Cell Therapy Using Human Induced Pluripotent Stem Cell-Derived Renal Progenitors Ameliorates Acute Kidney Injury in Mice

Abstract

Acute kidney injury (AKI) is defined as a rapid loss of renal function resulting from various etiologies, with a mortality rate exceeding 60% among intensive care patients. Because conventional treatments have failed to alleviate this condition, the development of regenerative therapies using human induced pluripotent stem cells (hiPSCs) presents a promising new therapeutic option for AKI. We describe our methodology for generating renal progenitors from hiPSCs that show potential in ameliorating AKI. We established a multistep differentiation protocol for inducing hiPSCs into OSR1+SIX2+ renal progenitors capable of reconstituting three-dimensional proximal renal tubule-like structures in vitro and in vivo. Moreover, we found that renal subcapsular transplantation of hiPSC-derived renal progenitors ameliorated the AKI in mice induced by ischemia/reperfusion injury, significantly suppressing the elevation of blood urea nitrogen and serum creatinine levels and attenuating histopathological changes, such as tubular necrosis, tubule dilatation with casts, and interstitial fibrosis. To our knowledge, few reports demonstrating the therapeutic efficacy of cell therapy with renal lineage cells generated from hiPSCs have been published. Our results suggest that regenerative medicine strategies for kidney diseases could be developed using hiPSC-derived renal cells.

Significance: This report is the first to demonstrate that the transplantation of renal progenitor cells differentiated from human induced pluripotent stem (iPS) cells has therapeutic effectiveness in mouse models of acute kidney injury induced by ischemia/reperfusion injury. In addition, this report clearly demonstrates that the therapeutic benefits come from trophic effects by the renal progenitor cells, and it identifies the renoprotective factors secreted by the progenitors. The results of this study indicate the feasibility of developing regenerative medicine strategy using iPS cells against renal diseases.

Keywords: Acute kidney injury; Cell- and tissue-based therapy; Induced pluripotent stem cells; Kidney; Nephrons; Renal progenitors; SIX2 protein.

Figure 1.

The generation of OSR1-GFP/SIX2-tdTomato double knock-in hiPSC lines. (A): A schematic representation of the targeting strategy using BAC-based vectors to produce OSR1-GFP/SIX2-tdTomato double knock-in hiPSC lines. The black boxes represent two exons of the SIX2 gene. (B): The TaqMan quantitative polymerase chain reaction (PCR) analyses of genomic DNA from the drug-resistant lines and the parental line (201B7). Note that a value of 1 indicates 2 intact SIX2 loci, and 0.5 suggests 1 intact and 1 targeted locus. 4A157 is a drug-resistant clone without homologous recombination. The primer pair indicated in (A) (red bars) was used. (C): Genomic PCR was used to confirm the removal of the Neo cassette by Cre recombination. The primer pair indicated in (A) (black bars) was used. (D): The CN of SIX2 gene loci was analyzed by determining the number of single-nucleotide polymorphisms (SNPs) in the 3 hiPSC lines: 4A6, 4A61, and 4A77. 4A77 is a drug-resistant transgenic line without homologous recombination. The red dots and y-axis represent the signal intensity of each SNP probe in each panel, and the CNs (black lines) were detected using a hidden Markov model-based algorithm implemented in the CNAG/AsCNAR software program (Cancer Genomics Project, University of Tokyo, Tokyo, Japan). The blue lines represent the moving average of total CNs. (E): Normal karyotypes were found for the 3 OSR1-GFP/SIX2-tdTomato double knock-in hiPSC lines (4A6C3-10, 4A61C4-16, and 4A61C4-20) in which the Neo cassette was excised at the flanking loxP sites by transient expression of Cre recombinase. Abbreviations: bp, base pairs; BAC, bacterial artificial chromosome; CN, copy number; Cre, creatinine; GFP, green fluorescent protein; hiPSC, human inducible pluripotent stem cell; IRES, internal ribosome entry site.

Figure 2.

