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# Establishment of induced pluripotent stem cells IRMBi005-A from a patient with sporadic Alzheimer's disease

C Clua Provost, L. Auboyer, Anne Rovelet-Lecrux, C. Monzo, Eliot Schob, S.

Lehmann, D. Wallon, C. Crozet

► To cite this version:

C Clua Provost, L. Auboyer, Anne Rovelet-Lecrux, C. Monzo, Eliot Schob, et al.. Establishment of induced pluripotent stem cells IRMBi005-A from a patient with sporadic Alzheimer's disease. Stem Cell Research, 2023, 72, pp.103216. 10.1016/j.scr.2023.103216. hal-04597809

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https://hal.science/hal-04597809

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# Establishment of induced pluripotent stem cells IRMBi005-A from a patient with sporadic Alzheimer's disease

C. Clua Provost<sup>a, 1</sup>, L. Auboyer<sup>b, 1</sup>, A. Rovelet-Lecrux<sup>c</sup>, C. Monzo<sup>a</sup>, Eliot Schob<sup>a</sup>, S. Lehmann<sup>a</sup>, D. Wallon<sup>d</sup>, C. Crozet<sup>a, b, *</sup>

## ABSTRACT

Alzheimer's disease (AD) is a progressive neurological disorder and the most common form of dementia worldwide. Sporadic Alzheimer's disease (sAD) cases are the main forms, over 95% of AD cases, but still poorly understood. Thereby there is a crucial need to develop *in vitro* models for studying this multifactorial disorder. Here, we report the reprogramming of skin fibroblasts from a 57-years-old male donor. The new generated iPSC cell line has a normal karyotype and, is pluripotent since it demonstrates the ability to differentiate *in vitro* into the three germ layers. This iPSC line will be used to understand pathological mechanisms of sAD.

## 1. Resource Table

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi005-A</td></tr><tr><td>Alternative name(s) of stem cell line</td><td>SAD-hiPSC</td></tr><tr><td>Institution</td><td>INM, Univ Montpellier, INSERM, Montpellier, France</td></tr><tr><td>Contact information of distributor</td><td>Carole CROZET carole.crozet@inserm.fr</td></tr><tr><td>Type of cell line</td><td>iPSC</td></tr><tr><td>Origin</td><td>Human</td></tr><tr><td>Additional origin info required for human ESC or iPSC</td><td>Age: 57 years' old<br/>Sex: Male<br/>Ethnicity if known: white</td></tr><tr><td>Cell Source</td><td>Fibroblasts</td></tr><tr><td>Clonality</td><td>Clonal</td></tr><tr><td>Method of reprogramming</td><td>Sendai virus Cytotune 2.0 kit (Thermofisher Scientific Inc.), hOCT4, hSOX2, hC-MYC, hKLF4</td></tr><tr><td>Genetic Modification</td><td>NO</td></tr><tr><td>Type of Genetic Modification</td><td>N/A</td></tr><tr><td>Evidence of the reprogramming transgene loss (including genomic copy if applicable)</td><td>Sendai virus and c-Myc PCR</td></tr><tr><td>Associated disease</td><td>Alzheimer's disease</td></tr><tr><td>Gene/locus</td><td>No Associated mutation</td></tr><tr><td>Date archived/stock date</td><td>07/26/2023</td></tr></table>

(continued on next column)

(continued)

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi005-A</td></tr><tr><td>Cell line repository/bank</td><td>https://hpscreg.eu/cell-line/I RMBi005-A</td></tr><tr><td>Ethical approval</td><td>DC-2015-2363 regional scientific ethical committee CPP Sud Med IV Informed consent was obtained from the patient.</td></tr></table>

## 2. Resource utility

The reported cell line may be useful to model and investigate the pathogenesis of sporadic Alzheimer's disease (sAD), the most common and complex form of the disease. It also constitutes a promising tool for drug screening.

## 3. Resource details

This paper describes the generation of an iPSC cell line from a patient with sporadic AD. Skin fibroblasts were obtained from a 57-year-old man diagnosed with sporadic Alzheimer's disease at 48. Fibroblasts were reprogrammed into iPSC with integration-free Sendai virus carrying gene sequences of the four Yamanaka factors OCT3/4, SOX2, KLF4

<footnote><sup>a</sup> INM, Univ Montpellier, INSERM, Montpellier, France</footnote>

<footnote><sup>b</sup> Institute for Regenerative Medicine and Biotherapy (IRMB), Univ Montpellier, INSERM, CHU Montpellier, Montpellier, France</footnote>

<footnote><sup>c</sup> Normandie Univ, UNIROUEN, INSERM U1245, Rouen University Hospital, Department of Genetics and CNR-MAJ, Normandy Center for Genomic and Personalized Medicine, F 76000 Rouen, France</footnote>

