Promoting Maturation of Human Pluripotent Stem Cell-Derived Renal Microtissue by Incorporation of Endothelial and Mesenchymal Cells

Abstract

Directed differentiation of human pluripotent stem cells (hPSCs) uses a growing number of small molecules and growth factors required for in vitro generation of renal lineage cells. Although current protocols are relatively inefficient or expensive. The first objective of the present work was to establish a new differentiation protocol for generating renal precursors. We sought to determine if inducer of definitive endoderm 1 (IDE1), a cost-effective small molecule, can be used to replace activin A. Gene expression data showed significantly increased expressions of nephrogenic markers in cells differentiated with 20 nM IDE1 compared with cells differentiated with activin A. Thus, renal lineage cells could be generated by this alternative approach. Afterward, we determined whether coculture of endothelial and mesenchymal cells could increase the maturation of three-dimensional (3D) renal structures. For this purpose, we employed a 3D coculture system in which hPSC-derived kidney precursors were cocultured with endothelial cells (ECs) and mesenchymal stem cells (MSCs), hereafter named RMEM (renal microtissue derived from coculture of renal precursors with endothelial and mesenchymal stem cells). hPSC-derived kidney precursors were cultured either alone [renal microtissue (RM)] or in coculture with human umbilical vein endothelial cells and human bone marrow-derived mesenchymal stem cells at an approximate ratio of 10:7:2, respectively. Immunofluorescent staining showed expressions of kidney-specific markers synaptopodin, LTL, and E-cadherin, as well as CD31 + ECs that were distributed throughout the RMEMs. Quantitative real-time polymerase chain reaction analysis confirmed a significant increase in gene expressions of the renal-specific markers in RMEMs compared with RMs. These findings demonstrated that renal precursors cocultured with endothelial and MSCs showed greater maturity compared with RMs. Moreover, ex ovo transplantation induced further maturation in the RMEM constructs. Our novel approach enabled the generation of RMEM that could potentially be used in high-throughput drug screening and nephrotoxicology studies.

Tissue-Specific Microparticles Improve Organoid Microenvironment for Efficient Maturation of Pluripotent Stem-Cell-Derived Hepatocytes

“…We assumed that the vascular structures that surrounded the BLOs through the highly porous 3D basket enhanced the maturation of the BLOs. Some studies have demonstrated the effect of in vivo and ex ovo vascularizations on the maturation of organoids or other tissues [33,34,[50][51][52].…”

“…We assumed that the vascular structures that surrounded the BLOs through the highly porous 3D basket enhanced the maturation of the BLOs. Some studies have demonstrated the effect of in vivo and ex ovo vascularizations on the maturation of organoids or other tissues [ 33 , 34 , 50 , 51 , 52 ]. Here, we found that the transplanted BLOs could rapidly perform hepatic functions such as ALB secretion; in addition, ALT and AST levels in BLO-transplanted mice were significantly decreased compared to the sham group.…”

Kidney organoids: current knowledge and future directions

“…Several EPC populations play a key role in the formation of de novo vessels and nephrogenesis during kidney development (Munro et al 2017). There is extremely limited generation of ECs in the majority of current differentiation methods (0.3% or less of total cells) (Wu et al 2018;Khoshdel-rad et al 2021); many of these ECs have immature phenotypic characteristics. In addition, glomerular capillary formation is rare (Czerniecki et al 2018).…”

A comprehensive survey on optimal components of extracellular matrix for kidney development and function

“…Matrigel, has used as a reconstituted basement membrane derived from the Engelbroth Holm-Swarm (EHS) mouse sarcoma [149] that mimics mechanical and biochemical properties of ECM. This agent used to facilitate cellular self-organization, successful implantation of artifi cial tissue include kidney and bladder in patients [150]. Atala and colleagues showed that artifi cial bladders are composed of autologous cells (isolated from the patient) seeded on collagen or composite collagen and polyglycolic acid scaffolds.…”

“…The using of decellularized biopsies of different regions of porcine kidneys including cortex, medulla, and papilla as the hydrogels and sheets of ECM showed that kidney stem cells had signifi cantly higher metabolic activity when cultured on papilla ECM [149]. In the intact ECM was observed potential to support proliferation of epithelial and endothelial cells, which consequently resulted in urine producing kidneys [150][151][152][153].…”