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Steroid-resistant nephrotic syndrome associated MYO1E mutations have differential effects on myosin 1e localization, dynamics, and activity

ABSTRACT: Patients deficient in myosin 1e and mice in which Myo1e is knocked out develop Focal Segmental Glomerlosclerosis. To study the impact of myo1e deficiency on cultured podocytes, we derived conditionally immortalized glomerular epithelial cells from crosses between the Myo1e-KO mice and a H-2Kb-tsA58 mice (Immortomouse) to obtain Myo1e- heterozygous and Myo1e-KO podocyte-derived cells. The purpose of this study was to analyze the transcriptome differences between wild-type Murine Podocyte Cells (MPC) originally described in PMID 9344605 and mantained in our lab for several passages, Myo1e Heterozygous cells, and Myo1e KO cells. RNAseq and differential gene expression analysis was performed.

ORGANISM(S): Mus musculus

PROVIDER: GSE202684 | GEO | 2022/08/24

REPOSITORIES: GEO

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Project description:Interstitial cells of Cajal (ICC) are electrical pacemakers and mediators of neuromuscular neurotransmission in the gastrointestinal tract. Studies in organotypic cultures of intact gastric muscular wall tissues identified Kit(low)Cd44(+)Cd34(+)Insr(+)Igf1r(+) cells as local ICC progenitors (Lorincz et al., Gastroenterology 2008;134:1083-1093). Subsequently, conditionally immortalized Kit(low)Cd34(+) cells were isolated by immunomagnetic and fluorescent cell sorting and serial cloning from the gastric muscular wall of homozygous, 14-day-old H-2Kb-tsA58 mice and shown to share other phenotypic characteristics with in-situ ICC precursors (2xSCS70 cells; Bardsley et al., Gastroenterology 2010;139:942-952). 2xSCS70 cells were also found to express the ICC marker Ano1, be able to extensively self-renew in the absence of the SV40 tsA58-TAg when cultured in NeuroCultÂ® Neural Stem Cell Proliferation Medium (NSCPM; STEMCELL Technologies) and differentiate into Kit(+) ICC both in vitro and in vivo. Wild-type cells with Kit(low)Ano1(+)Cd44(+)Cd34(+)Insr(+)Igf1r(+) phenotype (2xSCS2F10 cells) were prospectively isolated from primary cultures of dissociated gastric muscular wall tissues from 7-day-old C57BL/6J mice by serial cloning following 150-day expansion in NSCPM containing 5% cell-free chick embryo extract (CEE; Accurate Chemical). Long-term culturing of both cell lines was associated with further decline in Kit expression while expression of Ano1 and other markers of ICC precursors was maintained. Following spontaneous transformation, both 2xSCS70 and 2xSCS2F10 cells grafted into nude mice gave rise to tumors containing Kit(+)cells and otherwise resembling gastrointestinal stromal tumors (GIST), which originate from the ICC lineage (Bardsley et al., Gastroenterology 2010;139:942-952). This study utilized oligonucleotide microarray analysis to charaterize the transcriptome of non-transformed 2xSCS70 and 2xSCS2F10 cells relative to their source tissues. Total RNA from ICC progenitor cell lines and their source tissue (gastric tunica muscularis from C57BL/6J mice (source of 2xSCS2F10 cells) and H-2Kb-tsA58 mice (source of 2xSCS70 cells) was isolated. Oligonucleotide micorarray studies were performed using 2-3 Affymetrix Mouse Genome 430.2 GeneChips per sample.

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Steroid-resistant nephrotic syndrome associated MYO1E mutations have differential effects on myosin 1e localization, dynamics, and activity

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