<footnote><sup>d</sup> Normandie Univ, UNIROUEN, INSERM U1245, Rouen University Hospital, Department of Neurology and CNR-MAJ, Normandy Center for Genomic and Personalized Medicine, F 76000 Rouen, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote n="1">¹ Co-first author.</footnote>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

# Establishment of induced pluripotent stem cells IRMBi005-A from a patient with sporadic Alzheimer's disease

C. Clua Provost<sup>a,1</sup>, L. Auboyer<sup>b,1</sup>, A. Rovelet-Lecrux<sup>c</sup>, C. Monzo<sup>a</sup>, Eliot Schob<sup>a</sup>, S. Lehmann<sup>a</sup>, D. Wallon<sup>d</sup>, C. Crozet<sup>a,b,*</sup>

## ABSTRACT

Alzheimer's disease (AD) is a progressive neurological disorder and the most common form of dementia worldwide. Sporadic Alzheimer's disease (sAD) cases are the main forms, over 95% of AD cases, but still poorly understood. Thereby there is a crucial need to develop *in vitro* models for studying this multifactorial disorder. Here, we report the reprogramming of skin fibroblasts from a 57-years-old male donor. The new generated iPSC cell line has a normal karyotype and, is pluripotent since it demonstrates the ability to differentiate *in vitro* into the three germ layers. This iPSC line will be used to understand pathological mechanisms of sAD.

## 1. Resource Table

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi005-A</td></tr><tr><td>Alternative name(s) of stem cell line</td><td>SAD-hiPSC</td></tr><tr><td>Institution</td><td>INM, Univ Montpellier, INSERM, Montpellier, France</td></tr><tr><td>Contact information of distributor</td><td>Carole CROZET carole.crozet@inserm.fr</td></tr><tr><td>Type of cell line</td><td>iPSC</td></tr><tr><td>Origin</td><td>Human</td></tr><tr><td>Additional origin info required for human ESC or iPSC</td><td>Age: 57 years' old<br>Sex: Male<br>Ethnicity if known: white</td></tr><tr><td>Cell Source</td><td>Fibroblasts</td></tr><tr><td>Clonality</td><td>Clonal</td></tr><tr><td>Method of reprogramming</td><td>Sendai virus Cytotune 2.0 kit (Thermofisher Scientific Inc.), hOCT4, hSOX2, hC-MYC, hKLF4</td></tr><tr><td>Genetic Modification</td><td>NO</td></tr><tr><td>Type of Genetic Modification</td><td>N/A</td></tr><tr><td>Evidence of the reprogramming transgene loss (including genomic copy if applicable)</td><td>Sendai virus and c-Myc PCR</td></tr><tr><td>Associated disease</td><td>Alzheimer's disease</td></tr><tr><td>Gene/locus</td><td>No Associated mutation</td></tr><tr><td>Date archived/stock date</td><td>07/26/2023</td></tr></table>

(continued on next column)

(continued)

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi005-A</td></tr><tr><td>Cell line repository/bank</td><td>https://hpscreg.eu/cell-line/I RMBi005-A</td></tr><tr><td>Ethical approval</td><td>DC-2015-2363 regional scientific ethical committee CPP Sud Med IV Informed consent was obtained from the patient.</td></tr></table>

## 2. Resource utility

The reported cell line may be useful to model and investigate the pathogenesis of sporadic Alzheimer's disease (sAD), the most common and complex form of the disease. It also constitutes a promising tool for drug screening.

## 3. Resource details

This paper describes the generation of an iPSC cell line from a patient with sporadic AD. Skin fibroblasts were obtained from a 57-year-old man diagnosed with sporadic Alzheimer's disease at 48. Fibroblasts were reprogrammed into iPSC with integration-free Sendai virus carrying gene sequences of the four Yamanaka factors OCT3/4, SOX2, KLF4

<footnote><sup>a</sup> INM, Univ Montpellier, INSERM, Montpellier, France</footnote>

<footnote><sup>b</sup> Institute for Regenerative Medicine and Biotherapy (IRMB), Univ Montpellier, INSERM, CHU Montpellier, Montpellier, France</footnote>

<footnote><sup>c</sup> Normandie Univ, UNIROUEN, INSERM U1245, Rouen University Hospital, Department of Genetics and CNR-MAJ, Normandy Center for Genomic and Personalized Medicine, F 76000 Rouen, France</footnote>

