# Generation of induced pluripotent stem cell lines IRMBi003-A and IRMBi003-B from a healthy donor to model Alzheimer's disease

C. Clua Provost, L. Auboyer, Anne Rovelet-Lecrux, C. Monzo, E. Schob, F.

Andreux, C. Quittet, S. Lehmann, D. Wallon, C. Crozet

► To cite this version:

C. Clua Provost, L. Auboyer, Anne Rovelet-Lecrux, C. Monzo, E. Schob, et al. Generation of induced pluripotent stem cell lines IRMBi003-A and IRMBi003-B from a healthy donor to model Alzheimer's disease. Stem Cell Research, 2023, 73, pp.103250. 10.1016/j.scr.2023.103250. hal-04597795

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https://hal.science/hal-04597795

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# Generation of induced pluripotent stem cell lines IRMBi003-A and IRMBi003-B from a healthy donor to model 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>, E. Schob<sup>a</sup>, F. Andreux<sup>a</sup>, C. Quittet<sup>a</sup>, S. Lehmann<sup>a</sup>, D. Wallon<sup>d</sup>, C. Crozet<sup>a,*</sup>

## ABSTRACT

Induced Pluripotent Stem Cell (iPSC) lines derived from healthy individuals are helpful and essential tools for disease modelling. Here, we described the reprogramming of skin fibroblasts obtained from a healthy 59-year-old individual without Alzheimer's disease. The generated iPSC lines have a normal karyotype, expressed pluripotency markers, and demonstrated the ability to differentiate into the three germ layers. The iPSC lines will be used as controls to study Alzheimer's disease mechanisms.

### Resource Table.

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi003-A<br>IRMBi003-B</td></tr><tr><td>Alternative name(s) of stem cell line</td><td>CT1 and CT4</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</td><td>Age: 59 years' old<br>Sex: Male</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 Modification</td><td>N/A</td></tr><tr><td>Associated disease</td><td>N/A</td></tr><tr><td>Gene/locus</td><td>N/A</td></tr><tr><td>Method of modification</td><td>N/A</td></tr><tr><td>Name of transgene or resistance</td><td>N/A</td></tr><tr><td>Inducible/constitutive system</td><td>N/A</td></tr><tr><td>Date archived/stock date</td><td>07/26/2023</td></tr></table>

(continued on next column)

(continued)

<table><tr><td>Cell line repository/bank</td><td>https://hpscreg.eu/cell-line/IRMBi003-A and http://s://hpscreg.eu/cell-line/IRMBi003-B</td></tr><tr><td>Ethical approval</td><td>DC-2015-2363 regional scientific ethical committee<br>CPP Sud Med IV. Informed consent was obtained from the patient.</td></tr></table>

## 1. Resource utility

The reported cell lines may be used as healthy reference cell lines for *in vitro* studies or disease modelling for Alzheimer's disease (AD). They may also be genetically modified by gene editing to generate isogenic cell lines with mutations related to the pathology (Table 1).

## 2. Resource details

This paper details the generation of two iPSC clones (CT1 and CT4) from a healthy male without AD. They could be used as healthy control iPSC lines for AD studies to perform comparative analysis with AD iPSC cell lines (Auboyer et al., 2019a; Auboyer et al., 2019b). They may also provide cell sources for the generation of isogenic cell lines with various

<footnote><sup>a</sup> Institute for Neurosciences of Montpellier, Univ Montpellier, INSERM U1298, 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.
E-mail address: carole.crozet@inserm.fr (C. Crozet).</footnote>

<footnote n="1">¹ Co-first author.</footnote>
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

# Generation of induced pluripotent stem cell lines IRMBi003-A and IRMBi003-B from a healthy donor to model 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>, E. Schob<sup>a</sup>, F. Andreux<sup>a</sup>, C. Quittet<sup>a</sup>, S. Lehmann<sup>a</sup>, D. Wallon<sup>d</sup>, C. Crozet<sup>a,*</sup>

## ABSTRACT

Induced Pluripotent Stem Cell (iPSC) lines derived from healthy individuals are helpful and essential tools for disease modelling. Here, we described the reprogramming of skin fibroblasts obtained from a healthy 59-year-old individual without Alzheimer's disease. The generated iPSC lines have a normal karyotype, expressed pluripotency markers, and demonstrated the ability to differentiate into the three germ layers. The iPSC lines will be used as controls to study Alzheimer's disease mechanisms.

### Resource Table.

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi003-A<br>IRMBi003-B</td></tr><tr><td>Alternative name(s) of stem cell line</td><td>CT1 and CT4</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</td><td>Age: 59 years' old<br>Sex: Male</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 Modification</td><td>N/A</td></tr><tr><td>Associated disease</td><td>N/A</td></tr><tr><td>Gene/locus</td><td>N/A</td></tr><tr><td>Method of modification</td><td>N/A</td></tr><tr><td>Name of transgene or resistance</td><td>N/A</td></tr><tr><td>Inducible/constitutive system</td><td>N/A</td></tr><tr><td>Date archived/stock date</td><td>07/26/2023</td></tr></table>

(continued on next column)

(continued)

<table><tr><td>Cell line repository/bank</td><td>https://hpscreg.eu/cell-line/IRMBi003-A and http://s://hpscreg.eu/cell-line/IRMBi003-B</td></tr><tr><td>Ethical approval</td><td>DC-2015-2363 regional scientific ethical committee<br>CPP Sud Med IV. Informed consent was obtained from the patient.</td></tr></table>

## 1. Resource utility

The reported cell lines may be used as healthy reference cell lines for *in vitro* studies or disease modelling for Alzheimer's disease (AD). They may also be genetically modified by gene editing to generate isogenic cell lines with mutations related to the pathology (Table 1).

