CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System

Abstract

The aim of this study is to explore the possibility of modeling Gitelman’s disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman’s disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.

Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System

“…Therefore, the advantage of research using kidney organoids is that effects of FAN1 deficiency can be confirmed not only in proximal tubule cells, but also in various cells constituting the nephron. [22,24] As a result, we found co-localization of Ki-67 not only in proximal tubular epithelial cells, but also in podocyte and distal tubular cells, as shown in Figure 4A. In addition, healthy live cells were decreased in all nephron segmental cells in flowcytometry (Figure 4C,D).…”

“…Trilineage differentiation was performed as described previously [ 12 , 24 ] using a StemMACS™ Trilineage Differentiation Kit (#130-115-660; Miltenyi Biotec, Gaithersburg, MD, USA). Differentiated germ layers were confirmed by staining with antibodies: anti-PAX6 antibody for the ectoderm, anti-SM22A antibody for the mesoderm, and anti-FOXA2 antibody for the endoderm.…”

“…hiPSCs were differentiated into kidney organoids using the previously published protocol [ 21 , 22 , 23 , 24 , 25 ].…”

“…They show a native genomic context and cell-type heterogeneity within the kidney [ 18 , 19 , 20 ]. In addition, kidney organoids from genetically modified or patient-derived hiPSCs can successfully recapitulate the phenotype of various genetic kidney diseases [ 18 , 19 , 21 , 22 , 23 , 24 ].…”

“…For this, we created FAN1 -mutant hiPSCs using KIN patient-derived peripheral blood mononuclear cells and performed FAN1 gene editing using CRISPR/Cas9 in wild-type hiPSCs. We then differentiated them into kidney organoids using a well-established protocol [ 21 , 22 , 23 , 24 , 25 ]. After that, we investigated whether the disease phenotype of KIN was recapitulated in FAN1 -mutant kidney organoids in comparison with wild-type kidney organoids.…”