Experimental method for haplotype phasing across the entire length of chromosome 21 in trisomy 21 cells using a chromosome elimination technique

Ethics statement

This study was performed according to the Declaration of Helsinki and was approved by the Mie University Medical Research Ethics Committee (approval number: 1578). Selection of an individual with Down syndrome who has karyotype (47, XY, + 21) and the procedures for dermal sampling, isolation, and expansion of dermal fibroblasts were performed following an approved protocol. The purpose and content of this study were explained to the parents of the cell donors orally and in writing, and informed consent was obtained. All protocols used for animal experiments in this study were approved by the Animal Experimentation Committee of Mie University (approval number: 29–26). The study was conducted in compliance with the ARRIVE guidelines.

Reprogramming of skin fibroblasts and cell culture

Approximately 1 mm3 of dermal tissue was harvested from a boy with Down syndrome at the time of orthopedic surgery following informed consent. Dermal tissue was sandwiched between two coverslips in a 35 mm dish and cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Thermo Fisher Scientific, Massachusetts, USA) with 10% fetal bovine serum (PAA Laboratories GmbH, Upper Austria, Austria) at 37 °C and 5% CO2 until fibroblasts migrated out of the tissue. The fibroblasts were passaged weekly. At passage 5, 6 × 105 cells were electroporated with episomal plasmid vectors encoding hOCT4, hSOX2, hKLF4, hL-MYC, hLIN-28, short hairpin RNA for TP53 (shp53), and EBNA-1 (Addgene plasmids #27077, #27078, #27080, and #37624) using the Neon transfection system (Invitrogen, Massachusetts, USA) [30, 31]. On day 7, the cells were passaged, and 1 × 105 cells were plated onto a 100 mm tissue culture dish coated with 1.5 × 106 SNL76/7 feeder cells (DS Pharma Biomedical, Osaka, Japan) treated with mitomycin C (Wako, Osaka, Japan). The next day, the culture medium was replaced with Primate ES Cell Medium (Reprocell, Kanagawa, Japan). On day 25–31 post-transduction, embryonic stem cell-like colonies positively stained by rBC2LCN antibody (#180–02991, Wako, Osaka, Japan) were picked up and passaged onto new wells of a 24-well-plate in feeder-free conditions [32] with StemFit AK03 medium (Ajinomoto, Tokyo, Japan) containing 10 µM Y-27632 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and 0.4 µg/mL iMatrix-511 (Nippi, Tokyo, Japan). StemFit AK03 without Y-27632 and iMatrix-511 was used for the standard induced pluripotent stem cells (iPSC) culture. Of the 24 colonies, we selected one colony (original trisomy: T21) based on cell morphology, lower differentiation tendency, and staining properties with an anti-Tra-1–60 antibody (#09–0068, Stemgent, Cambridge, MA). The cells were cultured according to the protocols for human iPSC culture under feeder-free conditions provided by the Center for iPS Cell Research and Application (Kyoto University) [30].

Cell culture

The iPSCs generated from primary human dermal fibroblasts were maintained in 0.4 µg/mL iMatrix-511, using StemFit AK02N medium supplemented with 10 µM Y-27632. To subculture human iPSCs, cells were treated with TrypLE (Life Technologies, NY, USA) at 37 °C for 4 min and scraped off from the wells. After centrifugation at 200 × g for 5 min, the cells were seeded onto a new well with 0.4 µg/mL iMatrix-511 at a density of 5 × 104 cells/cm2. The cells were subcultured every 2–6 days.

Teratoma formation

The cells were washed with PBS, scraped, and collected. Approximately 2 × 106 cells in 100 µL Matrigel (BD Biosciences, New Jersey, USA) were intramuscularly injected into the thigh of a 9-week-old immunodeficient NOD/SCID mouse under 2% isoflurane (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and 100% oxygen anesthesia. Ten weeks after injection, the formed mass was dissected and fixed in 4% (w/v) paraformaldehyde (Merck Corporation, Tokyo, Japan). The tissue was embedded in paraffin, sectioned, and analyzed histologically by hematoxylin and eosin (H&E) staining [33]. Images were captured using an optical microscope (Keyence VHX-800; Osaka, Japan).