Confirmation of the ability of OSR1-GFP/SIX2-tdTomato double knock-in human inducible pluripotent stem cell lines to monitor SIX2 expression. (A): The flow cytometric analyses of tdTomato-positive cells on culture day 19 induced with or without the factors used in our previously reported differentiation protocol for the intermediate mesoderm [6]. The data from three independent experiments are presented as the mean ± SEM (n = 3). (B): The results of the reverse transcription-polymerase chain reaction analyses of the tdTomato-positive and tdTomato-negative cell populations isolated on day 19 shown in (A). (C): Immunostaining using antibodies against SIX2 on the tdTomato-positive and tdTomato-negative cells isolated on day 19. Alexa Fluor 647 was used as the secondary antibody to eliminate any overlap with the GFP or tdTomato signals. Scale bars = 50 μm. Abbreviations: APC, allophycocyanin; GFP, green fluorescent protein; Tx, treatment with factors described in [6].

Figure 3.

The establishment of methods for differentiating human iPSCs into OSR1⁺SIX2⁺ cells. (A): The differentiation method used to generate OSR1⁺SIX2⁺ cells with embryoid body-based three-dimensional cultures. (B): The differentiation efficiencies of OSR1⁺, SIX2⁺, and OSR1⁺SIX2⁺ cells were analyzed by flow cytometry on culture day 28. The treatments at stages 1, 2, and 3 were the same as those used in A. The factors and their concentrations used at stage 4 were as follows: 0.5 μM DMSO as a negative control, 5 ng/ml TGF-β1, 5 ng/ml TGF-β2, 5 ng/ml TGF-β3, 0.5 μM DMH1, 100 ng/ml Noggin, 0.5 μM DMH1, 0.5 μM LDN-193189, 50 μM IDE2, and 10 μM SB431542. Noggin, DMH1, LDN, and DMH1 are BMP inhibitors. IDE2 is a small molecule that activates TGF-β signaling. The data from five independent experiments are presented as the mean ± SEM (n = 5). (C): The periodic differentiation pattern of OSR1⁺SIX2⁺ cells was analyzed by flow cytometry. Upper: The two-dimensional distribution of the cell populations. Middle: The results of the time course analyses. The data from five independent experiments are presented as the mean ± SEM (n = 5). Day 1 indicates culture day 1 before treatment. Day 3 indicates day 3 after stage 1 treatment. Day 6 indicates day 6 after stage 2 treatment. On days 6–28, the cells were treated with stage 3 and 4 treatment or with DMSO at stages 3 and 4 (negative control). Representative anti-GFP and anti-DsRed (tdTomato) immunostaining images of OSR1⁺SIX2⁺ renal progenitors isolated on day 28 are shown in the lower panels. Scale bars = 50 μm. Abbreviations: BMP, bone morphogenetic protein; DMEM, Dulbecco’s modified Eagle’s medium; DMH1, dorsomorphin homolog 1; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GFP, green fluorescent protein; KSR, knockout serum replacement; PS, penicillin/streptomycin; TGF, transforming growth factor; Tx+, treatment with stage 3 and 4 treatment; Tx−, treatment with negative control.

Figure 4.

The expression of renal lineage marker genes in the differentiation culture of human induced pluripotent stem cells (hiPSCs) into OSR1⁺SIX2⁺ renal progenitors. (A): The time course analyses of mRNA expression in the differentiation culture. The graphs indicate the expression of each transcript relative to β-ACTIN. OCT3/4 and NANOG are markers for undifferentiated hiPSCs, BRACHYURY and TBX6 are markers for posterior nascent mesoderm, OSR1 is a marker for intermediate mesoderm, WT1, PAX2, SIX2, HOXA10, and HOXA11 are markers for the metanephric mesenchyme. The data from three independent experiments are presented as the mean ± SEM (n = 3). (B): The expression of marker genes for nephron progenitors in OSR1⁺SIX2⁺ cells differentiated from an OSR1-GFP/SIX2-tdTomato double knock-in hiPSC line, 4A6C3-10, on culture day 28. (C): Immunostaining images of OSR1⁺SIX2⁺ cells isolated on day 28 against SIX2, PAX2, WT1, and CDH6. (B, C): Representative data obtained from three independent experiments shown. Scale bars = 50 μm.

Figure 5.