<footnote><sup>d</sup> Normandie Univ, UNIROUEN, INSERM U1245, Rouen University Hospital, Department of Neurology and CNR-MAJ, Normandy Center for Genomic and Personalized Medicine, F 76000 Rouen, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote n="1">¹ Co-first author.</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 1.</figcaption></figure>

and c-MYC. Several iPSC colonies with normal morphology (Fig. 1A) were picked for expansion. The disappearance of Sendai virus was successfully established by RT-PCR analysis with Sendai virus specific primers in a selected iPS clone after 10 passages. The transgenes and viral sequences were also absent in the negative controls, non-transduced human healthy fibroblasts (hFF SAD Nt) and clone of interest, but present in fibroblasts freshly transduced with the Sendai virus (SAD Sendaï +) (Fig. 1B). Expression of endogenous pluripotency markers OCT4, SOX2, NANOG was confirmed by qPCR analysis (Fig. 1C) and immunofluorescent staining expression of the embryonic stem cell specific marker SSEA4 (Fig. 1A) and the nuclear localization of the two pluripotency factors OCT4 and SOX2 (Fig. 1A). Pluripotency of the clone was assessed through in vitro directed differentiation of iPS colonies to each germ layers, followed by immunofluorescence of ectodermal and endodermal markers, respectively PAX6 and Nestin and FOXA2 and SOX17, or RT-PCR of mesodermal makers, Brachyury and α-smooth

muscle actin (Fig. 1D). The SAD clone presents a normal karyotype 46, XY after 13 passages and no evidence of recurrent genomic abnormalities was detected (Fig. 1E) (Assou et al., 2020). The short tandem repeat (STR) profile established the 100% identity of the iPSC line to parental cells. The SAD clone was also shown to be mycoplasma free (Supplementary Data).

# 4. Materials and methods

## 4.1. Generation of iPSC

After amplification in DMEM (Gibco) supplemented with 10% FBS (Lonza), 1% NEAA, 0.1 mM β-mercaptoethanol, 1 mM glutamine and 1% penicillin/streptomycin (Gibco), fibroblasts were reprogrammed using the Cytotune-iPSC 2.0 Sendai Reprogramming kit (Life Technologies) (KOS MOI = 5, hc-Myc MOI = 5, hklf4 MOI = 3). The reprogramming
<figure><img src="image_5.png" /><figcaption>Fig. 1.</figcaption></figure>

and c-MYC. Several iPSC colonies with normal morphology (Fig. 1A) were picked for expansion. The disappearance of Sendai virus was successfully established by RT-PCR analysis with Sendai virus specific primers in a selected iPS clone after 10 passages. The transgenes and viral sequences were also absent in the negative controls, non-transduced human healthy fibroblasts (hFF SAD Nt) and clone of interest, but present in fibroblasts freshly transduced with the Sendaï virus (SAD Sendaï +) (Fig. 1B). Expression of endogenous pluripotency markers OCT4, SOX2, NANOG was confirmed by qPCR analysis (Fig. 1C) and immunofluorescent staining expression of the embryonic stem cell specific marker SSEA4 (Fig. 1A) and the nuclear localization of the two pluripotency factors OCT4 and SOX2 (Fig. 1A). Pluripotency of the clone was assessed through in vitro directed differentiation of iPS colonies to each germ layers, followed by immunofluorescence of ectodermal and endodermal markers, respectively PAX6 and Nestin and FOXA2 and SOX17, or RT-PCR of mesodermal makers, Brachyury and α-smooth

muscle actin (Fig. 1D). The SAD clone presents a normal karyotype 46, XY after 13 passages and no evidence of recurrent genomic abnormalities was detected (Fig. 1E) (Assou et al., 2020). The short tandem repeat (STR) profile established the 100% identity of the iPSC line to parental cells. The SAD clone was also shown to be mycoplasma free (Supplementary Data).

# 4. Materials and methods

## 4.1. Generation of iPSC

After amplification in DMEM (Gibco) supplemented with 10% FBS (Lonza), 1% NEAA, 0.1 mM β-mercaptoethanol, 1 mM glutamine and 1% penicillin/streptomycin (Gibco), fibroblasts were reprogrammed using the Cytotune-iPSC 2.0 Sendai Reprogramming kit (Life Technologies) (KOS MOI = 5, hc-Myc MOI = 5, hklf4 MOI = 3). The reprogramming
<table><caption>**Table 1**