## 2. Resource details

This paper details the generation of two iPSC clones (CT1 and CT4) from a healthy male without AD. They could be used as healthy control iPSC lines for AD studies to perform comparative analysis with AD iPSC cell lines (Auboyer et al., 2019a; Auboyer et al., 2019b). They may also provide cell sources for the generation of isogenic cell lines with various

<footnote><sup>a</sup> Institute for Neurosciences of Montpellier, Univ Montpellier, INSERM U1298, 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.
E-mail address: carole.crozet@inserm.fr (C. Crozet).</footnote>

<footnote n="1">¹ Co-first author.</footnote>
mutations of interest and known to be involved in the physiopathology of AD.

Skin fibroblasts were obtained from a 59-year-old man without diagnosed disease. Fibroblasts were reprogrammed into iPSC using integration-free Sendai viruses (Life Technologies) carrying gene sequences of the four Yamanaka factors: OCT3/4, SOX2, KLF4 and c-MYC. Several iPSC colonies with normal morphology (Fig. 1A) were picked for expansion. The disappearance of the Sendai virus was successfully established by RT-PCR analysis with Sendai virus specific primers (Fig. 1B), in selected iPSC colonies after at least 10 passages. The transgenes and viral sequences were also absent in the negative controls, non-transduced human healthy fibroblasts (hF Ct) and clones (CT1 and CT4) of interest, but present in freshly transduced (7 days) fibroblasts with the Sendai viruses (hF Ct Sendai). Expression of endogenous pluripotency factors OCT4, SOX2 and NANOG was confirmed by RT-qPCR analysis (Fig. 1C). Immunofluorescence labelling validated the expression of the embryonic stem cell specific marker SSEA4 and of the two pluripotency factors OCT4 and SOX2 (Fig. 1D). Pluripotency of the two clones was assessed following *in vitro* directed differentiation of iPSC colonies during 7 days (Fig. 1E), followed by immunofluorescence staining of ectodermal and endodermal markers, respectively βIII-tubulin and PAX6, HNF3beta/FOXA2 and SOX17, or RT-PCR for mesodermal markers, Brachyury and αSmooth muscle actin (Fig. 1E). CT1 and CT4 clones presented a normal karyotype 46,XY after 33 passages for CT1, and 35 passages for CT4. Genomic stability was also assessed by detection of recurrent genetic abnormalities using the iCS-digital™ PSC analysis provided as a service by Stem Genomics (https://www stems genomics.co m/), as described previously (Assou et al., 2020) and no evidence of recurrent genomic abnormalities was detected (Fig. 1F). The short tandem repeat (STR) profile established the 100 % identity of the iPSC line to parental cells (available upon request) and clones were shown to be mycoplasma free (Supplementary data).

## 3. Materials and methods

### 3.1. iPSC generation

Fibroblasts were amplified in 10 % FBS (Lonza), DMEM, 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). After 7 days, the freshly transduced cells were transferred on feeders (mytomycin-inactivated CF1 mouse

embryonic (E12.5) fibroblasts), (Charles River) on 0.1 % Gelatin (Sigma Aldrich) coated dishes in KODMEM/F-12 with 20 % KO serum, 1 % NEAA, 0.1 mM β-mercaptoethanol, 1 mM glutamax, 1 % penicillin/streptomycin (Gibco) and 5 ng/mL bFGF (Miltenyi Biotec). Several iPSC clones were passaged and expanded.

### 3.2. Pluripotent stem cell cultures

IPSC were first maintained on a feeder layer and they then adapted on 0.1 % Geltrex (Gibco)) coated dishes and maintained in mTeSR Plus medium (Stemcell Technologies). Colonies were mechanically passaged every 4–7 days into clumps. 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).

### 3.3. PCR analysis

Total RNA was extracted from fibroblasts and iPSCs (P20) with the RNAeasy micro kit (Qiagen). cDNA were obtained using the SupercriptIII kit (Invitrogen). Disappearance of the Sendai virus vectors and expression of mesodermal markers was assessed by PCR using specific primers (Table 2). PCR products were analysed on 2 % agarose gel and the picture was acquired using Ebox Vilber System. For the expression of the endogenous genes of pluripotency, quantitative PCR analysis was performed using the Light Cycler LC480 device (Roche Diagnostics) using SYBRGreen I Master Kit (Roche Diagnostics). The relative quantification was calculated using the 2-ΔΔCt method with non-transduced fibroblasts as controls and GAPDH as housekeeping gene (Primers are listed in Table 2) using H9 human embryonic stem cell mRNA as reference and positive control.

