Acquisition of chromosome 1q duplication in parental and genome-edited human-induced pluripotent stem cell-derived neural stem cells results in their higher proliferation rate in vitro and in vivo

Abstract

Objectives: Genetic engineering of human-induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC) may increase the risk of genomic aberrations. Therefore, we asked whether genetic modification of hiPSC-NSCs exacerbates chromosomal abnormalities that may occur during passaging and whether they may cause any functional perturbations in NSCs in vitro and in vivo. Materials and Methods: The transgenic cassette was inserted into the AAVS1 locus, and the genetic integrity of zinc-finger nuclease (ZFN)-modified hiPSC-NSCs was assessed by the SNP-based karyotyping. The hiPSC-NSC proliferation was assessed in vitro by the EdU incorporation assay and in vivo by staining of brain slices with Ki-67 antibody at 2 and 8 weeks after transplantation of ZFN-NSCs with and without chromosomal aberration into the striatum of immunodeficient rats. Results: During early passages, no chromosomal abnormalities were detected in unmodified or ZFN-modified hiPSC-NSCs. However, at higher passages both cell populations acquired duplication of the entire long arm of chromosome 1, dup(1)q. ZNF-NSCs carrying dup(1)q exhibited higher proliferation rate than karyotypically intact cells, which was partly mediated by increased expression of AKT3 located on Chr1q. Compared to karyotypically normal ZNF-NSCs, cells with dup(1)q also exhibited increased proliferation in vivo 2 weeks, but not 2 months, after transplantation. Conclusions: These results demonstrate that, independently of ZFN-editing, hiPSC-NSCs have a propensity for acquiring dup(1)q and this aberration results in increased proliferation which might compromise downstream hiPSC-NSC applications.