Characterization and validation.</caption><thead><tr><th>Classification</th><th>Test</th><th>Result</th><th>Data</th></tr></thead><tbody><tr><td rowspan="3">Morphology<br>Phenotype</td><td>Photography Bright field</td><td>Normal</td><td>Fig. 1A</td></tr><tr><td>Qualitative analysis (Immunocytochemistry)</td><td>Expression of pluripotency markers: Oct3/4, SOX2, SSEA-4</td><td>Fig. 1A</td></tr><tr><td>Quantitative analysis<br>(RT-qPCR)</td><td>Expression of Oct3/4, SOX2, NANOG</td><td>Fig. 1C</td></tr><tr><td rowspan="2">Genotype</td><td>Karyotype (G-banding) and resolution</td><td>46XY, Resolution 400</td><td>Fig. 1E</td></tr><tr><td>Recurrent genomic abnormalities</td><td>No recurrent genetic abnormalities detected</td><td></td></tr><tr><td rowspan="2">Identity</td><td>Microsatellite PCR (mPCR) OR<br>STR analysis</td><td>Not performed<br>4 matched sites</td><td>NA<br>Available with<br>authors</td></tr><tr><td>Mutation analysis (IF<br>APPLICABLE)</td><td>Sequencing<br>Southern Blot OR WGS</td><td>NA<br>NA</td><td>NA<br>NA</td></tr><tr><td>Microbiology and virology</td><td>Mycoplasma</td><td>Mycoplasma testing by luminescence. Negative</td><td>Supplementary<br>data</td></tr><tr><td>Differentiation potential</td><td>In vitro directed differentiation</td><td>Expression of PAX6, βIII-tubulin, SOX17 and FOXA2<br>by immunocytochemistry.<br>Expression of Brachyury and αSMA by RT-PCR.</td><td>Fig. 1D</td></tr><tr><td>List of recommended germ<br>layer markers</td><td>mRNA (RT-PCR) and protein (IF) levels, at least 2 markers need<br>to be shown per germ layer</td><td>Ectoderm: PAX6,TUBB3/TUJ1<br>Endoderm: SOX17, FOXA2,<br>Mesoderm: BRACHYURY, αSMA</td><td>Fig. 1D</td></tr><tr><td rowspan="3">Donor screening (OPTIONAL)<br>Genotype additional info<br>(OPTIONAL)</td><td>HIV 1 + 2 Hepatitis B, Hepatitis C</td><td>NA</td><td>NA</td></tr><tr><td>Blood group genotyping</td><td>NA</td><td>NA</td></tr><tr><td>HLA tissue typing</td><td>NA</td><td>NA</td></tr></tbody></table>

was performed on mytomycin-inactivated mouse embryonic fibroblasts from CF1 mouse (Charles River) on 0.1% Gelatin coated dishes and maintained, in KODMEM/F-12 with 20% knock-out serum, 1% NEAA, 0.1 mM β-mercaptoethanol, 1 mM glutamax, 1% penicillin/streptomycin and 5 ng/mL bFGF (Miltenyi Biotech). Several iPSC clones were picked up and expanded for the generation of different iPSC lines among which the present SAD line was selected.

## 4.2. Pluripotent stem cells culture

IPSC were maintained on feeder layers for several passages before adaptation to feeder-free culture condition, on 0.1% Geltrex (Gibco, Fisher Scientific) coated dishes and maintained in mTeSR Plus medium (Stemcell Technologies). Colonies were chemically passaged (1:10) 2 times a week as aggregates with enzymatic-free reagent ReLeSR (Stemcell Technologies). Cells were cultivated in a humidified incubator at 37 °C, 5% CO₂. The iPSC lines were frozen in liquid nitrogen in Cryostor and 10 µM of Rock Inhibitor (Stemcell Technologies). See Table 1.

## 4.3. PCR analysis

Total RNA was extracted from fibroblasts and the SAD clone with RNAeasy micro kit (Qiagen) and cDNA obtained using the Superscript III system (Invitrogen). Extinction of the expression of transgenes and expression of mesodermal markers was performed by conventional PCR with virus specific primers (Table 2). Expression of endogenous pluripotency genes was confirmed by quantitative PCR analysis using Syber-Green I Master kit (Roche Diagnostic) using H9 human embryonic stem cell mRNA as reference and positive control.

## 4.4. In vitro differentiation

iPS(p14) cells were differentiated into the three embryonal germ layers, mesoderm, ectoderm and endoderm with the StemMACS Trileneage Differentiation kit (Miltenyi) in 7 days, according to the supplier's recommendations.

## 4.5. Immunofluorescence staining

iPSC(p14) colonies or EB were fixed on coverlips in 4% PFA (15 min) and washed 3 times with DPBS (Gibco). Cells were permeabilized with 0.5% Triton X-100 (3x10min for nuclear staining, Sigma-Aldrich,). Saturation of non-specific binding sites was performed with 0.2% Bovine Serum Albumin (MP BIOMEDICALS) in DPBS (1 h, RT). Cells were incubated overnight with primary antibodies at 4 °C. Secondary antibodies were applied at RT for 1 h. Primary antibodies and secondary antibodies and their respective dilutions in 0.2% BSA-DPBS solution are listed in Table 2. Nuclei were labelled with DAPI in DPBS. After two washes in DPBS and water, coverslips were mounted with Fluorsave (Millipore). Samples were imaged with Upright Zeiss Apotome.

## 4.6. Karyotype and recurrent genomic abnormalities testing

G-banding karyotyping and iCS-digital PCS test (detection of recurrent genetic abnormalities) were performed by StemGenomics at passage p13.

## 4.7. STR analysis

Microsatellite analysis was performed on DNA extracted from parental fibroblasts and iPSCs at p13, using the following four short tandem repeats (STR): D1S439, D9S1784, D14S986 and D19S913.

