Ever since Dr. Joseph Murray’s Excelsior lecture in 1995 when he predicted a fourth “inductive phase” of surgical development in which replacement organs could be regrown or regenerated in vivo, and the first derivation of human pluripotent stem cells in 1998 by Dr. James Thomson, scientists have been expanding our knowledge of organ development and testing the potential of human pluripotent stem cells to differentiate into various cell types in the hopes of generating cells and tissues for transplantation. Reporting in Nature earlier this year, Yamaguchi et al1 synergized these 2 ideas to bring us 1 step closer to growing new organs and solving the organ shortage crisis. Building on prior work, they demonstrated the viable production of mouse-rat chimeric pancreata using the technique of blastocyst complementation by injecting mouse PSCs into PDX1 mutant rat blastocysts showing that isolated islets from these mouse-rat chimeric pancreata were capable of reversing streptozotocin induced diabetes in mice syngeneic to the original mouse PSC line, remarkably without long-term immunosuppression. As the ultimate goal of regenerative medicine seeks to generate replacement organs and tissues from the patient’s own cells, this study has profound implications.
Dr. Nakauchi’s laboratory has contributed with extensive work in the area of blastocyst complementation to construct chimeric organs, previously focusing on generating chimeric pancreata and kidneys in mice,2,3 pancreata in pigs,4 and endoderm and pancreas using Mixl1 inducible cells in rodents.5 These studies definitively demonstrated the requirement for a cellular niche created by disabling host organ development for ultimately generating donor PSC-derived organs, and that the host blastocyst species, not the PSC source species, determines organ size.
The current study builds and extends the authors previous work and investigates whether “autologous” organs (ie, mouse PSC-derived islets) would be susceptible to immune attack in syngeneic murine hosts1 (Figure 1). To test this, they first produced apancreatic rats using TALEN-mediated genome editing to modify the PDX1 locus. Then they injected enhanced green fluorescent protein (EGFP)-labeled rat PSCs into these PDX1mu/mu rat blastocysts, and observed homogeneous expression of pancreatic EGFP in the offspring. The pancreas exhibited normal histological architecture, normal islet endocrine composition, and reasonable but not perfect glucose disposal in glucose tolerance tests. This experiment showed that rat pancreata could be regrown using blastocyst complementation and that the PDX1mu/mu rat provided a developmental niche. Then, to generate interspecies chimeras, they injected EGFP-labeled mouse PSCs into PDX1mu/mu rat blastocysts and observed homogeneous pancreatic EGFP expression, suggesting that mouse PSCs contributed to all functional lineages of the pancreas. Although the PSCs seemed to contribute both structurally and functionally to pancreatic regrowth, the authors found sluggish glucose removal, suggesting suboptimal insulin production or responsiveness. In contrast, in heterozygous PDX1+/mu blastocyst-derived chimeras, they observed very limited contribution of EGFP-labeled cells to islets, once again indicating the requirement for a developmental niche. In their final experiment, the authors sought to determine whether islets isolated from mouse PSC-PDX1mu/mu rat chimeric pancreata could restore normoglycemia to streptozotocin-diabetic syngeneic C57BL/6 mouse recipients. They found that this was possible with as few as 100 islets transplanted under the kidney capsule, but required low-dose transient immunosuppression administered to the diabetic mice.
The attractiveness of this strategy extends well beyond islets and the treatment of diabetes. Patients with diseases in need of transplantable cells, tissues, or organs could donate a somatic cell, from skin or blood for example, then using standard reprogramming methods that generate patient-specific induced PSCs (iPSCs) in vitro. Using the blastocyst complementation technique, normal iPSCs, or “genetically corrected” iPSCs, could be injected into xenogeneic host blastocysts, such as from sheep, pigs, or nonhuman primates and allowed to develop into human chimeric organs. Combining patient-derived iPSCs with animal embryos to make chimeric organs and tissues has the potential to solve the organ shortage crisis and potentially eliminate or reduce the need for immunosuppression.
As the ultimate goal of regenerative medicine seeks to generate replacement organs and tissues from the patient’s own cells, this study has profound implications.
Translating this strategy to humans suggests several impediments: There are obvious ethical implications, and therefore ethical and regulatory frameworks would need to be established to accommodate such an approach. This strategy would also require producing homozygous mutant lines for each of the organs and tissues required in order to create the “developmental niche.” Although genome editing will certainly facilitate making these genomic modifications, and a porcine endogenous retrovirus (PERV)-free pig line has been created6 which could be used as a base organism to be further modified, this is still not trivial and remains inefficient (~5-10% of offspring are homozygous mutants). Moreover, blastocyst complementation efficiency is also low (2.7%) regardless of the source PSC line, as demonstrated in this study.
Another potential challenge is the fact that organs are comprised of other parenchymal cells such as endothelial cells, dendritic cells, fibroblasts, and so on, which would be derived from the xenogeneic blastocyst host and could elicit an immunological response. In fact, the authors showed that in the mouse-rat chimeras, there were a significant number of rat derived CD31+ endothelial cells in the isolated islets before transplantation, though they seemed to disappear in the graft by the end of the study. There are other nonpancreatic and nonendocrine cell types within native and isolated islets as well, but these were not evaluated in the study. Immunosuppression was administered, albeit transiently, to the murine recipients of “syngeneic” chimeric islet transplants. Although the amount of immunosuppression was not able to prevent rejection of fully xenogeneic grafts, prolonged survival of the chimeric islet grafts was achieved. Moreover, the mouse-rat interspecies combination used here represents a concordant xenograft and any clinical strategy may need to consider discordant human-pig chimeras to obtain larger organs. Indeed, in a more stringent discordant chimeric system, it is possible that one might find diminished human cellular contribution to the organ and/or a more aggressive rejection response which would need to be controlled. Future modifications of the pig genome leading to significant reductions in immunogenicity of porcine organs may support the clinical potential of this approach.
Another issue is that T1D is an autoimmune disease, and syngeneic transplants would likely be susceptible to recurrent immune attack with subsequent β cell destruction. Furthermore, it is concerning that chimeric animals developed hyperglycemia. The majority of chimeric animals exhibited clearly impaired glucose tolerance tests, and some animals even developed frank diabetes as adults with a lymphocytic infiltrate in their pancreas. The underlying mechanism of this important finding has not been identified and requires further investigation.
Nonetheless, this landmark study not only demonstrates feasibility of creating interspecies chimeric organs to study species specific developmental mechanisms but also addresses the therapeutic potential of “autologous” pluripotent stem cell-derived islets generated by blastocyst complementation in a xenogeneic host.
1. Yamaguchi T, Sato H, Kato-Itoh M, et al. Interspecies organogenesis generates autologous functional islets. Nature
2. Kobayashi T, Yamaguchi T, Hamanaka S, et al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell
3. Usui J, Kobayashi T, Yamaguchi T, et al. Generation of kidney from pluripotent stem cells via blastocyst complementation. Am J Pathol
4. Matsunari H, Nagashima H, Watanabe M, et al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc Natl Acad Sci U S A
5. Kobayashi T, Kato-Itoh M, Nakauchi H. Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation. Stem Cells Dev
6. Yang L, Guell M, George H, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science