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Open Access Publications from the University of California

Scholarly Works (2 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2023)

Usher Syndrome 1B (USH1B) is a severe genetic disorder characterized by congenital deafness and progressive vision loss due to retinitis pigmentosa. The role of myosin VIIa in the retinal pigment epithelium (RPE) is critical for photoreceptor function and survival, and mutations in the myosin VIIa gene (MYO7A) are the primary cause of USH1B. This work aims to advance therapeutic tools for USH1B and introduce tools that shed light on the functional roles of myosin VIIa within the RPE. We employed novel gene editing tools to correct Myo7a mutations using an engineered cell line. This cell line can be used as a cost-effective high-throughput approach for screening guide RNA sequences for producing precise gene edits. Additionally, we developed and optimized gene vectors for efficient delivery of the base editing system to disease models. This work focused on restoring functional myosin VIIa protein expression in the shaker-1 mouse, an animal model of USH1B. Our results highlight the promise of base editing for advancing approaches in gene therapy, and introduce a novel set of tools for better exploring the biological roles of myosin VIIa.

    Cover page: Advancing Tools for Treating Usher Syndrome 1B and Elucidating the Role of Myosin VIIa in the Retinal Pigment Epithelium
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2022)

    Aims

    We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations.

    Methods and results

    Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively.

    Conclusions

    We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects.
      Cover page: Isolation and characterization of human embryonic stem cell-derived heart field-specific cardiomyocytes unravels new insights into their transcriptional and electrophysiological profiles
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