## 4.8. Mycoplasma testing

Absence of mycoplasma contamination was assessed on cell culture supernatants of iPS cells (p13) using the MycoAlert® Mycoplasma Detection assay (Lonza).

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
<table><caption>**Table 1**

Characterization and validation.</caption><thead><tr><th>Classification</th><th>Test</th><th>Result</th><th>Data</th></tr></thead><tbody><tr><td rowspan="3">Morphology<br>Phenotype</td><td>Photography Bright field</td><td>Normal</td><td>Fig. 1A</td></tr><tr><td>Qualitative analysis (Immunocytochemistry)</td><td>Expression of pluripotency markers: Oct3/4, SOX2, SSEA-4</td><td>Fig. 1A</td></tr><tr><td>Quantitative analysis<br>(RT-qPCR)</td><td>Expression of Oct3/4, SOX2, NANOG</td><td>Fig. 1C</td></tr><tr><td rowspan="2">Genotype</td><td>Karyotype (G-banding) and resolution</td><td>46XY, Resolution 400</td><td rowspan="2">Fig. 1E</td></tr><tr><td>Recurrent genomic abnormalities</td><td>No recurrent genetic abnormalities detected</td></tr><tr><td rowspan="2">Identity</td><td>Microsatellite PCR (mPCR) OR</td><td>Not performed</td><td>NA</td></tr><tr><td>STR analysis</td><td>4 matched sites</td><td>Available with<br>authors</td></tr><tr><td rowspan="2">Mutation analysis (IF<br>APPLICABLE)</td><td>Sequencing</td><td>NA</td><td>NA</td></tr><tr><td>Southern Blot OR WGS</td><td>NA</td><td>NA</td></tr><tr><td>Microbiology and virology</td><td>Mycoplasma</td><td>Mycoplasma testing by luminescence. Negative</td><td>Supplementary<br>data</td></tr><tr><td>Differentiation potential</td><td>In vitro directed differentiation</td><td>Expression of PAX6, βIII-tubulin, SOX17 and FOXA2<br>by immunocytochemistry.<br>Expression of Brachyury and αSMA by RT-PCR.</td><td>Fig. 1D</td></tr><tr><td>List of recommended germ<br>layer markers</td><td>mRNA (RT-PCR) and protein (IF) levels, at least 2 markers need<br>to be shown per germ layer</td><td>Ectoderm: PAX6,TUBB3/TUJ1<br>Endoderm: SOX17, FOXA2,<br>Mesoderm: BRACHYURY, αSMA</td><td>Fig. 1D</td></tr><tr><td rowspan="3">Donor screening (OPTIONAL)<br>Genotype additional info<br>(OPTIONAL)</td><td>HIV 1 + 2 Hepatitis B, Hepatitis C</td><td>NA</td><td>NA</td></tr><tr><td>Blood group genotyping</td><td>NA</td><td>NA</td></tr><tr><td>HLA tissue typing</td><td>NA</td><td>NA</td></tr></tbody></table>

was performed on mytomycin-inactivated mouse embryonic fibroblasts from CF1 mouse (Charles River) on 0.1% Gelatin coated dishes and maintained, in KODMEM/F-12 with 20% knock-out serum, 1% NEAA, 0.1 mM β-mercaptoethanol, 1 mM glutamax, 1% penicillin/streptomycin and 5 ng/mL bFGF (Miltenyi Biotech). Several iPSC clones were picked up and expanded for the generation of different iPSC lines among which the present SAD line was selected.

## 4.2. Pluripotent stem cells culture

IPSC were maintained on feeder layers for several passages before adaptation to feeder-free culture condition, on 0.1% Geltrex (Gibco, Fisher Scientific) coated dishes and maintained in mTeSR Plus medium (Stemcell Technologies). Colonies were chemically passaged (1:10) 2 times a week as aggregates with enzymatic-free reagent ReLeSR (Stemcell Technologies). Cells were cultivated in a humidified incubator at 37 °C, 5% CO₂. The iPSC lines were frozen in liquid nitrogen in Cryostor and 10 µM of Rock Inhibitor (Stemcell Technologies). See Table 1.

## 4.3. PCR analysis

Total RNA was extracted from fibroblasts and the SAD clone with RNAeasy micro kit (Qiagen) and cDNA obtained using the Superscript III system (Invitrogen). Extinction of the expression of transgenes and expression of mesodermal markers was performed by conventional PCR with virus specific primers (Table 2). Expression of endogenous pluripotency genes was confirmed by quantitative PCR analysis using Syber-Green I Master kit (Roche Diagnostic) using H9 human embryonic stem cell mRNA as reference and positive control.

## 4.4. In vitro differentiation

iPS(p14) cells were differentiated into the three embryonal germ layers, mesoderm, ectoderm and endoderm with the StemMACS Trileneage Differentiation kit (Miltenyi) in 7 days, according to the supplier's recommendations.