Short tandem repeat analysis

To identify the origin of chromosome 21 in trisomy dermal fibroblasts, short tandem repeat (STR) analysis was adapted. Genomic DNA was extracted from the dermal fibroblasts with trisomy 21. Next, DNA samples were obtained by scraping the oral mucosa from the parents. The parental DNA samples were only used for STR analysis to determine the parental origin of chromosome 21 in the trisomy cell line. Genomic DNA was extracted using the QuickExtract DNA Extraction Solution (Lucigen, Wisconsin, USA) according to the manufacturer’s protocol. Multiplex PCR was performed using PrimeSTAR Max DNA polymerase (Takara Bio Inc., Shiga, Japan) with a primer set that amplified the STR loci Penta-D, D21S11, and D21S1411 [34, 35] on chromosome 21. For fragment analysis, we used capillary electrophoresis on the ABI 3130 Genetic Analyzer (Applied Biosystems, Massachusetts, USA). One microliter of PCR product was added to 8.5 µL Hi-Di Formamide (Invitrogen, Massachusetts, USA) and 0.5 µL of Internal Lane Standard 600 size standard (Promega, Wisconsin, USA). After data collection, samples were analyzed using GeneMapper v.4.0 software (Applied Biosystems, Massachusetts, USA). Supplementary Table 1 shows the primer sequences, PCR program, and product sizes used in this study.

Fabrication of disomy 21 iPSCs

Fabrication of disomy 21 iPSCs was accomplished by co-electroporating the targeting vector and sgRNA/Cas9 expression vector into trisomy 21 iPSCs, followed by positive/negative drug selection. An HSF2BP intron 3-specific CRISPR/Cas9 expression vector was constructed in eSpCas9(1.1)-pX330 containing expression cassettes for Streptococcus pyogenes Cas9 nuclease (Addgene #71814). The Cas9 target site on HSF2BP intron 3 (5′-GAGATTGCCTATCGTAGAGTGGGNGG-3′) is located 10 kb downstream of the Penta-D STR locus. In addition, we constructed a “chromosome elimination cassette” (2797 bp) containing a CAG promoter-driven puromycin-delta thymidine kinase (puroΔTK) flanked by inverted loxP sites to bear positive/negative drug selection markers [36, 37] (using puroΔTK fragment from Addgene #84036). The targeting vector (6451 bp DNA plasmid) designed with homology arms against HSF2BP intron 3 (973 and 982 bp in length) (Supplementary Fig. S1) was constructed using the NEBuilder HiFi DNA Assembly system (New England Biolabs Japan, Tokyo, Japan). On the day of transfection (day 0), 5 × 105 cells were dissociated with TrypLE, mixed with CRISPR/Cas9 expression vector (2 µg) and the targeting vector (6 µg), and then electroporated using the Neon Transfection System (Invitrogen, Massachusetts, USA). On day 4, drug selection with puromycin (0.5 µg/mL) was initiated, and the resulting colonies were selected on days 9–10. Junction PCR was performed to test for the elimination cassette integrated at the correct locus. The parental origin of the knocked-in allele was analyzed using STR analysis. The clones were then subjected to Cre recombinase-mediated chromosome elimination (Addgene #13775), followed by negative FIAU selection. After single cells were cloned by limiting dilution, we isolated three types of disomy 21 iPSCs: ΔPaternal chromosome (ΔP), ΔMaternal chromosome 1 (ΔM1), and ΔMaternal chromosome 2 (ΔM2). The isolated colonies were expanded for fluorescence in situ hybridization (FISH), STR, G-band karyotype analysis, NGS, Sanger sequencing, and multiplex ligation-dependent probe amplification (MLPA) analysis.

FISH

We used chromosome 21-specific probe 1 (BAC clone RP11–15E10) and probe 2 (BAC clone RP11–777J19), which were hybridized to 21q21.1 and 21q22.13, respectively. The cells were attached to a glass slide treated with pre-warmed denaturation buffer at 72 °C for 2 min and placed in 70% (w/v) ethanol at 4 °C, and 90% (w/v) and 100% (w/v) ethanol at room temperature (20 to 25 °C) for 5 min. Before hybridization, probes were denatured at 80 °C for 10 min, placed at 37 °C for 30 min, and then applied to the slides, which were then incubated at 37 °C for 16–20 h for hybridization. After washing, the slides were visualized under a microscope using an appropriate fluorescent filter.

