Whole-Genome DNA Methylation Analyses Revealed Epigenetic Instability in Tumorigenic Human iPS Cell-Derived Neural Stem/Progenitor Cells

Abstract

Although human induced pluripotent stem cell (hiPSC) derivatives are considered promising cellular resources for regenerative medicine, their tumorigenicity potentially limits their clinical application in hiPSC technologies. We previously demonstrated that oncogenic hiPSC-derived neural stem/progenitor cells (hiPSC-NS/PCs) produced tumor-like tissues that were distinct from teratomas. To gain insight into the mechanisms underlying the regulation of tumorigenicity in hiPSC-NS/PCs, we performed an integrated analysis using the Infinium HumanMethylation450 BeadChip array and the HumanHT-12 v4.0 Expression BeadChip array to compare the comprehensive DNA methylation and gene expression profiles of tumorigenic hiPSC-NS/PCs (253G1-NS/PCs) and non-tumorigenic cells (201B7-NS/PCs). Although the DNA methylation profiles of 253G1-hiPSCs and 201B7-hiPSCs were similar regardless of passage number, the methylation status of the global DNA methylation profiles of 253G1-NS/PCs and 201B7-NS/PCs differed; the genomic regions surrounding the transcriptional start site of the CAT and PSMD5 genes were hypermethylated in 253G1-NS/PCs but not in 201B7-NS/PCs. Interestingly, the aberrant DNA methylation profile was more pronounced in 253G1-NS/PCs that had been passaged more than 15 times. In addition, we identified aberrations in DNA methylation at the RBP1 gene locus; the DNA methylation frequency in RBP1 changed as 253G1-NS/PCs were sequentially passaged. These results indicate that different NS/PC clones have different DNA methylomes and that DNA methylation patterns are unstable as cells are passaged. Therefore, DNA methylation profiles should be included in the criteria used to evaluate the tumorigenicity of hiPSC-NS/PCs in the clinical setting. Stem Cells 2017;35:1316-1327.

In vivo monitoring of remnant undifferentiated neural cells following human induced pluripotent stem cell-derived neural stem/progenitor cells transplantation

“…Long‐term observation of transplanted cells, however, has revealed the existence of “unsafe” NS/PCs characterized by differentiation‐resistant properties that could cause abnormal cell growth in the injured spinal cords of rodents due to residual immature neuronal cells after hiPSC‐NS/PCs transplantation . Therefore, it is necessary to select “safe” and “unsafe” clones in the cell manufacturing process . Furthermore, since the extent of cellular differentiation and maturation depends on the host microenvironment, even “safe” clones, which can be purified for homogeneity and produced as high‐quality populations, may not be able to completely differentiate into neural cell types at the transplanted site.…”

“…In the present study, in order to determine the efficiency of PET with TSPO ligand, we used two different types of iPSC‐derived NS/PCs; “unsafe” 253G1‐NS/PCs known to have differentiation‐resistant proliferative properties and “safe” 414C2‐NS/PCs, which were reported as a nontumorigenic hiPSC‐NS/PCs . First, we examined differences in TSPO expression levels in each hiPSC‐NS/PCs before and after neuronal induction using immunohistochemistry, real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) and Western blot assays in vitro.…”

“…8,9 Therefore, it is necessary to select "safe" and "unsafe" clones in the cell manufacturing process. 7,10,11 Furthermore, since the extent of cellular differentiation and maturation depends on the host microenvironment, 12 even "safe" clones, which can be purified for homogeneity and produced as high-quality populations, may not be able to completely differentiate into neural cell types at the transplanted site. Therefore, clinical application of hiPSC-NS/PCs to central nervous system (CNS) disorders would benefit from a relatively noninvasive detection technique for monitoring the progress of cellulardifferentiation even after "safe" clone transplantation.…”

Selective Ablation of Tumorigenic Cells Following Human Induced Pluripotent Stem Cell-Derived Neural Stem/Progenitor Cell Transplantation in Spinal Cord Injury

“…HSVtk cDNA that had been modified by humanizing codon usage and eliminating all CpG dinucleotides was purchased from InvivoGen (San Diego, CA) and then cloned into the tetracycline (Tet)‐inducible lentiviral vector CSIV‐RfA‐TRE‐EF‐KT (Fig. A) . Recombinant lentiviral vector production and titer determination were performed as described previously .…”

“…253G1‐hiPSCs expressing Tet‐inducible HSVtk (HSVtk‐hiPSCs) were dissociated into single cells, seeded in 96‐well plates at a density of 5 × 10 3 cells/200 μl per well with or without 1 μg/ml doxycycline (DOX; Wako Pure Chemical Industries, Ltd., Osaka, Japan). After 3 days of incubation, the cell viability assay was performed using the Cell Counting Kit‐8 (Dojindo Molecular Technologies, Kumamoto, Japan) as described previously .…”