Derivation of two naturally isogenic iPSC lines (KAUSTi006-A and KAUSTi006-B) from a mosaic Klinefelter Syndrome patient (47-XXY/46-XY)

While Klinefelter Syndrome (KS) has a prevalence of 85–250 per 100,000 born males


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Isogenic KS 47-XXY and 46-XY iPSCs constitute a rare example of naturally isogenic lines derived from a 47-XXY/46-XY mosaic KS patient.They are characterized by a fully matched genetic background and provide a unique cellular platform to study the transcriptional mechanisms underlying KS pathogenesis during early development.

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KS etiopathology is mostly imputable to sex chromosome non-disjunction during gametogenesis at meiosis I or II (Bonomi et al., 2017).The non-disjunction event results in gametes with abnormal sex chromosome number that in turn leads to an XXY zygote.In rare cases, the non-disjunction occurs during early mitotic division of the zygote, thus leading to a mosaic form of KS. (Lanfranco et al., 2004;Bonomi et al., 2017;Anne Skakkebaek et al., 2020).Here, we performed somatic cell reprogramming using mosaic 47-XXY [70%] / 46-XY [30%] KS patient' fibroblasts and successfully isolated a 47-XXY iPSC clone and its isogenic healthy counterpart 46-XY (Table 1).
Somatic cell reprogramming was performed using a non-integrative and virus-free approach, thus mitigating the risks of genomic instability and exogenous factors integration.Specifically, we co-transfected patient fibroblasts with synthetic non-modified RNAs encoding the pluripotency-associated transcription factors OCT4, SOX2, KLF4, cMYC, NANOG, LIN28, combined with (i) the immune evasion mRNAs   cluster to enhance the reprogramming efficiency (Poleganov et al., 2015;Alowaysi et al., 2020) (Fig. 1A).
Between day 10 and day 13, emerging colonies with a typical ESCslike morphology were manually picked and expanded in Essential 8 media (Fig. 1A).The pluripotency of the established iPSC lines (KS6-iPSC#A and KS6-iPSC#B) (Table 1) was confirmed by assessing the expression of the endogenous nuclear markers SOX2, NANOG, OCT4 and surface marker SSEA4 by immunofluorescence (Fig. 1B) and measuring the mRNA levels of SOX2, NANOG and OCT4 via qRT-PCR (Fig. 1C).
The G-banding analysis of the isolated iPSC lines showed a 46-XY normal male chromosomal content in the KS6-iPSC#A and an abnormal 47-XXY karyotype in the KS6-iPSC#B (Fig. 1D).Short tandem repeats (STRs) analysis proved the perfect match of 12 loci between the established KS-iPSC lines and the patient fibroblasts.The analysis of the X-linked STRs also allowed us to demonstrate that the supernumerary X chromosome in this KS patient is consequent to an aberrant chromosomal segregation occurred during meiosis II resulting into two identical Xs (see Supplementary file).
Furthermore, the teratoma formation assay demonstrated that the generated iPSC lines successfully differentiate into derivatives of the three embryonic germ layers expressing: the muscle-specific intermediate filament protein Desmin (mesoderm); the Cytokeratin polypeptides specific of endodermal-epithelial structures (endoderm), and the calcium binding protein S100 found in virtually all nerve sheath tumors (ectoderm) (Fig. 1E and summarized in Table 2).

Immunocytochemistry
Cells were fixed in 3% (w/v) paraformaldehyde for 15 min, permeabilized in PBS containing 0.25% (v/v) Triton X-100 for 5 min, and subsequently blocked in PBS containing 0.2% Gelatin for 5 min.Cells were incubated with primary antibodies overnight at 4 °C and probed with the appropriate secondary antibodies for 45 min at room temperature (Thermo Fisher Scientific).Primary and secondary antibodies were resuspended in 0.2% Gelatin in PBS (Table 3).The nuclei were counterstained with 1 μg /mL DAPI nuclear staining (Thermo Fisher Scientific).Images were acquired on EVOS TM FL Auto 2 Imaging System (Thermo Fisher Scientific).

Quantitative Reverse Transcription PCR (RT-qPCR)
Total RNA was extracted with RNeasy Mini Kit (Qiagen) and was reverse transcribed using SuperScript® VILO cDNA Synthesis Kit (ThermoFisher scientific) according to manufacturer's instructions.Real-time qPCR reactions were run on QuantStudio 3 Real-Time PCR System (ThermoFisher scientific) using TaqMan Master Mix (ThermoFisher scientific) and 10 μM TaqMan® Gene Expression Probes (ThermoFisher scientific) (Table 3).Individual gene expression was normalized on TBP and performed in triplicates.

Karyotype analysis
For G banding karyotyping, iPSC lines were treated 0.3 μg/mL KaryoMAX™ Colcemid™ (1 μg) for 15 min, dissociated by TrypLE and incubated in hypotonic solution (75 mM potassium chloride) at 37 °C for 20 min.iPSCs were then fixed in methanol/glacial acetic acid 3:1 and stored at 4 °C.At least 50 metaphases were karyotyped at the cytogenetic laboratory (Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah).

X-Chromosome Short Tandem Repeat
PCR amplification for the 12 X-chromosomal short tandem repeat (STR) loci was performed according to the Investigator® Argus X-12 QS Kit (Qiagen) and were resolved on a 3730XL DNA analyzer (Thermo Fisher Scientific).Data were collected and analyzed with GeneMapper ID-X software v1.6 (Applied Biosystems).

Teratoma Formation
Approximately 1 × 10 6 iPS cells were dissociated around passages 13 by TryplE Express and resuspended in 60 μl of chilled Matrigel and PBS (v/v).Cells were injected subcutaneously into the dorsal flanks of 8-10 weeks old NSG mice.Teratomas of about 1 cm diameter were extracted, fixed and sectioned for staining with germ layers markers (Table 3).images were acquired on EVOS TM FL Auto 2 Imaging System (1 μg).

Mycoplasma detection
Mycoplasma contamination was assessed using LookOut® Mycoplasma PCR Detection Kit (SIGMA).

Table 1
Summary of lines. T

Table 2
Characterization and validation.