Characterization of Human iPSC RET Reporter Cell Line Differentiation to Kidney and Neural Crest Lineages
Authors: Salamon Rebecca Jean , Gong Bendi , Jain Sanjay 
Published: June 2021

Worldwide, congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of chronic renal disease in children and play a significant causative factor in pediatric end‐stage renal disease. Current research being conducted is reliant upon animal model systems to monitor embryonic kidney development; however, a system modeling the human kidney organogenesis is a necessity in order to elucidate the intertwined genetic and molecular networks related to CAKUT pathogenesis.One known genetic correlation to CAKUT is the mutation within the gene encoding for the RET receptor tyrosine kinase, which plays a crucial role in kidney development. Mutations within the gene encoding for RET affect cellular pathways correlated to congenital anomalies such as Hirschsprung's disease and renal agenesis, which are due to the maldevelopment of neural crest cells and kidney progenitor cells, respectively. These defects indicate that RET plays a unique role in cellular mechanisms related to the kidney and ureter maturation, allowing for RET to be utilized as a renal biomarker. Monitoring RET protein expression allows for the efficiency of differentiation protocols to generate cells of kidney and neural crest lineages to be validated. Furthermore, utilizing RET as a renal biomarker could elucidate signaling molecules needed for multipotent stem cell development into a complex urinary system.There is a necessity to characterize the proficiency of protocols for Human Induced Pluripotent Stem Cells (hiPSCs) differentiation into kidney organoid and Neural Crest Stem Cells (NCSCs), in order to advance the methods for obtaining functioning kidney lineages. A novel hiPSC RET reporter cell line was created via CRISPR‐Cas9 technology to determine the efficacy of current differentiation protocols. The hiPSC RET reporter cell line was an invaluable asset to the protocol characterization process due to the role of RET in collecting duct and enteric nervous system lineages. With the ability to detect RET utilizing the fluorescent marker, as well as via immunohistochemistry, the ability to produce ureteric bud progenitor cells and NCSCs was able to be reliably verified. The RNA and protein product was analyzed via qPCR and immunofluorescence microscopy, respectively, for the presence of several renal precursor and NCSC biomarkers.Analyzation of the RNA throughout differentiation provided strong evidence for the generation of kidney progenitor cells and NCSCs. Moreover, antibody staining of the kidney organoids and neural crest cells revealed the presence of protein biomarkers produced by ureteric bud and neuronal precursor cells, respectively. Kidney organoids and neural crest cells are a promising paradigm to monitor the pathogenesis of CAKUT. Currently, a model system of the human embryonic kidney development relies on discovery of additional biomarkers in order to verify cell identity. Ultimately, the potential of having hiPSCs as a renewable resource to produce NCSCs and organoids has clinical applications in drug screening, disease modeling, and stem cell therapies – all which may lead to a novel cure for CAKUT and neurocristopathies.Support or Funding InformationAmgen Scholars Program at Washington University in St. LouisThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.