Thermobrite system

Isolated trophoblast were centrifuged and incubated with 0.5% KCL and incubated at 37°C for 20 minutes. Subsequently, cells were treated with ice-cold fixative (one part acetic acid and three parts methanol) and incubated for 10 minutes at -20°C. Fixed cells were air-dried on glass slides at 42°C for 15 minutes and treated with pepsin (350 μl 0.5% Pepsin with 1 ml 1N HCl ad 100 ml H2O) for 15 min at 37°C. Slides were then washed in PBS and PBS/20mM MgCl2 each 5 minutes at room temperature. Subsequent to dehydration in a 10% formaldehyde solution, cells were incubated with a “ready to use” solution containing centromer specific probes for X- and Y-chromosomes (DXZ1 (green) and DYZ3 (red) (Leica Biosystems). Hybridization was performed using a ThermoBrite system (Leica) for 16 h at 37°C after 5 minutes denaturation at 75°C. Afterwards, cells were washed for two minutes with 0.4x SSC with 0.3% NP40 followed by a 1 minute wash step with 2x SSC with 0.1% NP40. Slides were air-dried and covered with Vectashield Mounting Medium containing DAPI (Vector Laboratories Burlingame, USA). FISH signals were detected using an Axioplane2 imaging system (Zeiss) equipped with “MetaSystems Isis” software version 5.3.18.

FISH analyses were carried on both metaphase arrested and cycling interphase nuclei of ESCs. The probes were purchased from Empire Genomics, USA (Catalog # MYBPC3-20GR and MYH7-20-OR). FISH probes specific for MYBPC3 (11p11.2 locus, ~188Kb) were labeled using Green-dUTP, and for MYH7 (14q11.2 locus, ~177 Kb) were labeled using Orange-dUTP. Briefly, ESCs were treated with KaryoMAX Colcemide (Life Technologies) at a final concentration of 200 ng/mL for 1.5 h at 37 °C. Treated cells were then detached by 0.25% trypsin/EDTA and incubated in hypotonic 0.075 M KCL for 20 min. Cells were next fixed with methanol: acetic acid (3:1 v/v) and dropped onto a slide and dried on a hot plate at 60 °C. The samples were dehydrated using ethanol (70, 85, and 100%) for 1 min in each and dried in air. Slides were applied with the probe mixture, covered with an 18 mm² coverslip, and incubated in a humidified Thermobrite® system (Leica) set at 73 °C for 2 min, and then 37 °C for 16 h. The incubated slides were rinsed with washing solution 1 (0.3% Igepal/0.4 × SSC) and washing solution 2 (0.1% Igepal/2 × SSC). Slides were mounted in ProLong™ Gold Antifade Mountant with DAPI (Life Technologies) and observed using a fluorescence microscopy equipped with a cooled CCD camera. Images were captured and analyzed by ISIS analysis software (MetaSystem GmbH).

Conventional G-banded chromosomal analysis was performed on unstimulated bone marrow aspiration or peripheral blood cultured for 47 h or 71 h using standard techniques. No fewer than 20 meta-phases were karyotyped. An abnormal karyotype (AK) was defined by the presence of at least one chromosomal abnormality, and a complex karyotype (CK) was defined by the presence of three or more chromosomal abnormalities in a single clone. Vysis probes (Abbott Molecular, Des Plaines, IL, USA) for CEP12, 11q22.3, 17p13.1, and 13q14.2 were used for FISH analysis. Hybridization and post-hybridization washes were performed by following the corresponding manufacturer’s protocol based on the ThermoBrite system (Leica Biosystems, Buffalo Grove, IL, USA), and the cells were counterstained with DAPI II (marrow aspiration and peripheral blood) or DAPI I (formalin-fixed paraffin-embedded tissue). The results were reported according to the International System for Human Cytogenetic Nomenclature (ISCN) 2016.