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.


| INTRODUC TI ON
Human-induced pluripotent stem cell-derived neural stem cells (hiP-SC-NSCs) have been used for developmental studies, 1 disease modelling, 2,3 drug screening, 4 toxicity testing 5 and in preclinical studies of neuroregenerative therapeutic approaches. 6 Genetic modification of stem cells is frequently utilized for lineage tracking, to modify the expression of a specific endogenous gene in order to study its biological role, or overexpress exogenous factors to monitor and/or enhance the engraftment and therapeutic efficacy of transplanted cells in regenerative approaches. [7][8][9][10] Genome engineering technologies such as zinc-finger nucleases (ZFN), 11 transcription activator-like effector nucleases (TALEN), 12 and the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system 13,14 enable DNA modifications in a highly precise manner and significantly lower the risks of various non-target effects that are associated with traditional genetic engineering techniques. 15 However, genome editing increases cell handling and cultivation time, which could affect their genomic stability and diminish their usefulness because the newly acquired genetic changes may be detrimental to the cell's viability, functionality and safety. [16][17][18][19][20] Many studies have demonstrated that different types of stem cells, including NSCs, acquire characteristic chromosomal aberrations during late and sometimes also in early passages in culture. [21][22][23] Comprehensive analysis of chromosomal aberrations in 58 adult human NSC samples and 39 human embryonic stem cell (hESC)-derived NSC samples identified a trisomy of chromosomes 7, 10,19 and 20q as well as a trisomy and monosomy of chromosome 18. 24 The overall frequency of aberrations in NSCs was about 9%. A similar frequency of samples with chromosomal aberrations was found in a separate analysis of hiPSC-derived NSCs (10%, 18 out of 182 samples) and adult NSCs (7%, 7 out of 100 samples). 25 In these samples, the most common were gains of chromosomes 1, 12 and 17, which also occur in undifferentiated human PSC cultures. 22,23,26,27 Targeting safe harbour areas like adeno-associated virus site 1 (AAVS1) by using gene editing methods have been used in various hESC 11,28 and hiPSC 9,29 lines and their differentiated derivatives, such as NSCs. [30][31][32] These studies demonstrated that cells modified To address these questions, we used the ZFN technology to integrate a cassette containing a human EF1-α promoter driving the expression of puromycin resistance gene (Pac) and enhanced GFP (EPG-cassette) into the AAVS1 locus in hiPSC-NSCs. SNP array-based karyotyping identified a duplication of the entire long arm of chromosome 1 [dup(1)q] in unmodified and ZFN-modified NSCs after prolonged passaging. Compared to ZFN-NSCs with an intact karyotype, cells that carried dup(1)q exhibited increased proliferation rate in vitro and in vivo after transplantation into the striatum of immunodeficient rats. The higher proliferation rate was partly mediated by overexpression of the proliferation promoting gene AKT3 located in duplicated area. These results show that dup(1)q occurs in high-passage hiP-SC-NSCs and demonstrate for the first time that its occurrence is not affected by ZFN-based editing but that it increases cell proliferation both in vitro as well as in vivo requiring strict quality control of cells before using them for applications in research and therapy.
in the R buffer from the Neon Transfection System (Life Technologies) together with 8 μg of the pAAVS1-EPG vector and 250 ng of mRNAs encoding ZFNs that target the ZFN cleavage site in the AAVS1 locus and were included in the CompoZr ® Targeted Integration Kit-AAVS1 (Sigma-Aldrich). Transfection was performed with the Neon Transfection System by using two 20 ms pulses at 1400 V. Transfected cells were plated onto a poly-l-ornithine/laminin-coated (both from Sigma) 6-well plate. Selection with 2 μg/mL puromycin began at day 7 after transfection. After 10 days, antibiotic-resistant cells were expanded and aliquots were cryopreserved for further studies. The procedures used for generation of single cell clones and identification of mono-and bi-allelic ZFN-NSC lines are described in the Supplemental material, Figure S1, and Tables S1 and S2.

| Molecular karyotyping
Karyotyping was performed by SNP array-based genotyping using

| Protein and gene expression analyses
Immunocytochemical analyses, cDNA synthesis and qPCR amplification of selected genes were carried out as described in the Supplemental material. PCR primers are listed in Table S3 and antibodies in Tables S4 and S5.

| Statistical analysis
For statistical analysis of differences between experimental groups, the independent two-tailed Studen's t test was performed by using the GraphPad Prism software (version 4.0). P values equal to or lower than .05 were considered statistically significant.

| Generation and characterization of hiPSC-NSCs
Neural stem cells used in this study were derived from hiPSC line R-iPSC4 as previously reported by us. 33 Transcriptome, proteome and immunocytochemical analyses of these hiPSC-NSCs revealed that they expressed typical NSC markers and exhibited robust differentiation potential to neurons, astrocytes and oligodendrocytes (see reference (33) and Figure 1A

| ZFN-mediated gene targeting into the AAVS1 locus in hiPSC-NSCs
Next, we sought to examine whether ZFN-based genome editing would affect the propensity of hiPSC-NSCs to acquire this or other chromosomal anomalies over prolonged passaging. To this end, the targeting vector pAAVS1-EPG and mRNAs encoding for a pair of ZFNs that target the genomic integration site of AAVS1 locus were co-transfected into R-iPSC4-NSCs at p14. This resulted in 77% eGFP-positive cells at day two after transfection ( Figure S3).

