TABLE 1.

Main protocols for generating kidney organoids from human pluripotent stem cells: differentiation stages and times, and characterization of final observed cells.

Kidney organoid differentiation protocols
References	Differentiation stages	Length	Cells obtained
Melissa H. Little group Takasato et al., 2014; Takasato et al., 2015; Forbes et al., 2018; Howden et al., 2019	Induction of primitive streak through CHIR99021, followed by FGF9 treatment to induce intermediate mesoderm and MM, plus 3D culture for generating organoids.	18–24 days	Proximal and distal tubule cells, podocytes, and collecting duct cells.
Nishinakamura group Taguchi et al., 2014; Taguchi and Nishinakamura, 2017; Yoshimura et al., 2019	The protocol starts with the formation of embryoid bodies in the presence of BMP4, followed by early mesoderm induction with Activin and Fgf2, and then a cocktail of BMP4, CHIR99021, Retinoic acid to induce intermediate mesoderm and MM. NPCs were then expanded with Fgf9 and CHR99021. For nephron maturation, 3D aggregation and coculture with mouse embryonic spinal cord was used. For inducing the ureteric bud specifically, after the initial mesoderm induction cells were treated with blockers of BMP/Activin signaling LDN193189 and SB43152. In both protocols UB or MM progenitors are sorted for further maturation in 3D culture.	22 days	Glomeruli with podocytes, proximal and distal tubule cells. In Taguchi and Nishinakamura, 2017, UB-derived collecting duct cells were generated.
Izpisua-Belmonte group Xia et al., 2014; Li et al., 2016; Low et al., 2019	These protocols are composed of an initial stage in monolayer with intermittent CHIR99021 treatment to induce intermediate mesoderm, followed by Fgf9 and CHIR99021 for NPCs induction. Then in a second stage, 3D culture of sorted SIX2 + NPCs with Fgf9 and CHIR is maintained, followed by basal media without growth factors. Ureteric bud differentiation is induced by monolayer culture in Fgf2 and Bmp4, followed by Bmp2, Activin A and retinoic acid.	21 days	Glomeruli with podocytes, proximal and distal tubule cells, as well as endothelial cells.
Bonventre and Freedman group Freedman et al., 2015; Morizane et al., 2015; Morizane and Bonventre, 2017	Primitive streak is induced by CHIR treatment in monolayer culture. Then Activin is added to induce intermediate mesoderm, and NPCs are expanded at the presence of Fgf9. Finally cells are aggregated and cultured for 3 days at the presence of Fgf9 and CHIR99021, and then switched to basic media without growth factors and inhibitors.	28–35 days	Podocytes, proximal and distal tubule like cells were observed.
Przepiorski et al., 2018	This protocol is based on the formation and growth of embryoid bodies from hiPSCs, in a bioreactor culture system, The only supplemented used is CHIR99021. The expansion of NPCs within the embryoid bodies is achieved with Knockout serum replacement media instead of Fgf9 as the majority of the differentiation protocols.	14–26 days	Podocytes, proximal and distal tubules, and stromal cells.