## 4.5. Immunofluorescence staining

iPSC(p14) colonies or EB were fixed on coverlips in 4% PFA (15 min) and washed 3 times with DPBS (Gibco). Cells were permeabilized with 0.5% Triton X-100 (3x10min for nuclear staining, Sigma-Aldrich,). Saturation of non-specific binding sites was performed with 0.2% Bovine Serum Albumin (MP BIOMEDICALS) in DPBS (1 h, RT). Cells were incubated overnight with primary antibodies at 4 °C. Secondary antibodies were applied at RT for 1 h. Primary antibodies and secondary antibodies and their respective dilutions in 0.2% BSA-DPBS solution are listed in Table 2. Nuclei were labelled with DAPI in DPBS. After two washes in DPBS and water, coverslips were mounted with Fluorsave (Millipore). Samples were imaged with Upright Zeiss Apotome.

## 4.6. Karyotype and recurrent genomic abnormalities testing

G-banding karyotyping and iCS-digital PCS test (detection of recurrent genetic abnormalities) were performed by StemGenomics at passage p13.

## 4.7. STR analysis

Microsatellite analysis was performed on DNA extracted from parental fibroblasts and iPSCs at p13, using the following four short tandem repeats (STR): D1S439, D9S1784, D14S986 and D19S913.

## 4.8. Mycoplasma testing

Absence of mycoplasma contamination was assessed on cell culture supernatants of iPS cells (p13) using the MycoAlert® Mycoplasma Detection assay (Lonza).

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
<table><caption>**Table 2**
Reagents details.</caption><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="4">Antibodies used for immunocytochemistry/flow-cytometry</th></tr><tr><th>Antibody</th><th>Dilution</th><th>Company Cat #</th><th>RRID</th></tr></thead><tbody><tr><td rowspan="2">Pluripotency Markers</td><td>Mouse anti-OCT3-4 (C-10)</td><td>1:500</td><td>Santa Cruz<br>Biotechnology<br>Cat# sc-5279</td><td>RRID:<br>AB_628051</td></tr><tr><td>Rabbit anti-SOX2</td><td>1:100</td><td>Bethyl<br>Cat#301-739A</td><td>RRID:<br>AB_1211354</td></tr><tr><td rowspan="2">Differentiation Markers</td><td>Mouse anti-SSEA4 (MC-813-70)</td><td>1:500</td><td>Thermo Fisher<br>Scientific Cat# MA1-021</td><td>RRID:<br>AB_2536687</td></tr><tr><td>Mouse anti-Beta-Tubulin III, Clone TUJ1</td><td>1:200</td><td>STEMCELL<br>Technologies<br>Cat#60052</td><td>RRID:AB<br>2,313,773</td></tr><tr><td rowspan="4">Secondary antibodies</td><td>Rabbit anti-PAX6</td><td>1:20</td><td>Thermo Fisher<br>Scientific Cat# PA5-25970</td><td>RRID:<br>AB_2543470</td></tr><tr><td>Rabbit anti-HNF3beta/FOXA2</td><td>1:75</td><td>Millipore Cat# 07-633</td><td>RRID:<br>AB_390153</td></tr><tr><td>Mouse anti-SOX17</td><td>1:75</td><td>R&amp;D Systems<br>Cat# MAB1924</td><td>RRID:<br>AB_2195646</td></tr><tr><td>Donkey Anti-Mouse IgG, Alexa Fluor 555-conjugated Goat anti-Rabbit IgG, Alexa Fluor 488-conjugated Donkey anti-Rabbit IgG, Alexa Fluor 488-conjugated Donkey anti-Goat IgG, Alexa Fluor 555-conjugated Donkey anti-Mouse IgG, Alexa Fluor 488-conjugated</td><td>1:1000</td><td>Molecular<br>Probes Cat# A21424<br><br>Molecular<br>Probes Cat# A-11008<br><br>Molecular<br>Probes Cat# A21206<br><br>Thermo Fisher<br>Scientific Cat# A21432<br><br>Molecular<br>Probes Cat# A21202</td><td>RRID:<br>AB_141780<br><br>RRID:<br>AB_2535792<br><br>RRID:<br>AB_2535853<br><br>RRID:<br>AB_141607</td></tr><tr><td>Transgenes from SeV vector (PCR)</td><td>SeV</td><td>181 bp</td><td>Forward: GGA TCA CTA GGT<br>GAT ATC GAG C<br>Reverse: ACC AGA CAA GAG<br>TTT AAG AGA TAT GTA TC</td><td>RRID:<br>Forward: TAA CTG ACT AGC<br>AGG CTT GTC G<br>Reverse: TCC ACA TAC AGT CCT<br>GGA TGA TGA TG</td></tr><tr><td rowspan="2">Mesodermal markers (PCR)</td><td>c-Myc</td><td>532 bp</td><td>Forward: TTC AAT GTC CCA<br>GCC ATG TA<br>Reverse: GAA GGA ATA GCC<br>ACG CTC AG</td><td>RRID:<br>Forward: ACC CAG TTC ATA<br>GCG GTG AC<br>Reverse: CAA TTG TCA TGG<br>GAT TGC AG</td></tr><tr><td>GAPDH</td><td>300 bp</td><td>Forward: TTC AAT GTC CCA<br>GCC ATG TA<br>Reverse: GAA GGA ATA GCC<br>ACG CTC AG</td><td>RRID:<br>Forward: TGT ACT CCT CGG<br>TCC CTT TC<br>Reverse: TCC AGG TTT TCT TTC<br>CCT AGC</td></tr><tr><td rowspan="2">Pluripotency Markers (qPCR)</td><td>OCT4</td><td>150 bp</td><td>Forward: ATG CAC CGC TAC<br>GAC GTG A<br>Reverse: CTT TTG CAC CCC TCC<br>CAT TTC</td><td>RRID:<br>Forward: TGC CTC ACA CGG<br>AGA CTG TC</td></tr><tr><td>SOX2</td><td>144 bp</td><td>Forward: ATG CAC CGC TAC<br>GAC GTG A<br>Reverse: CTT TTG CAC CCC TCC<br>CAT TTC</td><td>RRID:<br>Forward: TGC CTC ACA CGG<br>AGA CTG TC</td></tr><tr><td></td><td>NANOG</td><td>354 bp</td><td>Forward: TGC CTC ACA CGG<br>AGA CTG TC</td><td>RRID:<br>Forward: TGC TAT TCT TCG GCC<br>AGT TG<br>Reverse: CCA TGG<br>TGT CTG<br>AGC GAT<br>GT</td></tr></tbody></table>