Selection with puromycin yielded a pure population of ZFN-edited
NSCs that stably expressed eGFP over at least 23 passages in

| Characterization of gene modified hiPSC-NSCs
To determine whether genetic modification and subcloning af-

| Molecular karyotyping of ZFN-edited hiPSC-NSCs
Next, we sought to determine which chromosomal aberrations occur in hiPSC-NSCs that underwent the ZFN modification procedure. SNP genotyping of ZFN-NSCs that were kept in culture for four passages after transfection (p14 + 4) revealed no chromosomal aberrations in these cells ( Figure 3A and Figure S9). Chromosomal abnormalities were also not detected after clonal selection of ZFN-NSCs as shown at p14 + 14 for the clone 44 harbouring bi-allelic insertion of the transgene cassette ( Figure 3B and Figure S10).
However, extended passaging of clonal ZFN-NSCs reproducibly led to the acquisition of a dup(1)q aberration as shown for the biallelic clone 44 at p14 + 20 ( Figure 3C and Figure S11) and p14 + 36 ( Figure 3D and Figure S12), as well as for the mono-allelic clone 138 analysed at p14 + 11 ( Figure 3E and Figure S13). Cultivation

| Effect of the dup(1)q on the proliferation rate of ZFN-NSCs in vitro
The Chr1q region harbours the genes which are involved in the regulation of cell survival, proliferation and differentiation, such as AKT3, PIK3C2B, MDM4 and NOTCH2NLA. Therefore, we used the EdU incorporation assay to determine whether dup(1)q affects the proliferation rate of hiPSC-NSCs. This analysis showed that ZFN-mediated genetic modification of NSCs does not affect their proliferative activity in comparison to parental hiPSC-NSCs ( Figure 4A,B). However, there was a significantly higher percentage of EdU-positive ZFN-NSCs in cells with dup(1)q compared to those without this aberration (P < .0001) ( Figure 4C), suggesting that dup(1)q increases the proliferation of hiPSC-NSCs.
Next, we used RT-qPCR analysis to assess whether ZFN-NSCs with and without dup(1)q differ in expression of the above-mentioned genes. These analyses revealed significant upregulation of the AKT3 (55-fold), PIK3C2B (30-fold), MDM4 (24-fold) and NOTCH2NLA (14-fold) transcripts in NSCs carrying dup(1)q compared to their genetically intact counterparts (n = 3, P < .0001) ( Figure 4D). In contrast, expression of DNMT3B, which is located on Chr20 and served as a negative control for gene dosage, did not significantly differ between these cell lines. These data show that dup(1)q in NSCs leads to perturbations in expression of genes located on Chr1q and suggest that genes, such as AKT3 or PIK3C2B, might be responsible for their increased proliferation rate.
F I G U R E 1 Generation and karyotype analysis of hiPSC-NSCs. A, R-iPSC4-hiPSC colonies growing on Matrigel. B, Embryoid bodies (EBs) formed after digestion of hiPSCs with collagenase IV. C, Rosette-like structures appeared 7-10 d after plating of EBs treated with TGFβinhibitor SB421543 and BMP-inhibitor dorsomorphin onto poly-l-ornithine-and laminin-coated plates. D, Neuroectodermal cells were obtained by dissociating rosette-like structures and plating on poly-l-ornithine-and laminin-coated plates. E, F, These cells expressed the NSC marker Nestin (E) and differentiated to neurons expressing microtubule-associated protein 2 (MAP2) at day 30 of differentiation (F). Nuclei were stained with Hoechst 33342 (blue). Scale bars: 100 µm. G, H, Whole-genome SNP array-based karyotyping of hiPSC-NSCs. B-allele frequencies (upper panels) and log 2 R ratios (lower panels) are plotted for each chromosome for all SNPs on the array located in this region. Each point is an SNP. While cells at passage 10 (p10) did not show any major karyotype abnormalities (G), hiPSC-NSCs at p16 exhibited duplication of the entire long arm of the chromosome 1, dup (1) Figures S9-S14. A, Analysis of the heterogeneous population of ZFN-NSCs at p4 after genome modification (p14 + 4) did not reveal any major chromosomal abnormalities (see also Figure S9). B, Early after clonal selection at p14 + 14, the bi-allelic ZFN-NSC clone 44 did not show any detectable aberrations in Chr1 or any other chromosomes ( Figure S10). C-E, Extended passaging of clonally selected ZFN-NSCs led to the acquisition of a dup(1)q abnormality (asterisks) as shown for two different batches of clone 44 (batch A at p14 + 20 shown in panel C and Figure S11; batch B at p14 + 36 shown in panel D and Figure S12), and for the mono-allelic clone 138 at p14 + 11 (panel E and Figure S13). F, ZFN-NSC expansion for an even longer period led to the acquisition of additional chromosomal abnormalities as exemplified by the duplication of the 10 Mbp telomeric end in the long arm of chromosome 9 observed in clone 44 at p14 + 44 (arrow see also Figure S14) without dup(1)q ( Figure 4E,F). However, the AKT3 inhibitor MK2206 reduced the proliferation of NSCs carrying dup(1)q to a significantly greater extent than the proliferation of karyotypically normal cells ( Figure 4G). In contrast, the inhibitory effect of PIK3C2B inhibitor NU7441 did not differ significantly between NSCs with and without dup(1)q ( Figure 4G), indicating that AKT3 but not the PIK3C2B signalling pathway at least partly mediates the higher proliferation rate of NSCs with dup(1)q as a consequence of the increased gene dosage.

