Creating a kidney in vitro is challenging, as kidney development involves complex interactions between nephron progenitor cells, stromal progenitor cells and the precursors of the collecting duct system (that is, the ureteric bud and its precursor, the Wolffian duct). Previous studies using dissociated cells from the embryonic mouse kidney successfully recapitulated kidney structure in vitro but, in studies using pluripotent stem cells (PSCs), nephrons were not correctly organized in 3D by the collecting duct system owing to the lack of a method to induce a functional ureteric bud. Now, Taguchi and Nishinakamura have established conditions under which mouse embryonic stem cells (mESCs) differentiate into two of the three progenitors required to generate kidney organoids in vitro, which, together with embryo-derived stromal progenitors, recapitulate embryonic kidney architecture.
To identify cues that induce differentiation of the ureteric bud cell lineage in normal development, the researchers carried out gene expression array analyses of Wolffian duct maturation (embryonic day (E) 8.75–E10.5) and development of the ureteric bud from the Wolffian duct (E10–E11.5). Components of the retinoic acid, WNT and fibroblast growth factor (FGF) signalling pathways were upregulated in the developing Wolffian duct in vivo — using these molecules, together with glial cell line-derived neurotrophic factor (GDNF; an established ureteric bud attractant), the researchers successfully differentiated mouse embryo-derived Wolffian duct progenitor cells (at E8.75) into E11.5 ureteric bud-like tissue in vitro.
Next, the researchers identified CXC-chemokine receptor 4 (CXCR4) and KIT from their in vivo expression array data as molecules that are specifically expressed on the cell surface of committed Wolffian duct progenitor cells (at E8.75). They then used these markers to optimize the growth factors required to differentiate mESCs into the epiblast/mesoderm (which include activin, bone morphogenetic protein (BMP) and WNT) and the source of the Wolffian duct progenitors, the intermediate mesoderm (which include retinoic acid, FGF9 and transforming growth factor-β (TGFβ) inhibitor). Applying these factors, followed by the Wolffian duct progenitor maturation factors (retinoic acid, WNT, FGF9 and GDNF), to mESCs induced their differentiation into ureteric buds. Additionally, this combined induction protocol that they developed in mice could successfully induce ureteric bud formation by human induced PSCs (iPSCs).
“integration of induced ureteric buds into the kidney organoid ... enabled the reconstitution of organotypic architecture”
Importantly, the researchers show that “ureteric buds induced from mESCs and human iPSCs can develop branching ureteric epithelium. Furthermore, the mESC-derived ureteric buds fulfil innate functional criteria, including the capacity to induce differentiation of nephron progenitor cells, but also to maintain their progenitor state,” explains lead author Atsuhiro Taguchi. “Indeed, integration of induced ureteric buds into the kidney organoid (together with induced nephron progenitor cells and embryo-derived stromal progenitor cells) enabled the reconstitution of organotypic architecture — namely, the higher-order structure of the embryonic kidney, which has never been obtained before. Our selective induction study also suggests that nephron progenitor cells and the ureteric bud acquire mutually distinct cellular characteristics from a very early stage of kidney development. Our method is also an important tool to investigate the lineage-specific roles of genes involved in human kidney development.”
In future studies, the authors are hoping to “induce the third renal progenitor, stromal progenitor cells, from mESCs and human iPSCs,” concludes Ryuichi Nishinakamura. “We want to generate a human kidney (not an 'organoid') that we can transplant into an animal model to test its functionality.”
Taguchi, A. & Nishinakamura, R. Higher-order kidney organogenesis from pluripotent stem cells. Cell Stem Cell http://dx.doi.org/10.1016/j.stem.2017.10.011 (2017)
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