Browsing by Subject "Cell therapy"

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    Gene Correction of Limb Girdle Muscular Dystrophy Type 2A Patient-Specific Induced Pluripotent Stem Cells
    (2019-09) Selvaraj, Sridhar
    Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes invariably display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease typically does not manifest in the embryonic muscle, but at more mature stages. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by increased expression of fetal, neonatal and adult myosin heavy-chain isoforms. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle bundles, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells. We applied this maturation model for in vitro validation of Calpain 3 (CAPN3) protein expression. CAPN3 mutations are associated with Limb Girdle Muscular Dystrophy type 2A (LGMD2A), which is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. Using a gene knock-in approach, here we applied CRISPR-Cas9 mediated genome editing to induced pluripotent stem (iPS) cells from three LGMD2A patients carrying three different CAPN3 mutations, to enable correction of mutations in the CAPN3 gene. CAPN3 protein rescue upon gene correction was validated in myotube-derivatives in vitro following the small molecule treatment. Transplantation of gene corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof concept for the integration of genome editing and iPS cell technologies to develop a novel autologous cell therapy for LGMD2A.
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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.