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.

A Personalized Glomerulus Chip Engineered from Stem Cell-Derived Epithelium and Vascular Endothelium

“…During glomerulogenesis, VEGF-A is secreted by the metanephric mesenchyme to direct the migration of endothelial cells towards the developing glomerulus [ 36 ]. Mature kidney podocytes produce VEGF-A throughout their life span to help maintain homeostasis and the function and phenotype of the capillary endothelial cells [ 37 , 38 ]. We quantified VEGF-A secretion from the differentiated podocytes, and found significantly high levels of VEGF-A (VEGF-165 isoform) secretion ( p = 0.0169), with the most optimal secretion timepoint occurring on day 23 ( Figure 2 e).…”

Enhancing Kidney Vasculature in Tissue Engineering—Current Trends and Approaches: A Review

“…In addition to the vascularization of organoids, microfluidics has the potential to further optimize the organoid differentiation protocol and mass produce highly controlled organoids in vitro. Glass et al recently optimized a microbioreactor procedure for generating kidney organoids from hPSCs under perfusion [ 88 ]. The microfluidic device was fabricated by soft lithography on silicon wafers using layers of polydimethylsiloxane (PDMS), and organoids were developed from human ESCs.…”

Kidney Organoids and Tubuloids

“…Another approach is organoid implementation in microfluidics systems. Superfusion enhanced the number of endothelial vessels and improved podocyte characteristics [87,93]. Besides in vitro approaches, xenograft transplantation to mice resulted in improved maturation of organoid tissue (e.g., expression of Na-Cl cotransporter and aquaporin 2) [94,95].…”

“…High-throughput screens were developed that expedite improvement of differentiation in terms of growth factor concentrations, timing and duration. Minor concentration changes in factors such as CHIR99021 or FGF9 have major effects on the proportion of UB, MM, and early proximal and distal nephron cells [ 80 , 87 ]. To better understand and characterize complex cell fate dynamics of human kidney development in organoids, genetic tools were established during the past years [ 82 , 88 , 89 , 90 , 91 , 92 ].…”