Browsing by Subject "Blastocyst complementation"

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    Formation Of Exogenic Pituitary, Eye Lens, And Midbrain Dopaminergic Neurons In Pigs Via Blastocyst Complementation
    (2016-12) Mikkila, Jennifer
    Thousands of patients die waiting for an organ transplant every year, while those that do obtain a transplant may run into complications such as Graft vs. Host disease. One reason for this is that there is a lack of organ donors who can biologically match specific patients. A solution to this problem would be to generate human organs in pigs using the patient’s own cells, and then to transplant these organs once they are grown. Blastocyst complementation is the perfect method to do this as it is involves injecting donor cells into a pig embryo, which can then contribute to growing to an organ in the pig if the pig’s own genetic code does not allow its own cells to form that very same tissue. Blastocyst complementation was previously done using PITX3 KO pig embryos and human induced pluripotent stem cells (hiPSCs) or human umbilical cord blood stem cells (hUCBSCs) in the Low Lab to create exogenic pituitaries, eye lenses, and midbrain dopaminergic neurons made of human cells. Human Nuclear Antigen staining of pituitaries in this study show positive signals. The human cells failed to integrate into the eye lens and midbrain dopamine neurons, despite the fact that the dopaminergic neurons were complemented and able to grow into functional cells. This failure could have been due to either incorrect stage-matching of donor cells to the host embryo, or knocking out a gene that was only partially responsible for cellular development. A second gene, LMX1A, was therefore knocked out together with PITX3 to try and get complementation with GFP-labelled porcine blastomeres. This study shows that the eye lenses of these embryos are similar in terms of a round, circular morphology to that of wild-type pig lenses. GFP is found to be present in the pituitary of a PITX3/LMX1A KO embryo injected with porcine blastomeres, meaning the donor cells successfully integrated into the organ.
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    The Potential to Generate Exogenic Interneurons for Alzheimer’s Disease via Blastocyst Complementation
    (2022-12) Johnson, Sether
    Alzheimer’s disease (AD) currently affects millions of patients worldwide, and to date the development of effective therapies has been slow. In AD, numerous types of neural cells become dysfunctional and are susceptible to degeneration, leading to cognitive deficits. One particular cell type affected are GABAergic inhibitory interneurons. Normally, these cells function as modulators of neural circuits, and are associated with maintenance of network synchrony and oscillatory signaling important for memory encoding. Impairments in short term memory, electrophysiological abnormalities such as neural hyperactivity and epileptiform spikes, and loss of interneurons are seen in AD patients and AD mouse models. These observations suggest that degeneration and dysfunction of interneurons contributes to cognitive deficits in AD. Thus, restoring interneuron activity is one potential approach to treat AD. The generation of exogenic interneurons via blastocyst complementation is one promising method to generate these cells. In this method, interspecies chimeras are created by genetic editing in a host blastocyst, which establishes a developmental niche to be filled during expansion of the progeny of donor pluripotent stem cells (PSCs) injected into the blastocyst. Blastocyst complementation has several advantages compared to in-vitro directed differentiation of stem cells, namely that development occurs in an in-vivo context. Thus, progenitor cells are exposed to all the inductive cues needed for differentiation to the appropriate cell phenotype of interest, and therefore may more faithfully recapitulate the intended cell-type specific gene networks and biomolecular characteristics of those cells. Studies have shown that this technique can be applied for CNS tissues including specific brain regions. Specifically, previous work from the Low Lab at the University of Minnesota has shown that targeting the homeobox gene HHEX establishes a niche for the formation of various organs from donor cells including liver, pancreas, and brain (Ruiz-Estevez et al., 2021). HHEX may be a viable target gene for the generation of exogenic interneurons as previous work has indicated that knockout of HHEX impairs development of the medial ganglionic eminence (MGE), a developmental structure enriched in GABAergic interneuron progenitors (Martinez-Barbera et al., 2000). In addition, many studies have demonstrated that engraftment of MGE cells can reduce cognitive and electrophysiological deficits in AD mouse models. This suggests that the transplant of exogenic interneurons may be a feasible strategy to restore interneuron activity and reduce cognitive deficits in AD. While the generation of human-animal brain chimeras is controversial, recent surveys indicate the public is amenable to the concept for research and therapeutic use (Crane et al., 2020). Thus, future translation of this approach using human-porcine chimeras may provide exogenic human interneurons to treat AD patients. This thesis will describe the scientific background and rationale for exogenic interneuron generation by HHEX KO/blastocyst complementation as a potential approach to treat AD. It will also show preliminary analysis of HHEX KO/complemented mice, and show testing of a primary antibody for Lhx6 in wild type mouse tissue prior to the antibody being used to search for donor-derived interneuron progenitors in chimeras.