ISOLATION AND REPROGRAMMING OF URINARY PROGENITOR CELLS FROM A PATIENT WITH MUTATION IN GENE CYFIP2 THAT CAUSES INTELLECTUAL DISABILITY

Abstract

<h3>Background</h3> West syndrome (WS) is an infantile neural disorder that causes a delay in development and infantile spasms. Mutations in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene were recently described as possible causes of WS. CYFIP2 has a role in actin polymerization through WAVE regulatory complex (WRC), especially on dendritic spines and synaptosomes. To study the molecular mechanism of mutation in CYFIP2 and to propose a possible therapy, this study aimed to establish a cell model based on induced pluripotent stem cells (iPSC), through a non-invasive cell sample collected from the patient and its subsequent reprogramming. <h3>Methods</h3> The urine sample was collected for a 2 years old Brazilian female patient characterized with the R87C variant of CYFIP2, with the parent's consent. For isolation, the sample was washed, centrifuged, and the cellular pellet was cultivated until confluence. For reprogramming, the episomal vectors expressing hSK, hOCT4, shp53, hUL, and EBNA1 were electroporated in cells by Nucleofector. Cells were cultivated until reprogrammed cell colonies appeared. Colonies were labeled with anti-TRA-1-60 and anti-TRA-1-81 antibodies on days 15 and 24 to follow up. <h3>Results</h3> Isolation and expansion of urine-derived cells lasted 21 days and based on its morphology and growth rate, cells were classified as type II urinary progenitor cells (UPC), according to the literature. After 15 days of reprogramming, pluripotent stem cells-like colonies can be visualized in culture. The TRA-1-60 and TRA-1-81 markers were even more evident in colonies on day 24, compared to day 15. On day 26 after reprogramming, colonies were manually harvested, and the cells were expanded until confluence. <h3>Conclusions</h3> The isolation and reprogramming of UPC collected from a patient with the CYFIP2 R87C variant were possible, even with the possibility of CYFIP2 mutation impaired cell growth or reprogramming. Also, the reprogrammed cells expressed two of the pluripotent markers, TRA-1-60 and TRA-1-81. The next step is the characterization of the reprogrammed cells, through expression analysis for different pluripotent markers, differentiation in the three germ layers, and karyotype. After the establishment of the iPSC as a study model for CYFIP2, cells will be differentiated into neural cells, making possible the study of the molecular mechanisms and drug screening.