| Dup(1)q increases the proliferation rate of ZFN-NSCs in vivo
To determine whether the proliferation-enhancing effect of dup (1) q is also retained in vivo, equal numbers of GFP-expressing ZFN-NSCs (clone 44) with or without this chromosomal aberration were transplanted into the striatum of immunodeficient rat brains (n = 3 in each group). Their engraftment and mitotic fractions in the graft area were analysed 2 weeks and 2 months after transplantation.
The 2-week analysis of brain slices revealed that transplanted ZFN-NSCs in both groups formed well-delineated grafts which expressed GFP as well as the human-specific marker TRA-1-85 ( Figure 5A,E). and chromosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. 40 They suggested that similar mechanism may also operate during initiation of chromosomal instability in diploid hPSCs. In addition, Zhang and coworkers reported that loss-of-function mutations in pro-apoptotic genes or upregulation of anti-apoptotic genes that may occur in hPSC desensitize them to mitotic stress and enable aneuploid cell survival. 41 F I G U R E 4 Assessment of the proliferation rate and expression of genes located on chromosome 1q in hiPSC-NSCs. A, Fluorescence microscopy of EdU-labelled hiPSC-NSCs (upper panels) and ZFN-NSCs (clone 44, lower panels). Cells were incubated with EdU for 2 h and then stained with EdU antibodies to visualize positive nuclei (red). Only transgenic ZFN-NSCs expressed GFP (green). Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 100 µm. B, Quantification of the percentage of EdU-positive NSCs in the experiment shown in panel A based on the scoring of 3087 and 2369 nuclei in non-modified hiPSC-NSCs and ZFN-NSCs (clone 44), respectively (data obtained from two independent experiments, each performed in triplicate). C, The proliferation rate of ZFN-NSCs (clone 44) with (p14 + 44) and without dup(1)q (p14 + 13) as determined by the EdU incorporation assay. The percentage of EdU-positive cells was determined in two independent experiments each performed in triplicate. D, RT-qPCR analysis of PIK3C2B, AKT3, MDM4 and NOTCH2NLA gene expressions localized on Chr 1q in comparison to DNMT3B localized on Chr 20 in ZFN-NSC clone 44 without (p14 + 11) and with dup(1)q (p14 + 41). E,F, ZFN-NSCs (clone 44) without (E) and with dup(1)q (F) were cultured for 24 h in the absence (CTRL) and presence of AKT3 inhibitor (AKT3i) MK2206 or PIK3C2B inhibitor (PIK3i) NU7441 (both at 1 μmol/L). The proliferation rate was assessed by the EdU incorporation assay as described for panel A. G, Comparison of the extent of inhibition of EdU incorporation into ZFN-NSCs with and without dup(1)q after treatment with AKT3i and PIK3i (calculated from data shown in panels E and F). Treatment with AKT3i, but not PIK3i, exerted a significantly stronger inhibition on the proliferation rate of ZFN-NSCs with dup(1)q than on genetically intact ZFN-NSCs. n.s.: Non-significant, **P < .01 and ***P < .001

Immunohistochemistry revealed that most injected cells in both
Other studies identified POLD3 and ZSCAN10 as factors involved in maintenance of genomic stability in PSCs. POLD3 is a gene encoding for the accessory subunit of DNA polymerase delta 3, and its loss results in replicative stress, DNA repair impairment, micronucleation and aneuploidy in ESCs. 42 The embryonic stem cell-specific transcription factor ZSCAN10 has been shown to protect hPSCs from accumulation of chromosomal structural abnormalities, and defects in apoptosis and in the DNA damage response. 43 The mechanism which is specifically responsible for the acquisition of dup(1)q in hiP-SC-NSCs will be explored in future studies.
Gains of chromosome 1 have been detected by other groups both in NSCs 25,44 as well as in human ESCs and iPSCs. 22,23,27,45 Among them were whole chromosome 1 duplications (trisomy) or unbalanced translocations and interstitial duplications of different segments in its long arm. For example, Weissbein and coworkers observed duplication of the whole chromosome 1q in human PSC-NSCs but they did not assess its functional consequences. 25 In contrast,

Varela and coworkers detected amplification of a segment of 1q and
its translocation onto the telomeric ends of chromosomes 5p, 8q and 13q in long-term cultured hESC-NSCs. 44 They also showed that neuronal differentiation of two aberrant NSC lines was decreased in vitro but this was not systematically observed in all lines that were It is worth noting that the human chromosome 1q corresponds to mouse chromosomes 1 and 3. Interestingly, gain of the entire chromosome 1 was observed in long-term cultured NSCs derived from mouse ESCs or adult and foetal mouse brain. 46,47 In the study with mouse foetal brain-derived NSCs, cells carrying trisomy 1 exhibited increased proliferation and decreased neural differentiation capacity in vitro. 47 Aberrations in 1q are also one of the most common abnormalities reported among human neoplasms, including haematologic malignancies [48][49][50] and paediatric brain tumours, [51][52][53] suggesting that they might be associated with advantages in cell proliferation and survival. Indeed, the proliferation rate of hiPSC-NSCs carrying dup(1)q in our study was higher than that of karyotypically normal NSCs both in vitro and in vivo.
The identification of specific driver gene(s) on chromosome 1q responsible for this effect in NSCs or cancer cells is difficult because more than one gene could be involved in conveying the growth advantage to aneuploid cells. However, number of genes located on chromosome 1q, such as AKT3, PIK3C2B, MDM4 and NOTCH2NL, are known to be associated with the control of cell proliferation, survival, migration, stress response, oncogenic transformation, neuronal differentiation and intracellular protein trafficking. [54][55][56][57] Interestingly, these genes were found to be overexpressed in ZFN-NSCs with dup(1)q and inhibitor studies suggested that AKT3 pathway may be at least partially responsible for their increased proliferation rate. In conclusion, we show that an isolated duplication of chromosome 1q occurs in unmodified and ZFN-modified hiPSC-NSCs after prolonged passaging, that this aberration increases NSC proliferation rate in vitro, and that these changes still persist in transplanted cells in vivo. Our preliminary data suggest that the higher proliferation rate of aberrant NSCs is partly mediated by overexpression of the proliferation promoting gene AKT3 located in duplicated area, but additional studies are required to elucidate the exact mechanism responsible for this phenomenon. It should be noted that in some prior studies no chromosomal abnormalities were observed in the long-term cultured hPSC-NSCs. 59,60 This indicates that acquisition of such abnormalities in cultured cells is not an inevitable event and that conditions might be selected that F I G U R E 5 In vivo engraftment and proliferation rate of ZFN-NSCs with and without dup(1)q. A, E, Representative images of brain slices stained with antibodies against transgenic eGFP and human cell marker TRA-1-85 2 weeks after transplantation of ZFN-NSCs (clone 44) without dup(1)q (A) and with dup(1)q (E) into the striatum of RNU rats. B, F, Expression of Nestin (red) in engrafted ZFN-NSCs indicates that, independently of karyotype status, most transplanted cells had not differentiated to neural cells. C, G, Mitotic NSCs in the graft area were detected by staining for the proliferation marker Ki-67 and counterstaining with haematoxylin and eosin (D,H). I, The percentage of Ki-67-positive ZFN-NSCs was significantly higher in engrafted dup(1)q cells than in grafts containing karyotypically normal NSCs. The total number of nuclei counted in animals transplanted with NSCs with and without dup(1)q was 2991 and 452, respectively. Hoechst 33342 was used to label the nuclei (blue). *P < .05. Scale bars: 100 µm prevent their occurrence as it was demonstrated in several previous studies. 43,[61][62][63][64][65] Nevertheless, the genomic integrity of all cell products used for regenerative approaches should be carefully assessed and monitored to ensure that they are safe and therapeutically effective.

ACK N OWLED G M ENTS
This project was supported by funds from the Deutsche

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.