G-banding karyotyping

The cells were treated with 0.02 µg/mL colcemid (Gibco, NY, USA) for 2 h to enrich metaphase cells, exposed to buffered hypotonic solution (Genial Helix, Flintshire, UK) for 15 min at 37 °C, and fixed thrice with 3:1 methanol: acetic acid for 5 min each at room temperature. The fixed cells were sent to Chromocenter, Inc. (Tottori, Japan) for high-resolution G-banded karyotyping.

Whole-genome sequencing (WGS)

Genomic DNA was extracted from cell pellets using the QIAprep Spin Miniprep Kit (QIAGEN, Venlo, The Netherlands) and sent to Hokkaido System Science Co. (Hokkaido, Japan). Paired-end 150 base-pair read lengths were sequenced on an Illumina HiSeq X next-generation sequencer, achieving 30-fold coverage on average per sample. The Burrows–Wheeler Aligner (BWA version. 0.7.8-r455) was used to map the paired-end clean reads to the human reference genome (b37, ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/b37/human_g1k_v37_decoy.fasta.gz). The BAM files were viewed using Integrative Genomics Viewer [38], and the SNPs on chromosome 21 were identified using Samtools (version. 1.0) [39]/BCFtools (version. 1.2) [40]. SnpEff [41] was used for annotating genetic variants and creating mutation table data.

Comparison of the combination of SNP data between each corrected disomy cell

Four cellular strains with different combinations of chromosome 21 (T21, ΔP, ΔM1, and ΔM2) were subjected to whole-genome sequencing. Based on the aligned data (BAM format) for four samples obtained from WGS, multi-sample variant calling focusing on chromosome 21 was performed. Among the four samples, SNV locations covered by at least 10 or more reads per sample were extracted. In addition, from the variant data of chromosome 21, variants detected as insertions and deletions (indel) were excluded. In an effort to resolve their haplotypes across chromosome 21, we first calculated a ratio of bases (base allele frequency) at each heterogeneous SNV locus for each cell line, based on the number of the aligned sequence reads. From the variant data, only the variant information in which some bases were detected at a ratio of 0.06 or more in T21 cells with three chromosomes (P, M1, and M2) was extracted to filter base call errors. Theoretically, at any given position, bases specific to the allele disappear only in the cell line in which the allele has been deleted. Theoretically, at any position, bases specific to the allele disappear only in the cell line that the allele has been deleted. Therefore, we selected allele-specific bases for each chromosome 21 using conditions summarized in Supplementary Table 2. By simply comparing the base sequence at the same position, we determined the DNA sequence on the erased chromosome and performed phasing. For example, the base at SNP1 of each T21, ΔM1, and ΔM2 cell has two types of bases: adenine (A) and guanine (G), while ΔP cells have only G, suggesting that SNP1 of P allele is A and those of M1 and M2 alleles are G (Fig. 1B).

Sanger validation and segregation analysis

The primers were designed to include the identified SNP regions using Primer3 online software [42]. PCR was performed using PrimeSTAR Max, according to the manufacturer’s protocol. The PCR products (product size: 306–1 509 bp) were analyzed by Sanger sequencing using an ABI 3130XL DNA Analyzer. The sequencing data were used to compare trisomy cells with those of the corrected disomy cells.

MLPA analysis

Genomic DNA was analyzed using the SALSA MLPA probe-mix P095/Aneuploidy kit (MRC-Holland, Amsterdam, Netherlands), according to the manufacturer’s instructions. The product fragments were separated by capillary electrophoresis on an ABI 3130XL DNA analyzer. MLPA data were analyzed using Coffalyser.Net v.140721.1958 (MRC-Holland, Amsterdam, Netherlands). Following the manufacturer’s instructions, a trisomy was indicated if at least four of the eight relative probe ratios were ≥1.30 when compared with the control for a certain chromosome [43].

Depositing resources

We deposited the trisomy 21 iPS cell line [47, XY, + 21] (identification number HPS4270) and three types of corrected disomy 21 iPS cell line [46,XY] (identification numbers HPS4271, HPS4272, and HPS4273) at the RIKEN BioResource Research Center (Ibaraki, Japan).

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