Pluripotent Stem Cell Derived Endoderm in Interspecies Blastocyst Complementation
2019-02
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Pluripotent Stem Cell Derived Endoderm in Interspecies Blastocyst Complementation
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2019-02
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Every year in the United States approximately 4.5 million people are living with liver disease. Of those, an estimated 45,000 will die due to complete liver failure. This accounts for the 12th leading cause of death in the United States. There is currently no way to prevent death once the liver has failed and the only therapeutic option is transplantation. While 8,250 transplants occur every year clearly there exists an organ shortage that needs to be addressed (faststats @cdc.org 2019 a,b) (Liver Disease Stories @ liverfoundation.org, 2019). Several techniques are being used to try to address this organ shortage. Of these whole organ blastocyst complementation may hold significant promise. This technique allows for the generation of chimeric animals with target organs that are derived from a donor cell population. The therapeutic goal of whole organ complementation is to produce human organs in large animal surrogates. However, both a species and developmental timing barrier exist which may preclude human stem cells from efficiently contributing to the body plan of mammalian species such as the pig. There are also ethical concerns that must be considered when performing these techniques. This study addresses the species and developmental timing barriers associated with interspecies blastocyst complementation as well as current ethical concerns. In this work donor cells engineered for increased cell survivability were prepared for injection and cells from the non-human primate Callithrix jacchus (marmoset) was used as non-human primate substitute. For this study pluripotent stem cells from three species were used, human induced pluripotent stem cells (hiPSCs), marmoset embryonic stem cells (ESCs), and mouse induced pluripotent stem cells (miPSCs). Definitive endoderm was differentiated from these three cell types. In addition, the hiPSCs and the marmoset ESCs were engineered to constitutively express the either the reporter protein GFP alone or the anti-apoptotic BCL2 protein and the reporter protein GFP. Human iPSC BCL2/GFP-derived endoderm, marmoset ESC and ESC BCL2/GFP-derived endoderm, miPSC and miPSC-derived endoderm were prepared for injection into mouse embryos with a homozygous Hhex KO background. This studied aimed to investigate if preventing cellular apoptosis allows donor cells to overcome the species and developmental timing barriers and enables their progeny to complement a developmental niche and generate liver in a host embryo. Endoderm was successfully induced in all three species, with varying degrees of purity, and miPSC, human iPSC-derived endoderm, and marmoset ESC-derived endoderm were injected into mouse embryos with an Hhex KO background. Those embryos were then implanted into pseudo pregnant surrogate hosts. At e9.5 the remaining implanted embryos were harvested and analyzed. Only embryos injected with miPSCs displayed chimerism between the host and donor cells with no contribution was seen in embryos injected with either human iPSC or marmoset ESC-derived endoderm.
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University of Minnesota M.S. thesis.January 2019. Major: Stem Cell Biology. Advisor: James Dutton. 1 computer file (PDF); vi, 42 pages.
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Denman, Noah. (2019). Pluripotent Stem Cell Derived Endoderm in Interspecies Blastocyst Complementation. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/219283.
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