Browsing by Subject "Induced pluripotent stem cells"

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    Effect of Medium Supplements on Mesenchymal Stromal Cell Development and Permissiveness to Transformation by EWSR1-FLI1 Fusion Protein
    (2020-01) Lan, Tianxia
    Ewing sarcoma is the second most frequent childhood bone tumor and is characterized by FET-ETS translocations. In ~ 85% of cases, the translocation fuses EWSR1 (a member of the FET gene family) with FLI1 (a member of the ETS family). Accurate models are required to study the mechanisms underlying tumorigenesis driven by EWSR1-FLI1, however no such models exist for Ewing sarcoma at present. The main obstacle to establishing a model for Ewing sarcoma is that the cell of origin remains undefined. A growing amount of evidence suggests that Ewing sarcoma originates from a small subset of mesenchymal stromal cells (MSC) permissive to the normally toxic EWSR1-FLI1 fusion protein. Characterizing this permissive subset is hindered by the substantial heterogeneity of MSCs. We sought to address this problem using induced pluripotent stem cells as a model system. It has been shown that some cytokines are capable of influencing the differentiation from iPSCs to MSCs, and the portion of different subpopulations of MSCs can be altered by changing the combination of cytokines that are involved in the differentiation. Herein, we characterized MSC derived from iPSC treated with different combinations of cytokines during differentiation with the ultimate goal of characterizing specific subsets of MSC that are permissive to EWSR1-FLI1.
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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.