Generation and characterization of induced pluripotent stem cells from breast cancer patients carrying ATM mutations

We generated two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of breast cancer patients carrying germline ATM mutations, a gene associated with a 7% prevalence in breast cancer. These iPSC lines displayed typical morphology, expressed pluripotency markers, maintained a stable karyotype, and retained the ability to differentiate into the three germ layers. These patient-specific iPSC lines hold great potential for mechanistic investigations and the development of drug screening strategies aimed at addressing ATM-related cancer.


Resource utility
Germline mutations in the ATM gene are known to cause the autosomal recessive Ataxia Telangiectasia disease and also confer an increased risk of developing breast cancer.Generating iPSC lines carrying ATM variants provide an unlimited source for disease modeling, gene therapy, and screening of compounds for potential therapeutic effects (Table 1).

Resource details
The Ataxia Telangiectasia Mutated (ATM) gene, also known as ATM serine/threonine kinase, is a crucial tumor suppressor gene that belongs to the phosphatidylinositol 3-kinaserelated protein kinase (PIKK) superfamily.It plays a pivotal role in DNA repair and cell cycle control (Moslemi et al., 2021).Mutations in the ATM gene are the primary cause of Ataxia Telangiectasia, an autosomal recessive neurodegenerative disorder.Recent studies have revealed a significant association between ATM variants and the risk of multiple types of cancers, particularly breast cancer (Stucci et al., 2021).Carriers of pathogenic ATM variants have a 2 to 4-fold increased risk of developing breast cancer (McDuff et al., 2021), especially early-onset cancer and bilateral breast cancer (Renwick et al., 2006).To understand these genetic associations, we successfully generated and characterized iPSC lines derived from female donors carrying specific ATM variants: c.4143dup and c.5697C > A, respectively.These iPSC lines serve as renewable and genetically relevant cellular models for investigating disease pathology and conducting drug screening for precision medicine.
We recruited two breast cancer patients, a 72-year-old White female who developed breast cancer, stage II (T2N1M0), of the left breast and a 23-year-old East Asian female who developed breast cancer, stage IV (T3N3M1), of the left breast.Genetic testing demonstrated that they carried the pathogenic variants ATM c.4143dup (ClinVar ID: 181880) and c.5697C > A (ClinVar ID: 421488), respectively.Using a Sendai Virusbased vector carrying the Yamanaka factors OCT4, SOX2, KLF4, and c-MYC (Mondéjar-Parreño et al., 2021), we successfully generated iPSCs from the patients' peripheral blood mononuclear cells (PBMCs), named SCVIi083-A and SCVIi084-A.Both lines exhibited typical stem cell morphology when observed under bright field microscope (Fig. 1A).These cells expressed pluripotent markers (SOX2, NANOG, and POU5F1) and lost the expression of Sendai virus vector (SEV), as shown by quantitative RT-PCR (Fig. 1C).The iPSCs were further analyzed for pluripotency markers using immunofluorescence staining (Fig. 1B).The iPSC lines tested negative for mycoplasma (Fig. 1D).Karyotype analysis exhibited normal female chromosomes (Fig. 1E).Sanger sequencing demonstrated a heterozygous mutation of c.4143dup in SCVIi083-A and c.5697(C > A) in SCVIi084-A (Fig. 1F).Short tandem repeat (STR) analysis of the parental PBMCs and derived iPSCs confirmed clonal identity (Submitted in the archive with journal).Both lines were successfully differentiated into the three germ layers (Fig. 1G).

Cell culture
The iPSCs were passaged at 90 % confluency using Gentle Cell Dissociation Reagent (STEMCELL ™ technologies).Detached cells were resuspended in Brew medium with 5 μM ROCK1 inhibitor (SelleckChem), and replated onto Matrigel-coated (1:500) 6-well plates.Cells were cultured for 24 hr at 37 °C with 5 % CO 2 , and after that the media was replaced with Brew medium every two days.

RNA extraction and RT-qPCR
Total RNA was extracted from iPSCs at passage 13 using the miRNeasy Micro Kit (Qiagen), then cDNA was synthesized using the iScript ™ Reverse Transcription Supermix (BIO-RAD).Target genes were examined using the TagMan ™ Universal PCR Master Mix (ThermoFisher Scientific) and probes (Table 2).

Immunofluorescence staining
Cells at passage 18 were fixed with 4 % paraformaldehyde for 20 min.After two washes with DPBS, the cells were permeabilized using 0.1 % Triton X 100 in DPBS for 10 min.
Subsequently, blocking was performed with 10 % goat serum in DPBS for 1 hr.Cells were then incubated with primary antibodies (Table 2) overnight at 4 °C.On day 2, the cells were washed with DPBS and incubated with the corresponding secondary antibodies (Table 2) for 1 hr at room temperature.The nuclei were counterstained with NucBlue Probes (ThermoFisher Scientific).

Karyotyping
The Karyotyping was performed on iPSCs at passage 12 using the KaryoStat ™ assay (ThermoFisher Scientific).

Targeted sequencing
The genomic DNA was extracted using the QuickExtract ™ DNA extraction solution (Biosearch Technologies).The PCR assay was performed using the PrimeSTAR GXL DNA Polymerase (Clontech) and the primers (Table 2) under the following conditions: 98 °C for 5 s 60 °C for 15 s, 72 °C for 30 s for 35 cycles.The PCR products were purified, and the sequencing was performed at Stanford Protein and Nuclear Acid (PAN) facility.

Mycoplasma detection
The mycoplasma test was conducted on iPSCs at passage 13 using the MycoAlertTM Detection Kit (Lonza), following the manufacturer's instructions.

Trilineage differentiation
iPSCs at passage 17 were used for all three-germ layer differentiation.The StemXVivo Ectoderm kit (R & D systems) and the StemDiff ™ Definitive Endoderm differentiation kit (STEMCELL ™ Technologies) were used to derive the ectoderm and the endoderm, respectively.Mesoderm differentiation was induced by 6 μM CHIR-99021 (Selleck Chemicals) in RPMI media supplemented with B27 minus Insulin for 48 hr.

Short tandem repeat (STR) analysis
Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen).PCR and capillary electrophoresis were performed using the CLA IdentiFiler ™ Direct PCR Amplification Kit and ABI3130xl at the Stanford PAN facility.Characterization of two iPSC lines derived from breast cancer patients carrying ATM mutations.
Table 1 Characterization and validation.

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Photography brightfield