Multivariate patterning of human pluripotent cells under perfusion reveals critical roles of induced paracrine factors in kidney organoid development

Abstract

Creating complex multicellular kidney organoids from pluripotent stem cells shows great promise. Further improvements in differentiation outcomes, patterning, and maturation of specific cell types are, however, intrinsically limited by standard tissue culture approaches. We describe a novel full factorial microbioreactor array-based methodology to achieve rapid interrogation and optimization of this complex multicellular differentiation process in a facile manner. We successfully recapitulate early kidney tissue patterning events, exploring more than 1000 unique conditions in an unbiased and quantitative manner, and define new media combinations that achieve near-pure renal cell type specification. Single-cell resolution identification of distinct renal cell types within multilayered kidney organoids, coupled with multivariate analysis, defined the definitive roles of Wnt, fibroblast growth factor, and bone morphogenetic protein signaling in their specification, exposed retinoic acid as a minimal effector of nephron patterning, and highlighted critical contributions of induced paracrine factors on cell specification and patterning.

Fig. 1. A schematic of the early developing nephron and some key cell types: Schematic of nephron development.

A small budding region begins to form on the UE (blue-green). MM (red) begins to condense into clusters around the budding UE structures. These clusters of MM begin forming comma-like structures and begin to show distal (EDN, blue) and proximal polarization (EPN, magenta). This structure migrates and merges with the tubular structure of the collecting duct. Stromal cells (SM, green) also begin to appear as a step toward vascularization.

Fig. 2. Day 12 end point MBA stained for GATA3, WT1, and ECAD with the factorial input combination of FGF9, BMP7, and RA.

(A) The experimental time course setup for the day 12 bioreactors. Here, cells were cultured in well plates for the first 6 days and in the MBA system for the following 6 days and stained for DNA (white), GATA3 (green), WT1 (red), and ECAD (blue). (B) A representation of two of the kidney cell subtypes being investigated: the MM (WT1⁺) and the UE (GATA3⁺ and ECAD⁺) and their immunofluorescent representation. (C) The input of the MBA and subsequent immunofluorescence of each well. Even at a mesoscale view, it is clear that FGF9 is critical in the differentiation of IM cells toward kidney lineages. Furthermore, several cellular assemblies can be seen, which change in organization depending on the position within the MBA. (D) A zoomed-in view of nine wells along the fourth row of wells representing the intermediate concentration of FGF9 [red box shown in (C)]. Here, the effect of BMP7 and RA can be seen, which causes the condensation of the UE in tightly packed structures. (E). A zoomed-in view of nine sequential wells representing the intermediate concentration of FGF9, BMP7, and RA [blue box shown in (C)]. Here, similar changes in structures condensation of structures can be viewed as paracrine and autocrine factor accumulation occurs.

Fig. 3. Image cytometry comparison for the first row of the day 9 and day 12 bioreactors.

(A) The immunofluorescent imaging of the first row of a day 9 MBA array with the variation of FGF9, BMP7, and RA as indicated and the corresponding image cytometry. Wells are stained with GATA3 (green), WT1 (red), and ECAD (blue). Here, the UE (GATA3⁺ECAD⁺), MM (WT1⁺), EPNs (WT1⁺ECAD⁺), EDNs (ECAD⁺), and mesangial cells (GATA3⁺) can be identified, and small perturbations in the relative amounts of each phenotype can be seen. (B) The immunofluorescent imaging of the first row of a day 12 MBA array with the variation of FGF9, BMP7, and RA as indicated and the corresponding image cytometry with immunostaining as above.

Fig. 4. Pareto chart of the standard effects and main effects plots from a full factorial design of experiment multivariate analysis day 12 MBAs.

Pareto charts of all factors (row number, FGF9, BMP7, and RA) and their combinations and main effects of each input factor for the day 12 MBAs (n = 4). Phenotypical cell assessment included the cell count (A), UE (B), MM (C), EPN (D), EDN (E), and SM (F). The dashed line on each Pareto chart represents (P = 0.05), whereas the dashed line on the main effect plots represents the global mean of that parameter. *P < 0.5, **P < 0.01, and ***P < 0.001. ns, not significant.

Fig. 5. Analysis of the effect of continued Wnt signaling and RA activity after variation in the initial duration of canonical Wnt patterning (4 days of initial CHIR, day 4 to 9 MBAs).

(A) Experimental plan for 4-day CHIR induction with 5-day factorial analysis (day 4 to 9 MBAs). FGF9 was present across the entire 5-day period during the factorial perfusion. (B) The working concentrations of CHIR, AGN, and RA used for both MBA experiments (day 3 to 8 and day 4 to 9). (C) The full scan of each well of day 4 to 9 MBAs stained for GATA3 (green), WT1 (red), and ECAD (blue). (D) Representative wells from row 3 representing the effect of CHIR and RA with constant AGN. These wells are highlighted about in red on the overall scan. (E) Representative wells from column 14 representing the induced paracrine signaling. These wells are highlighted in blue on the overall scan.

Fig. 6. Main effects plots from a full factorial design of experiments multivariate analysis for day 3 to 8 and day 4 to 9 MBAs.

Pareto charts of all factors (row number, FGF9, BMP7, and RA) and their combinations and main effects of each input factor for the day 4 to 9 MBAs (n = 4). Phenotypical cell assessment included the cell count (A), UE (B), MM (C), EPN (D), EDN (E), and SM (F). The dashed line on each Pareto chart represents (P = 0.05), whereas the dashed line on the main effect plots represents the global mean of that parameter. *P < 0.5, **P < 0.01, and ***P < 0.001.