Browsing by Author "Tutrow, Kaylee D."

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    A highly reproducible and efficient method for retinal organoid differentiation from human pluripotent stem cells
    (National Academy of Sciences, 2024) Harkin, Jade; Peña, Kiersten H.; Gomes, Cátia; Hernandez, Melody; Lavekar, Sailee S.; So, Kaman; Lentsch, Kelly; Feder, Elyse M.; Morrow, Sarah; Huang, Kang-Chieh; Tutrow, Kaylee D.; Morris, Ann; Zhang, Chi; Meyer, Jason S.; Pharmacology and Toxicology, School of Medicine
    Human pluripotent stem cell (hPSC)-derived retinal organoids are three-dimensional cellular aggregates that differentiate and self-organize to closely mimic the spatial and temporal patterning of the developing human retina. Retinal organoid models serve as reliable tools for studying human retinogenesis, yet limitations in the efficiency and reproducibility of current retinal organoid differentiation protocols have reduced the use of these models for more high-throughput applications such as disease modeling and drug screening. To address these shortcomings, the current study aimed to standardize prior differentiation protocols to yield a highly reproducible and efficient method for generating retinal organoids. Results demonstrated that through regulation of organoid size and shape using quick reaggregation methods, retinal organoids were highly reproducible compared to more traditional methods. Additionally, the timed activation of BMP signaling within developing cells generated pure populations of retinal organoids at 100% efficiency from multiple widely used cell lines, with the default forebrain fate resulting from the inhibition of BMP signaling. Furthermore, given the ability to direct retinal or forebrain fates at complete purity, mRNA-seq analyses were then utilized to identify some of the earliest transcriptional changes that occur during the specification of these two lineages from a common progenitor. These improved methods also yielded retinal organoids with expedited differentiation timelines when compared to traditional methods. Taken together, the results of this study demonstrate the development of a highly reproducible and minimally variable method for generating retinal organoids suitable for analyzing the earliest stages of human retinal cell fate specification.
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    Heart failure is not a determinant of central sleep apnea in the pediatric population
    (Wiley, 2021) Wheeler, Jonathan A.; Tutrow, Kaylee D.; Ebenroth, Eric S.; Gaston, Benjamin; Bandyopadhyay, Anuja; Medical and Molecular Genetics, School of Medicine
    Background/objectives: Adults with heart failure (HF) have high prevalence of central sleep apnea (CSA). While this has been repeatedly investigated in adults, there is a deficiency of similar research in pediatric populations. The goal of this study was to compare prevalence of CSA in children with and without HF and correlate central apneic events with heart function. Methods: Retrospective analysis of data from children with and without HF was conducted. Eligible children were less than 18 years old with echocardiogram and polysomnogram within 6 months of each other. Children were separated into groups with and without HF based on left ventricular ejection fraction (LVEF). Defining CSA as central apnea-hypopnea index (CAHI) more than 1/hour, the cohort was also classified into children with and without CSA for comparative study. Results: A total of 120 children (+HF: 19, -HF: 101) were included. The +HF group was younger, with higher prevalence of trisomy 21, muscular dystrophy, oromotor incoordination, and structural heart disease. The +HF group had lower apnea-hypopnea index (median: 3/hour vs. 8.6/hour) and lower central apnea index (CAI) (median: 0.2/hour vs. 0.55/hour). Prevalence of CSA was similar in both groups (p = .195). LogCAHI was inversely correlated to age (Pearson correlation coefficient: -0.245, p = .022). Children with CSA were younger and had higher prevalence of prematurity (40% vs. 5.3%). There was no significant difference in LVEF between children with and without CSA. After excluding children with prematurity, relationship between CAHI and age was no longer sustained. Conclusions: In contrast to adults, there is no difference in prevalence of CSA in children with and without HF. Unlike in adults, LVEF does not correlate with CAI in children. Overall, it appears that central apneic events may be more a function of age and prematurity rather than of heart function.
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    Utilization of a Human Induced Pluripotent Stem Cell Model of Microglial Fractalkine Signaling Dysfunction in Alzheimer's Disease
    (2025-03) Tutrow, Kaylee D.; Meyer, Jason; Bissel, Stephanie; Kim, Jungsu; Lasagna-Reeves, Cristian
    Dysfunctional microglial activity has been identified as a potential mechanism leading to accumulation of amyloid beta and pTau and subsequent neurodegeneration in Alzheimer's Disease (AD). The CX3CR1/fractalkine axis serves as a mechanism for bidirectional communication between microglia and neurons, respectively, to promote an anti-inflammatory microglial state. Previous studies have demonstrated that deficiency in CX3CR1 signaling leads microglia to develop a more pro-inflammatory phenotype, induces phagocytic deficits, and increases susceptibility of neurons to cell death. The CX3CR1-V249I polymorphism was recently identified as a potential risk allele for AD. However, the role offractalk:ine dysfunction in human cells and the mechanisms by which microglia with the CX3CR1-V249I SNP contribute to neurodegeneration remain unclear. To address this shortcoming, we utilized human induced pluripotent stem cells and CRISPR/Cas9 technology to elucidate the effects of the V249I polymorphism on human microglia-like cells (hMGLs). We demonstrate effective differentiation from paired isogenic control and CX3CR1-V249I backgrounds into hMGLs. Transcriptional profiling via RNA-seq analyses demonstrated alterations in pathways such as apoptosis, toll-like receptor signaling, and the inflammasome due to the CX3CR1-V249I SNP. Both heterozygous and homozygous microglia bearing the V249I allele demonstrated decreased phagocytosis of amyloid beta in vitro compared to controls, with this effect modulated by the presence of fractalkine in heterozygous but not homozygous V249I hMGLs. Both heterozygous and homozygous V249I microglia exhibited increased stress-induced cell death compared to controls, with homozygous hMGLs demonstrating increased cell death at earlier time points. These findings suggest that the CX3CR1-V249I polymorphism may confer a dysfunctional microglia phenotype, which may subsequently contribute to neuronal dysfunction. Further investigation of microglia in neuron co-culture models demonstrated that the CX3CR1-V249I variant conferred altered neuronal excitability. Collectively, the results of this study highlight the importance of understanding CX3CR1 function in AD pathology to identify targetable mechanisms for intervention.