Protocol for the Growth and Maturation of hiPSC‐Derived Kidney Organoids using Mechanically Defined Hydrogels

Abstract

AbstractWith recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC‐derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney‐like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self‐organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self‐assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi‐synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)‐derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC‐derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self‐assembling peptide hydrogels (SAPHs)Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels.Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture.Support Protocol 2: Organoid fixation with paraformaldehyde (PFA)Basic Protocol 2: Whole‐mount immunofluorescence imaging of kidney organoids.Basic Protocol 3: Immunofluorescence of organoid cryosections