Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells

Abstract

Kidney 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.

Cellular and Molecular Mechanisms of Kidney Development: From the Embryo to the Kidney Organoid

“…RNA sequencing analysis indicated that Takasato organoids were very similar to the first trimester kidney . Several studies utilized the Takasato protocol to conduct developmental studies (Bantounas et al, 2018), disease modeling (Forbes et al, 2018), in vivo transplantation (Koning et al, 2018), and a scale-up of organoid generation (Kumar et al, 2019). Another study was conducted by Bonventre's laboratory.…”

Kidney-based in vitro models for drug-induced toxicity testing

“…In these cultures, typical proximal tubule cell characteristics have been demonstrated, such as albumin uptake, expression of tight junction proteins, expression of OAT1, microvilli, and apical and basolateral membrane polarization. In addition, cytotoxicity was demonstrated using cisplatin (Czerniecki et al 2018;Freedman et al 2015;Kaminski Michael et al 2016;Morizane et al 2015;Takasato et al 2015), gentamicin (Freedman et al 2015;Kaminski Michael et al 2016;Morizane et al 2015), tacrolimus (Kaminski Michael et al 2016), and adriamycin (Kumar et al 2019). As earlier stated, kidney tubuloids grown from adult stem cells (ASCs) are another promising approach as they allow for long-term culturing and better maturation without losing chromosomal stability (Schutgens et al 2019).…”

Kidney organoids: accurate models or fortunate accidents

“…Indeed, with time in culture the center of organoids show deposition of extracellular matrix and a decline in endothelial populations . Several approaches to generating kidney organoids in suspension show clear evidence of cyst formation and fibrotic expansion with time (Przepiorski et al 2018;Kumar et al 2019). This suggests that while we have managed to replicate early patterning events, we have not created an environment suitable for tubular proliferation and continued development.…”

“…What is possible outside of the embryo is to modify culture format to optimize such models for one or other application, even when this does not necessarily involve creating a perfect model of the developing organ. A variety of studies have now claimed improvements in scale up or relative maturation, including the use of dis-tinct culture formats and changes in culture materials (Table 2; Cruz et al 2017;Czerniecki et al 2018;Przepiorski et al 2018;Garreta et al 2019;Gupta et al 2019;Kumar et al 2019). In addition, protocols for the generation of specific renal cell subtypes, such as podocyte or collecting duct epithelium, are being reported (Musah et al 2017;Taguchi and Nishinakamura 2017;Hale et al 2018;Yoshimura et al 2019).…”

“…Cruz et al (2017) reported that this allowed for the presentation of epithelial cysts within cultures derived from stem cell lines into which autosomal dominant polycystic kidney disease gene mutations (homozygous PKD1 mutations) were engineered (Table 2). Two more recent adaptations of the Takasato protocol have cultured early EBs, or micro-organoids generated from IM in suspension cultures, achieving a significant scale up in terms of final cell number (Table 2; Przepiorski et al 2018;Kumar et al 2019). However, both of these studies reported inappropriate stromal expansion and epithelial cyst formation even within organoids generated from control cell lines, either suggesting that these cultures failed to progress without pathological change or that they did not pattern as accurately when cultured in this format.…”