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Developing Renal Organoids Using Human Embryonic Stem Cells
Author(s) -
Torres Collazo Bryan J.,
CebrianLigero Cristina
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.877.9
Subject(s) - organoid , mesenchyme , embryonic stem cell , microbiology and biotechnology , stem cell , biology , mesoderm , cellular differentiation , embryoid body , regenerative medicine , adult stem cell , mesenchymal stem cell , genetics , gene
Stem cells have the remarkable potential to develop into all the different cell types in the body. They are unspecialized cells capable of self‐renewing and, under certain physiologic or experimental conditions, they can be induced to become tissue‐ or organ‐specific cells with unique functions. Human Embryonic Stem Cells (hESCs) are derived from the inner cell mass of a blastocyst and can be induced to differentiate into specialized cell types with defined culture conditions. This ability to grow human tissues from stem cells in culture has the potential to revolutionize the drug discovery process and regenerative medicine. One of these approaches relies in the generation of organoids from hESCs. An organoid is 3‐dimensional structure that mimics the cellular composition and function of the organ of study. The mammalian kidney is derived from the intermediate mesoderm (IM). During embryonic development, the IM differentiates into the ureteric bud(UB) epithelia and into the metanephric mesenchyme. The UB will branch and become the renal collecting system while the mesenchyme will epithelialize and differentiate into nephrons. The primary goal of this work is to differentiate UM63.1 hESCs into renal organoids, specifically organoids composed of UB epithelia. Several signaling pathways have been identified as critical for mouse kidney development, and specifically for UB epithelial growth. We hypothesized that by recapitulating those signaling pathways in culture, UM63.1 cells can be induced to differentiate into kidneys organoids. Our differentiation protocol can be divided into two phases. On phase one cells are grown as a monolayer and treated with decreasing concentrations of an agonist (CHIR99021) to induce intermediate mesoderm. After 7 days in culture, clusters of cells (spheroids) develop. On phase two we growth those spheroids in a 3‐dimensional matrix in the presence of fibroblast growth factors (FGFs) 1 and 9, glial cell‐line derived neurotrophic factor (GDNF) and Retinoic acid. Previous research has shown that these growth factors are involved in mouse embryonic kidney development. After a few days in culture, the spheroids grow into structures resembling kidney organoids. We have imaged the progression of these organoids over time and we have also conducted immunofluorescence assays to detect markers specific for the renal ureteric bud. Our experiments demonstrate that the UM63.1 hESC‐derived organoids are positive for the following UB‐specific markers: TROMA, PAX2 and GATA3 indicating their identity as UB epithelia.