Successful disease-specific induced pluripotent stem cell generation from patients with kidney transplantation

Abstract

Introduction: End-stage renal disease (ESRD) is a major public health problem. Although kidney transplantation is a viable therapeutic option, this therapy is associated with significant limitations, including a shortage of donor organs. Induced pluripotent stem (iPS) cell technology, which allows derivation of patient-specific pluripotent stem cells, could provide a possible alternative modality for kidney replacement therapy for patients with ESRD.

Methods: The feasibility of iPS cell generation from patients with a history of ESRD was investigated using lentiviral vectors expressing pluripotency-associated factors.

Results: In the present article we report, for the first time, generation of iPS cells from kidney transplant recipients with a history of autosomal-dominant polycystic kidney disease (ADPKD), systemic lupus erythematosus, or Wilms tumor and ESRD. Lentiviral transduction of OCT4, SOX2, KLF4 and c-MYC, under feeder-free conditions, resulted in reprogramming of skin-derived keratinocytes. Keratinocyte-derived iPS cells exhibited properties of human embryonic stem cells, including morphology, growth properties, expression of pluripotency genes and surface markers, spontaneous differentiation and teratoma formation. All iPS cell clones from the ADPKD patient retained the conserved W3842X mutation in exon 41 of the PKD1 gene.

Conclusions: Our results demonstrate successful iPS cell generation from patients with a history of ESRD, PKD1 gene mutation, or chronic immunosuppression. iPS cells from autosomal kidney diseases, such as ADPKD, would provide unique opportunities to study patient-specific disease pathogenesis in vitro.

Figure 1

Characterization of human induced pluripotent stem cells generated from patient-specific keratinocytes. Patient-specific induced pluripotent stem (iPS) cell clones were generated from keratinocytes of Wilms tumor (WT), systemic lupus erythematosus (SLE) and autosomal-dominant polycystic kidney disease (ADPKD) patients by ectopic expression of lentiviral vectors expressing OCT3/4, SOX2, KLF4 and c-MYC. iPS cell clones from keratinocytes of the WT patient were termed WT-iPS, similarly for SLE-iPS and ADPKD-iPS. Two clones each were analyzed for human embryonic stem (ES) characteristics (WT#5J, WT#20G, SLE#100H, SLE#20J, ADPKD#50B and ADPKD#10D). WT-iPS, SLE-iPS and ADPKD-iPS clones were generated and maintained under feeder-free conditions, exhibiting morphology similar to human ES cells. (A) All of the iPS cell clones expressed alkaline phosphatase (AP). (B) WT-iPS, SLE-iPS and ADPKD iPS clones expressed cell surface markers SSEA4, TRA-1-60 and TRA-1-81, whereas no notable staining was observed for SSEA1. The nucleus was revealed by counterstaining with 4,6-diamidino-2-phenylindole. Phase-contrast images and AP images are 10× magnification. Immunofluorescence images were obtained at 40× magnification.

Figure 2

Patient-specific induced pluripotent stem cell clones were analyzed for pluripotency-associated gene expression. (A) Wilms tumor (WT)-induced pluripotent stem (iPS) cell, systemic lupus erythematosus (SLE)-iPS cell and autosomal-dominant polycystic kidney disease (ADPKD)-iPS cell clone expression of stemness-associated genes was analyzed by immunofluorescence and RT-PCR analysis. WT-iPS, SLE-iPS and ADPKD-iPS clones expressed high levels of OCT4, SOX2, KLF4 and NANOG. Immunofluorescence images were obtained at 40× magnification. (B) Total cellular RNA from WT-iPS, SLE-iPS and ADPKD-iPS clones was isolated and analyzed for endogenous pluripotency genes by RT-PCR. WT-iPS, SLE-iPS and ADPKD-iPS clones expressed gene transcripts of OCT4, SOX2, KLF4, NANOG, GDF3, hTERT and c-MYC. (C) For detection of silencing of exogenous pluripotency genes, BJ fibroblasts were infected with lentiviral vectors expressing OCT4, SOX2, KLF4 and c-MYC. Three days after viral vector transduction, RNA was isolated from infected fibroblasts, control uninfected fibroblasts, and WT-iPS, SLE-iPS and ADPKD-iPS clones. RT-PCR was performed for transgenes KLF4, c-MYC and OCT4. GAPDH gene transcript was amplified as an internal RNA control. No template (water) samples were included as controls.

Figure 3

Induced pluripotent stem cell clone spontaneous differentiation into cells of embryonic germ layers. Wilms tumor (WT)-induced pluripotent stem (iPS) cell, systemic lupus erythematosus (SLE)-iPS cell and autosomal-dominant polycystic kidney disease (ADPKD)-iPS cell clones were allowed to spontaneously differentiate as embryoid bodies in suspension, followed by adherent culture for 10 to 14 days. Differentiated cells were immunostained for markers of ectoderm, endoderm and mesoderm lineages β-III-tubulin (green), FOXA2 (red) and CD31 (PECAM-1) (green), respectively. Nuclei were stained with 4,6-diamidino-2-phenylindole. All images were obtained at 40× magnification.

Figure 4

Wilms tumor and systemic lupus erythematosus induced pluripotent stem cells form teratoma in immunodeficient mice. Wilms tumor (WT)-induced pluripotent stem (iPS) cell, systemic lupus erythematosus (SLE)-iPS cell and autosomal-dominant polycystic kidney disease (ADPKD)-iPS cell clones were injected subcutaneously into SCID-Beige mice. Tumors were detected from the site of injection and harvested after 3 to 5 months, examined for the presence of cells of three embryonic germ layers. H & E-stained teratoma showed multiple differentiated tissues including cells of ectoderm, neuronal rosette-like structures (indicated *), endoderm (gut tube-like structures, intestinal epithelial cells) and mesoderm (adipose-like tissue, muscle-like tissue). Magnification 10×.

Figure 5

Sequence analysis confirms PKD1 mutation in autosomal-dominant polycystic kidney disease-specific induced pluripotent stem cells. Autosomal-dominant polycystic kidney disease (ADPKD) patient DNA and ADPKD- induced pluripotent stem (iPS) cell DNA were analyzed by sequencing analysis. Sequence of exon 41 of PKD1 showing the 11,525G > A change resulting in W3842X in the ADPKD-iPS cells (center) and the patient's germline DNA (top) but not in the Wilms tumor (WT)-iPS cells (bottom).