Abstract
ABSTRACTKidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types in vitro. We describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method. This suspension culture micro-organoid methodology resulted in a three- to fourfold increase in final cell yield compared with static culture, thereby representing an economical approach to the production of kidney cells for various biological applications.
Highlights
The directed differentiation of human pluripotent stem cells, including both induced pluripotent stem cells and embryonic stem cells, to distinct cellular endpoints has enabled the generation of complex organoid models for a variety of human tissues, including the kidney
At day 7, the monolayer cultures of intermediate mesoderm (IM) cells were exposed to EDTA or TrypLE Select and the resulting cell suspension was subjected to low speed (60 rpm) swirling on an orbital shaker in the presence of differentiation media with 0.1% polyvinyl alcohol (PVA) and methyl cellulose (MC) to form cell aggregates using low adhesion 6 cm2 cell culture dishes
All human pluripotent stem cells (hPSCs) lines uniformly responded to the protocol and patterned to kidney micro-organoids (Fig. S1F)
Summary
The directed differentiation of human pluripotent stem cells (hPSCs), including both induced pluripotent stem cells (iPSCs) and embryonic stem cells (hESCs), to distinct cellular endpoints has enabled the generation of complex organoid models for a variety of human tissues, including the kidney. As for other hPSC directed differentiation protocols, the resulting component cell types represent a foetal stage of differentiation, for which transcriptional profiling suggests equivalence to trimester 1 human kidney development (Takasato et al, 2015). Such protocols represent a source of human kidney cell types for disease modelling, drug screening, cellular therapy or tissue bioengineering, these protocols generate low numbers of final cells at a relatively high cost and have not been optimised for scale-up to the extent that will be required for potential cellular therapy or high-content screening approaches. Improvements in culture methods are needed to facilitate scale-up of kidney cells types in vitro for biomedical applications
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