Jaap Mulder1, Sazia Sharmin2, Theresa Chow3,4, Deivid Carvalho Rodrigues2, Matthew R Hildebrandt2, Robert D'Cruz2,5, Ian Rogers3,4,6, James Ellis2,7, Norman D Rosenblum8,9,10. 1. Division of Nephrology, Department of Pediatrics, The Hospital for Sick Children, Toronto, ON, Canada. 2. Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada. 3. Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, ON, Canada. 4. Department of Physiology, University of Toronto, Toronto, ON, Canada. 5. Faculty of Medicine, University of Toronto, Toronto, ON, Canada. 6. Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada. 7. Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. 8. Division of Nephrology, Department of Pediatrics, The Hospital for Sick Children, Toronto, ON, Canada. norman.rosenblum@sickkids.ca. 9. Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada. norman.rosenblum@sickkids.ca. 10. Faculty of Medicine, University of Toronto, Toronto, ON, Canada. norman.rosenblum@sickkids.ca.
Abstract
BACKGROUND: Human induced pluripotent stem cells (iPSCs) are a promising tool to investigate pathogenic mechanisms underlying human genetic conditions, such as congenital anomalies of the kidney and urinary tract (CAKUT). Currently, iPSC-based research in pediatrics is limited by the invasiveness of cell collection. METHODS: Urine cells (UCs) were isolated from pediatric urine specimens, including bag collections, and reprogrammed using episomal vectors into urinary iPSCs (UiPSCs). Following iPSC-quality assessment, human kidney organoids were generated. RESULTS: UCs were isolated from 71% (12/17) of single, remnant urine samples obtained in an outpatient setting (patients 1 month-17 years, volumes 10-75 ml). Three independent UCs were reprogrammed to UiPSCs with early episome loss, confirmed pluripotency and normal karyotyping. Subsequently, these UiPSCs were successfully differentiated into kidney organoids, closely resembling organoids generated from control fibroblast-derived iPSCs. Importantly, under research conditions with immediate sample processing, UC isolation was successful 100% for target pediatric CAKUT patients and controls (11/11) after at most two urine collections. CONCLUSIONS: Urine in small volumes or collected in bags is a reliable source for reprogrammable somatic cells that can be utilized to generate kidney organoids. This constitutes an attractive approach for patient-specific iPSC research involving infants and children with wide applicability and a low threshold for participation.
BACKGROUND:Human induced pluripotent stem cells (iPSCs) are a promising tool to investigate pathogenic mechanisms underlying human genetic conditions, such as congenital anomalies of the kidney and urinary tract (CAKUT). Currently, iPSC-based research in pediatrics is limited by the invasiveness of cell collection. METHODS: Urine cells (UCs) were isolated from pediatric urine specimens, including bag collections, and reprogrammed using episomal vectors into urinary iPSCs (UiPSCs). Following iPSC-quality assessment, human kidney organoids were generated. RESULTS: UCs were isolated from 71% (12/17) of single, remnant urine samples obtained in an outpatient setting (patients 1 month-17 years, volumes 10-75 ml). Three independent UCs were reprogrammed to UiPSCs with early episome loss, confirmed pluripotency and normal karyotyping. Subsequently, these UiPSCs were successfully differentiated into kidney organoids, closely resembling organoids generated from control fibroblast-derived iPSCs. Importantly, under research conditions with immediate sample processing, UC isolation was successful 100% for target pediatric CAKUT patients and controls (11/11) after at most two urine collections. CONCLUSIONS: Urine in small volumes or collected in bags is a reliable source for reprogrammable somatic cells that can be utilized to generate kidney organoids. This constitutes an attractive approach for patient-specific iPSC research involving infants and children with wide applicability and a low threshold for participation.
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