of the species barrier is the difference in developmental speed between species. How species-specific developmental timing is controlled is largely unknown. Experiments have shown that developmental speed may be species-specific and cell-autonomous (top). Some reports have suggested that developmental timing can be at least modestly modulated. In order for interspecies chimerism to occur, it will be necessary to achieve coordinated morphogenesis between human cells and animal host tissue (bottom). B. Engineering developmental compatibility across species. The existence of viable adult interspecies chimeras between mice and rats suggests the feasibility of generating interspecies chimeras using human cells. Choosing a host that is evolutionarily closer to humans, such as non-human primates (NHP), may help increase the degree of chimaerism by donor human PS cells. It may be possible to use human-primate chimeras to gain insight into the mechanisms underlying interspecies chimeric compatibility (compatible signaling environment). Using these insights, one can genetically “humanize” compatible large animal hosts (incompatible signaling environment) using multiplexed CRISPR/Cas9 gene editing. If successful, appropriately targeted genetic interventions will result in a more compatible signaling environment for higher efficiency interspecies chimerism.