<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2"></th><th colspan="4">Antibodies used for immunocytochemistry/flow-cytometry</th></tr><tr><th>Antibody</th><th>Dilution</th><th>Company Cat #</th><th>RRID</th></tr></thead><tbody><tr><td rowspan="2">House-Keeping Genes (qPCR)</td><td rowspan="2">GAPDH</td><td rowspan="2">NA</td><td>Reverse: TGC TAT TCT TCG GCC<br>AGT TG</td></tr><tr><td>Forward:<br>CGC TCT<br>CTG CTC<br>CTC CTG TT<br>Reverse:<br>CCA TGG<br>TGT CTG<br>AGC GAT<br>GT</td></tr></tbody></table>

**Data availability**

Data will be made available on request.

**Acknowledgment**

We thank the FRM and Fondation plan Alzheimer for their financial support.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2023.103216.

**References**

Assou, S., Girault, N., Plinet, M., Bouckenheimer, J., Sansac, C., Combe, M., Mianné, J., Bourguignon, C., Fieldes, M., Ahmed, E., Commes, T., Boureux, A., Lemaître, J.-M., De Vos, J., 2020. Recurrent genetic abnormalities in human pluripotent stem cells: definition and routine detection in culture supernatant by targeted droplet digital PCR. Stem Cell Rep. 14 (1), 1-8.
<table><caption>**Table 2**
Reagents details.</caption><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="4">Antibodies used for immunocytochemistry/flow-cytometry</th></tr><tr><th>Antibody</th><th>Dilution</th><th>Company Cat #</th><th>RRID</th></tr></thead><tbody><tr><td rowspan="2">Pluripotency Markers</td><td>Mouse anti-OCT3-4 (C-10)</td><td>1:500</td><td>Santa Cruz<br>Biotechnology<br>Cat# sc-5279</td><td>RRID:<br>AB_628051</td></tr><tr><td>Rabbit anti-SOX2</td><td>1:100</td><td>Bethyl<br>Cat#301-739A</td><td>RRID:<br>AB_1211354</td></tr><tr><td rowspan="2">Differentiation Markers</td><td>Mouse anti-SSEA4 (MC-813-70)</td><td>1:500</td><td>Thermo Fisher<br>Scientific Cat# MA1-021</td><td>RRID:<br>AB_2536687</td></tr><tr><td>Mouse anti-Beta-Tubulin III, Clone TUJ1</td><td>1:200</td><td>STEMCELL<br>Technologies<br>Cat#60052</td><td>RRID:AB<br>2,313,773</td></tr><tr><td rowspan="4">Secondary antibodies</td><td>Rabbit anti-PAX6</td><td>1:20</td><td>Thermo Fisher<br>Scientific Cat# PA5-25970</td><td>RRID:<br>AB_2543470</td></tr><tr><td>Rabbit anti-HNF3beta/FOXA2</td><td>1:75</td><td>Millipore Cat# 07-633</td><td>RRID:<br>AB_390153</td></tr><tr><td>Mouse anti-SOX17</td><td>1:75</td><td>R&amp;D Systems<br>Cat# MAB1924</td><td>RRID:<br>AB_2195646</td></tr><tr><td>Donkey Anti-Mouse IgG, Alexa Fluor 555-conjugated Goat anti-Rabbit IgG, Alexa Fluor 488-conjugated Donkey anti-Rabbit IgG, Alexa Fluor 488-conjugated Donkey anti-Goat IgG, Alexa Fluor 555-conjugated Donkey anti-Mouse IgG, Alexa Fluor 488-conjugated</td><td>1:1000</td><td>Molecular<br>Probes Cat# A21424<br><br>Molecular<br>Probes Cat# A-11008<br><br>Molecular<br>Probes