Kidney Organoids: A Translational Journey Ryuji Morizane, Joseph V. Bonventre  Trends in Molecular Medicine  Volume 23, Issue 3, Pages 246-263 (March 2017) DOI: 10.1016/j.molmed.2017.01.001 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Mesoderm Patterning from the Primitive Streak in Mammals. The location of cells in the primitive streak defines the subsequent differentiation into paraxial mesoderm, intermediate mesoderm (IM), and lateral plate mesoderm. The early-stage primitive streak cells migrate anteriorly and form mesoderm tissues at the anterior part of the embryo, while the late-stage primitive streak cells form the posterior mesoderm. The metanephros (containing nephron progenitor cells, NPCs) is derived from the late-stage mid primitive streak cells. Trends in Molecular Medicine 2017 23, 246-263DOI: (10.1016/j.molmed.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Comparison of Human Nephron Progenitor Cell Differentiation Protocols. The schematic summarizes recent protocols that have been used to differentiate nephron progenitor cells from hPSCs. Inducing factors, concentrations, and treatment duration in each step of differentiation protocols are shown with colored boxes. The culture method (the suspension or adhesion culture) is indicated at the bottom of each protocol. The cell types induced by each step of differentiation are shown on the top of the protocols. Abbreviations: CHIR, CHIR99021; BMP, bone morphogenetic protein; DMH1, 4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline, 4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline, a selective inhibitor of BMP ALK2 receptor; FBS, fetal bovine serum; FGF, fibroblast growth factor; HOXD11, homeobox D11; IM, intermediate mesoderm; KSR, knockout serum replacement; OSR1, odd-skipped related transcription factor 1; PAX2, paired box 2; RA, retinoic acid; SALL1, spalt-like transcription factor 1; SIX2, SIX homeobox 2; TGF, transforming growth factor; TTNPB, 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid, a retinoic acid receptor agonist; WNT3A, wingless-type MMTV integration site family, member 3A; WT1, Wilms tumor 1; Y27632, a selective p160ROCK inhibitor. Trends in Molecular Medicine 2017 23, 246-263DOI: (10.1016/j.molmed.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Key Figure: Methods for Generating Kidney Organoids The diagram depicts recent protocols used to generate kidney organoids from human pluripotent stem cells. Taguchi [6], Morizane [3] and colleagues generated kidney organoids from nephron progenitor cells (NPCs) while Freedman [19], Takasato [7] and colleagues differentiated mesenchyme cells or intermediate mesoderm cells into kidney organoids. Red lines, podocyte-like cells; light-blue lines, proximal tubule-like cells; yellow lines, loops of Henle-like cells; light-green lines, distal nephron-like cells. Abbreviations: AQP1, aquaporin 1; CD31, cluster of differentiation 31; CDH1, cadherin 1; CDH2, cadherin 2; CDH6, cadherin 6; CHIR, CHIR99021; CUBN, cubilin; FGF, fibroblast growth factor; GATA3, GATA binding protein 3; GF, growth factor; hiPSCs, human induced pluripotent stem cells; LAM, laminin; LHX1, LIM homeobox 1; LTL, lotus tetragonolobus lectin; LRP2, LDL receptor-related protein 2; NPCs, nephron progenitor cells; NPHS1, nephrosis 1, nephrin; PAX2, paired box 2; PAX8, paired box 8; PODXL, podocalyxin-like; SALL1, spalt-like transcription factor 1; SYNPO, synaptopodin; TUJ1, neuron-specific class 3 β-tubulin; UMOD, uromodulin; WT1, Wilms tumor 1; vWF, von Willebrand factor. Trends in Molecular Medicine 2017 23, 246-263DOI: (10.1016/j.molmed.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Human Kidney Organoids in Culture. Representative micrograph images of human kidney organoids in 3D culture. (A) Human kidney organoids were generated in 96-well ultra-low attachment plates with U-shaped bottom on day 24 after initiation of differentiation. Scale bar, 500μm. Images were taken with an inverted microscope (Nikon, Eclipse Ti) at 40×magnification. (B) Human kidney organoids were generated in six-well plates with micro-space (Elplasia, #RB900700NA6) on day 22 after initiation of differentiation. Scale bars, 5mm (left) and 1mm (right). Photographs were taken with an inverted microscope (Nikon, Eclipse Ti) at 40×magnification. The left picture was generated by merging 40×pictures to show a single well of a six-well plate. Trends in Molecular Medicine 2017 23, 246-263DOI: (10.1016/j.molmed.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions