Autonomous trisomic rescue of Down syndrome cells

Down syndrome is the most frequent chromosomal abnormality among live-born infants. All Down syndrome patients have mental retardation and are prone to develop early onset Alzheimer’s disease. However, it has not yet been elucidated whether there is a correlation between the phenotype of Down syndrome and the extra chromosome 21. In this study, we continuously cultivated induced pluripotent stem cells (iPSCs) with chromosome 21 trisomy for more than 70 weeks, and serendipitously obtained revertant cells with normal chromosome 21 diploids from the trisomic cells during long-term cultivation. Repeated experiments revealed that this trisomy rescue was not due to mosaicism of chromosome 21 diploid cells and occurred at an extremely high frequency. We herewith report the spontaneous correction from chromosome 21 trisomy to disomy without genetic manipulation, chemical treatment or exposure to irradiation. The revertant diploid cells will possibly serve a reference for drug screening and a raw material of regenerative medicinal products for cell-based therapy.


Introduction
Recently, attention to prenatal diagnosis is increasing due to the higher average age of pregnant women. Due to the availability of diagnostic techniques such as non-invasive prenatal genetic testing and improvement of imaging technology, congenital diseases including chromosomal abnormalities are possible to diagnose earlier than before [1,2]. On the other hand, there are few genetic disorders in which early diagnosis contributes to the improvement of the prognosis of children. Down syndrome is the most frequent chromosomal abnormality among live-born infants. All Down syndrome patients have mental retardation and are prone to develop early onset Alzheimer's disease. In addition, leukemia, cardiac malformation, hearing disorders, and vision disorders are also seen at a high rate. Hyperkeratosis of the skin is occasionally observed [3]. Ninety percent of Down syndrome cases are due to an extra copy of chromosome 21 and the remainder exhibit imbalanced translocation or mosaicism. Triplication of specific regions of chromosome 21, band 21q22, causes various physical and cognitive phenotypes of Down syndrome, and the causative genes include amyloid beta precursor protein (APP) related to Alzheimer's disease, and superoxide dismutase 1 (SOD 1) involved in the onset of amyotrophic lateral sclerosis [4,5]. In addition, dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) and Down syndrome critical region gene 1 (DSCR1) on chromosome 21 are related to neurogenesis [6]. DYRK1A has attracted attention as a target for normalizing the phenotype of Down syndrome [7,8]. DYRK1A inhibitor as a therapeutic agent for Down syndrome has been widely studied and developed and has been tested in clinical trials [9]. Additionally, low molecular weight molecules that improve the phenotype of Down syndrome have also been tested [10].
To develop drugs for Down syndrome, murine models for Down syndrome or trisomy 21 have been developed. Since the distal part of mouse chromosome 16 is orthologous to a large portion of human chromosome 21, mouse models, in particular the chromosome 16 segmental trisomies, Ts65Dn and Ts1Cje, were produced [11,12]. These models are used for exploration of the etiology of Down syndrome and drug development [13][14][15]. Ts65Dn mice mimic the human condition, including developmental delay [16] and memory deficit, and may therefore be used for drug development with the aim of improving cognitive function [7]. Likewise, Ts1Cje carries a segmental trisomy of mouse chromosome 16 [12] and shows Down syndromerelated abnormalities such as craniofacial alterations [17] and spatial learning deficits [12]. Maternal supplementation of low molecular weight molecules such as epigallocatechin-3-gallate, fluoxetine, neuroprotective peptide, and choline during pregnancy improve function of these model mice [8,10,18,19]. Neural stem cell-based therapy was also attempted with neonatal Down syndrome mice [20]. Further studies are necessary in order to determine the efficacy of these therapies.
Immortality of induced pluripotent stem cells (iPSCs) makes it possible to obtain a large number of cells from a small specimen, and pluripotency enables differentiation into various cell types [21][22][23][24]. Therefore, they are widely used to clarify disease etiology and test therapeutic drugs [25][26][27][28]. Attempts to normalize chromosomal abnormalities have been drawing intense research interest in the study of Down syndrome using iPSCs. In order to determine the mechanism of development of Down syndrome, normal cells are needed as controls. In a previous study, a comparison between monozygotic twins discordant for trisomy 21 had been performed [29]. Previous studies have reported normalization with using genome editing techniques and spontaneous correction during reprogramming to iPSCs [30][31][32][33]. In this study, iPSCs with the normal karyotype, i.e., chromosome 21-diploid cells, was detected at a high frequency in the process of culturing iPSCs derived from a patient with Down syndrome. In order to investigate the properties of trisomy 21 cells, we have characterized disomic and trisomic subclones that are isogenic with the exception of chromosome 21.

Human cells
Amniotic fluid was obtained from a fetus with Down syndrome associated with polyhydroamnios. It was collected at 29 weeks of gestation for the purpose of reducing amniotic fluid. Cells were incubated in 4 mL of Amnio-MAX-II complete medium (Invitrogen, catalog number (#) 11269-016). Cell clusters appeared 6 to 7 days after seeding. Nonadherent cells were discarded and the medium replaced every 2 days. When the culture reached subconfluence, cells were harvested with a trypsin-EDTA solution (Wako, #209-16941) and re-plated at a 1:8 ratio in a 60-mm dish.

Real-time qPCR
RNA was extracted from cells using the RNeasy Mini kit (Qiagen, #74104). An aliquot of total RNA was reversetranscribed using an oligo (dT) primer (Invitrogen, #18418-020). For the thermal cycle reactions, the cDNA template was amplified (Applied Biosystems Quantstudio 12 K Flex Real-Time PCR System) with gene-specific primer sets (Table 1) using the Platinum SYBR Green qPCR SuperMix-UDG with

Karyotypic analysis
Karyotypic analysis was performed at the Chromosome Science Labo Inc. Chromosome spreads were Giemsa banded and photographed. Twenty metaphase spreads were analyzed for each sample and karyotyped using a chromosome imaging analyzer system (Applied Spectral Imaging).

Short tandem repeat analysis
STR analysis was conducted at BEX facility. Genomic DNA was used and 16 microsatellite markers were amplified by PCR using microsatellite specific primers.

Microarray analysis
RNA extraction and microarray analysis were performed at DNA Chip Research Inc. RNA extraction was performed using the Qiagen RNeasy mini kit (Qiagen, #74104) and cRNA synthesis was carried out according to the manufacturer's protocol using Low Input Quick Amp Labeling Kit (Agilent, #5190-2305). Hybridization was performed using SurePrint G3 Human Gene Expression 8 × 60 K v3 (Agilent, G4858A #072363).

Fluorescence-activated cell sorting analysis
The expression of cell-surface markers was analyzed by BD LSR Fortessa (BD Biosciences). Primary antibodies were incubated for 1 h in PBS with 1% BSA. After washing with PBS, cells were incubated with fluorescently coupled secondary antibodies; anti-rabbit IgG conjugated with Alexa 488 (1:1000) for 30 min at room temperature.

Comparison of T21-iPSCs and D21-iPSCs
We examined T21-iPSC#12 and D21-iPSC#2 to investigate difference of the growth rate. The growth rates of these two clones were comparable (Fig. 2a). We performed gene chip analysis on T21-iPSC#12, D21-iPSC#1, and D21-iPSC#2 to investigate gene expression levels. Expression levels of genes on chromosome 21 and all chromosomes are shown in heat map and hierarchical clustering analysis (Fig. 2b, c). D21-iPSC#1 and −2, i.e., two independent iPSC subclones with normal chromosome 21, are categorized into the same group. Up-regulated and down-regulated genes at 10-fold difference are listed in Tables 3 and 4. We then performed qRT-PCR analysis of the genes on Chromosome 21, based on the results of the gene chip analysis (Fig. 2d-h). The expression levels of the genes for APP (Alzheimer's disease marker), DYRK1A, DSCR1 (Down-syndrome critical region 1), ETS2 and SOD1, all of which are located in chromosome 21, decreased to two-thirds in D21-iPSC#2, compared to T21-iPSC#12, implying that the revertant cells regained the gene expression levels of intact iPSCs. *p < 0.05. e Quantitative RT-PCR analysis for expression of DYRK1A in T21-iPSC#12 and D21-iPSC#2. Data shown are mean ± SD of the expression from three independent experiments. *p < 0.05. f Quantitative RT-PCR analysis for expression of DSCR1 in T21-iPSC#12 and D21-iPSC#2. Data shown are mean ± SD of the expression from three independent experiments. *p < 0.05. g Quantitative RT-PCR analysis for expression of SOD1 in T21-iPSC#12 and D21-iPSC#2. Data shown are mean ± SD of the expression from three independent experiments. *p < 0.05. h Quantitative RT-PCR analysis for expression of ETS2 in T21-iPSC#12 and D21-iPSC#2. Data shown are mean ± SD of the expression from three independent experiments. N.S., not significant

Discussion
In this study, we introduced a spontaneous trisomy rescue in Down syndrome-derived iPSCs. Chromosome 21 trisomy has been reported to be mostly due chromosomal nondisjunction during meiosis I in the maternal egg. In contrast, paternal chromosomal non-disjunction occurs during meiosis II (spermatidogenesis). Non-disjunction of chromosomes during meiosis I and meiosis II result in heterologous pair of chromosomes and duplicated homologous pair of chromosomes, respectively. Presence of three different STR patterns in T21-iPSC#12 generated in this study suggests chromosomal non-disjunction during meiosis I. Possibility of other cell contamination can also be excluded by the same STR pattern between the trisomic and disomic cells. Furthermore, mosaicism of trisomic and disomic cells from the donor can also be excluded because the amniotic fluid-derived parental cells exhibit chromosome 21 trisomy exclusively and disomic cells have been reproducibly obtained from 100% trisomy cells. Proliferation rates of trisomic cells are considered to be tissue-dependent; the proliferation rate of trisomic cells is low in mouse embryonic fibroblasts and in fibroblasts from Down syndrome fetuses, but high in hematopoietic cells [41][42][43][44]. Trisomy 21 cells are highly responsive to a variety of external stimuli acting through cell-surface receptors, such as genes encoding the interferon-α/β receptor and IFN-γ receptor on chromosome 21 [44]. The differential growth rates of iPSCderived keratinocytes and NSCs in this study were consistent with this tissue-dependent Trisomy 21 cell growth.
Trisomy rescue arises from mitotic or meiotic nondisjunction, and the nondisjunction of chromosome 21 occurs more often in trisomic cells than in normal cells [45,46]. Likewise, trisomic rescue during iPSC cultivation in this study can possibly be attributed to chromosomal nondisjunction. Interestingly, the proportion of trisomic cells in blood cells is lower than that in skin fibroblasts cultured from the same individual, and the frequency of trisomic rescue is tissue-dependent [44]. Elucidating the conditions in which aneuploid iPSCs produce revertant cells may facilitate the development of treatments for various chromosomal abnormalities.
Differences between trisomic and disomic cells are of interest because the genetic background of these cells is the same. The benefit of Down syndrome iPSC availability is pluripotency and immortality. Additionally, Down syndrome iPSCs can efficiently differentiate into neural and hematopoietic cells that are associated with mental retardation and leukemogenesis, respectively. Expression levels of Down syndrome-related genes such as SOD1, DYRK1A, ETS2, APP, and DSCR1 in chromosome 21 are comparable with the gene number, i.e., three 21 chromosomes. Cells rescued from trisomy serve as a good control for Down syndrome cells due to the same/similar genetic background. These differentiated cells can contribute to drug development for Down syndrome in two ways: (1) Correction of gene expression levels, i.e., ets2, dyrk1a, dscr1, and app; (2) Increased frequency of trisomic rescue. Drug re-profiling is most practical to fetus with Down syndrome as well as Down syndrome patients. Predicted usage may include treatment of mental retardation and prevention of leukemogenesis. Low molecular weight molecules have been clinically investigated in Down syndrome patients [9]. However, no studies have yet reported correction of mental retardation. Neurogenesis continues in the ventricular and subventricular zones of the cerebral cortex in the third trimester of pregnancy [47], and therapeutic intervention in fetus could therefore be possible. Drug may restore neurogenesis, enhance cortical growth, and improve the neurodevelopmental outcome of Down syndrome.
Acknowledgements The authors would like to thank Kenji Miyado for the fruitful discussion.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical statement The protocol for using human cells in this study was approved by the Institution Review Board of the National Research Institute for Child Health and Development of Japan and was fully compliant with the Ethical Guidelines for Clinical Studies (Ministry of Health, Labor, and Welfare).
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