Cat# A21206<br><br>Thermo Fisher<br>Scientific Cat# A21432<br><br>Molecular<br>Probes Cat# A21202</td><td>RRID:<br>AB_141780<br><br>RRID:<br>AB_2535792<br><br>RRID:<br>AB_2535853<br><br>RRID:<br>AB_141607</td></tr><tr><td>Transgenes from SeV vector (PCR)</td><td>SeV</td><td>181 bp</td><td>Forward: GGA TCA CTA GGT<br>GAT ATC GAG C<br>Reverse: ACC AGA CAA GAG<br>TTT AAG AGA TAT GTA TC</td><td>RRID:<br>Forward: TAA CTG ACT AGC<br>AGG CTT GTC G<br>Reverse: TCC ACA TAC AGT CCT<br>GGA TGA TGA TG</td></tr><tr><td rowspan="2">Mesodermal markers (PCR)</td><td>c-Myc</td><td>532 bp</td><td>Forward: TTC AAT GTC CCA<br>GCC ATG TA<br>Reverse: GAA GGA ATA GCC<br>ACG CTC AG</td><td>RRID:<br>Forward: ACC CAG TTC ATA<br>GCG GTG AC<br>Reverse: CAA TTG TCA TGG<br>GAT TGC AG</td></tr><tr><td>GAPDH</td><td>300 bp</td><td>Forward: TTC AAT GTC CCA<br>GCC ATG TA<br>Reverse: GAA GGA ATA GCC<br>ACG CTC AG</td><td>RRID:<br>Forward: TGT ACT CCT CGG<br>TCC CTT TC<br>Reverse: TCC AGG TTT TCT TTC<br>CCT AGC</td></tr><tr><td rowspan="2">Pluripotency Markers (qPCR)</td><td>OCT4</td><td>150 bp</td><td>Forward: ATG CAC CGC TAC<br>GAC GTG A<br>Reverse: CTT TTG CAC CCC TCC<br>CAT TTC</td><td>RRID:<br>Forward: TGC CTC ACA CGG<br>AGA CTG TC</td></tr><tr><td>SOX2</td><td>144 bp</td><td>Forward: ATG CAC CGC TAC<br>GAC GTG A<br>Reverse: CTT TTG CAC CCC TCC<br>CAT TTC</td><td>RRID:<br>Forward: TGC CTC ACA CGG<br>AGA CTG TC</td></tr><tr><td></td><td>NANOG</td><td>354 bp</td><td>Forward: TGC CTC ACA CGG<br>AGA CTG TC</td><td>RRID:<br>Forward: TGC TAT TCT TCG GCC<br>AGT TG<br>Reverse: CCA TGG<br>TGT CTG<br>AGC GAT<br>GT</td></tr></tbody></table>

<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2"></th><th colspan="4">Antibodies used for immunocytochemistry/flow-cytometry</th></tr><tr><th>Antibody</th><th>Dilution</th><th>Company Cat #</th><th>RRID</th></tr></thead><tbody><tr><td rowspan="2">House-Keeping Genes (qPCR)</td><td rowspan="2">GAPDH</td><td rowspan="2">NA</td><td>Reverse: TGC TAT TCT TCG GCC<br>AGT TG</td></tr><tr><td>Forward:<br>CGC TCT<br>CTG CTC<br>CTC CTG TT<br>Reverse:<br>CCA TGG<br>TGT CTG<br>AGC GAT<br>GT</td></tr></tbody></table>

## Data availability

Data will be made available on request.

## Acknowledgment

We thank the FRM and Fondation plan Alzheimer for their financial support.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2023.103216.

## References

Assou, S., Girault, N., Plinet, M., Bouckenheimer, J., Sansac, C., Combe, M., Mianné, J., Bourguignon, C., Fieldes, M., Ahmed, E., Commes, T., Boureux, A., Lemaître, J.-M., De Vos, J., 2020. Recurrent genetic abnormalities in human pluripotent stem cells: definition and routine detection in culture supernatant by targeted droplet digital PCR. Stem Cell Rep. 14 (1), 1–8.