Developmental potential of OSR1⁺SIX2⁺ renal progenitors to differentiate into renal lineage cells or tissues. (A): The presented macroscopic views show the aggregates of OSR1⁻SIX2⁻ cells on culture day 5 (upper left) and OSR1⁺SIX2⁺ cells on day 7 (upper right) cocultured with E11.5 mouse spinal cord tissue in organ cultures. Lower panels: Immunostaining images of the histological sections of the OSR1⁺SIX2⁺ cell aggregates after 7 days of coculture with spinal cord tissue. LTL is a marker of the proximal renal tubules; LAMININ, a marker of the polarized epithelia; CDH1, an epithelial marker; CDH6, an early proximal tubule marker. (B): Coculture experiments with NIH3T3 fibroblasts expressing Wnt4. Macroscopic views of the aggregates of OSR1⁻SIX2⁻ cells on day 5 (upper left) and OSR1⁺SIX2⁺ cells on day 7 (upper right) and immunostaining images of the histological sections of the OSR1⁺SIX2⁺ cell aggregates after 7 days of coculture (lower). Note that the lower left two panels in (A) and (B) are of the same sections. (C): Immunostaining images of the organ culture of the OSR1⁺SIX2⁺ cells and E11.5 metanephric cells. (D): Immunostaining images of the OSR1⁺SIX2⁺ cell aggregates transplanted into the epididymal fat pads of the immunodeficient mice (NOD. CB17-Prkdc^scid/J) after 30 days of transplantation. Scale bars = 50 μm. Abbreviations: AG, OSR1⁻SIX2⁻ or OSR1⁺SIX2⁺ cell aggregate; HuNu, human nuclei; LTL, Lotus tetragonolobus lectin; SP, spinal cord.

Figure 6.

Cell therapy using human iPSC (hiPSC)-derived renal progenitors for the mouse acute kidney (AKI) injury models. (A): The time course analyses of the BUN and plasma Cre levels in the I/R AKI mice that received the renal subcapsular transplantation of hiPSC-derived renal progenitors (n = 6; iPSC-RPs, triangle), undifferentiated hiPSCs (n = 7; iPSCs, square), or saline (n = 11; circle). Statistical significance at the p < .05 level after multiple testing adjustment: ∗∗∗, p < .001 versus saline; ††, p < .01 versus iPSCs; †††, p < .001 versus iPSCs. Least square mean and 95% confidence intervals were estimated according to the mixed effects model for repeated measures. (B): Sections of representative kidney samples from the host mice that received transplantation of iPSC-RPs, iPSCs, or saline were stained with HE, PAS, or MT on day 12 after I/R and transplantation. Representative findings of tubular necrosis, urinary casts, tubular dilatation, loss of tubular borders, and interstitial fibrosis in each treatment group are shown. The arrows indicate the representative area of each finding. Scale bars = 20 μm. (C): Histological scoring of the areas with tubular necrosis, urinary casts, tubular dilatation, loss of tubular borders and interstitial fibrosis and total scores in the host kidneys on day 12 after I/R and transplantation (n = 5 for iPSC-RPs, n = 7 for iPSCs, n = 7 for saline). ∗, p < .05 versus saline; †, p < .05 versus iPSCs. The data are presented as the mean ± SEM in (A) and (C). Abbreviations: BUN, blood urea nitrogen; Cre, creatinine; HE, hematoxylin and eosin; I/R, ischemia/reperfusion; iPSC, induced pluripotent stem cell; iPSC-RP, OSR1⁺SIX2⁺ renal progenitor cells; MT, Masson’s trichrome; PAS, periodic acid-Schiff.

Figure 7.

OSR1⁺SIX2⁺ renal progenitors secrete the growth factors known to be renoprotective factors. (A): The RNA expression of renoprotective factors in the iPSC-RPs compared with that observed in the iPSCs using a microarray analysis. (B): Protein section of renoprotective factors analyzed by the human magnetic luminex screening assay in medium only (medium) and cell culture supernatant of iPSCs and iPSC-RPs. The data from 3 independent experiments are presented as the mean ± SEM (n = 3). Note that the secretion levels of ANG-1, VEGF-A, and HGF in the medium and those of ANG-1 and HGF in the iPSCs were so low that the histogram bars are at the baseline level. Abbreviations: ANG-1, angiopoietin 1; CCL2, C-C motif chemokine 2; CXCL12, C-X-C motif chemokine 12; FGF-9, fibroblast growth factor 9; HGF, hepatocyte growth factor; IGF1, insulin-like growth factor 1; IGF2, insulin-like growth factor 2; iPSCs, inducible pluripotent stem cells; iPSC-RPs, OSR1⁺SIX2⁺ renal progenitor cells; TGF-B2, transforming growth factor-β2; VEGF-A, vascular endothelial growth factor A; VEGF-C, vascular endothelial growth factor C; VEGF-D, vascular endothelial growth factor D.