### 3.4. *In vitro* directed differentiation

IPS cells (p24) were dissociated as single cells (Gentle Dissociation Reagent, Stemcell Technologies) and differentiated into each germ layers (mesoderm, endoderm, ectoderm) in parallel with StemMACS Trilineage Differentiation Kit (Miltenyi). After 7 days, cells were fixed for immunocychemistery.

### 3.5. Immunofluorescence

iPSC colonies (p22) or EB were fixed on coverslips in 4 % PFA during 15 min and washed 3 times with DPBS (Gibco). Cells were permeabilized

<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>Morphology</td><td>Photography Bright field</td><td>Normal</td><td>Fig. 1A</td></tr><tr><td rowspan="2">Phenotype</td><td>Qualitative analysis (Immunocytochemistry)</td><td>Positive for OCT4, SOX2, SSEA4</td><td>Fig. 1D</td></tr><tr><td>Quantitative analysis (RT-qPCR)</td><td>Expression of pluripotency markers: OCT4, SOX2, NANOG</td><td>Fig. 1C</td></tr><tr><td>Genotype</td><td>Karyotype and resolution</td><td>46XY, Resolution: 400</td><td>Fig. 1F</td></tr><tr><td rowspan="2">Identity</td><td>Recurrent genomic abnormalities</td><td>No recurrent genetic abnormalities detected</td><td rowspan="2">NA</td></tr><tr><td>Microsatellite PCR (mPCR) OR STR analysis</td><td>Not performed</td></tr><tr><td></td><td></td><td>Matched to parental STR</td><td>Available with authors</td></tr><tr><td>Mutation analysis (IF APPLICABLE)</td><td>Sequencing</td><td>NA</td><td>NA</td></tr><tr><td rowspan="2">Microbiology and virology</td><td>Southern Blot OR WGS</td><td>NA</td><td>NA</td></tr><tr><td>Mycoplasma</td><td>Mycoplasma testing by luminescence. Negative</td><td>Supplementary data</td></tr><tr><td>Differentiation potential</td><td>In vitro directed differentiation</td><td>Expression of PAX6, βIII-tubulin, SOX17 and FOXA2 by immunocytochemistry. Expression of Brachyury and αSMA by RT-PCR.</td><td>Fig. 1E</td></tr><tr><td>List of recommended germ layer markers</td><td>mRNA (RT-PCR) and protein (IF) levels, at least 2 markers need to be shown per germ layer</td><td>Ectoderm: PAX6, TUBB3/TUJ1<br>Endoderm: SOX17, FOXA2,<br>Mesoderm: BRACHYURY, αSMA</td><td>Fig. 1E</td></tr><tr><td>Donor screening (OPTIONAL)</td><td>HIV 1 + 2 Hepatitis B, Hepatitis C</td><td>NA</td><td>NA</td></tr><tr><td rowspan="2">Genotype additional info (OPTIONAL)</td><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>
mutations of interest and known to be involved in the physiopathology of AD.

Skin fibroblasts were obtained from a 59-year-old man without diagnosed disease. Fibroblasts were reprogrammed into iPSC using integration-free Sendai viruses (Life Technologies) carrying gene sequences of the four Yamanaka factors: OCT3/4, SOX2, KLF4 and c-MYC. Several iPSC colonies with normal morphology (Fig. 1A) were picked for expansion. The disappearance of the Sendai virus was successfully established by RT-PCR analysis with Sendai virus specific primers (Fig. 1B), in selected iPSC colonies after at least 10 passages. The transgenes and viral sequences were also absent in the negative controls, non-transduced human healthy fibroblasts (hF Ct) and clones (CT1 and CT4) of interest, but present in freshly transduced (7 days) fibroblasts with the Sendai viruses (hF Ct Sendai). Expression of endogenous pluripotency factors OCT4, SOX2 and NANOG was confirmed by RT-qPCR analysis (Fig. 1C). Immunofluorescence labelling validated the expression of the embryonic stem cell specific marker SSEA4 and of the two pluripotency factors OCT4 and SOX2 (Fig. 1D). Pluripotency of the two clones was assessed following in vitro directed differentiation of iPSC colonies during 7 days (Fig. 1E), followed by immunofluorescence staining of ectodermal and endodermal markers, respectively βIII-tubulin and PAX6, HNF3beta/FOXA2 and SOX17, or RT-PCR for mesodermal markers, Brachyury and αSmooth muscle actin (Fig. 1E). CT1 and CT4 clones presented a normal karyotype 46,XY after 33 passages for CT1, and 35 passages for CT4. Genomic stability was also assessed by detection of recurrent genetic abnormalities using the iCS-digital™ PSC analysis provided as a service by Stem Genomics (https://www stems genomics.co m/), as described previously (Assou et al., 2020) and no evidence of recurrent genomic abnormalities was detected (Fig. 1F). The short tandem repeat (STR) profile established the 100 % identity of the iPSC line to parental cells (available upon request) and clones were shown to be mycoplasma free (Supplementary data).

## 3. Materials and methods

### 3.1. iPSC generation

Fibroblasts were amplified in 10 % FBS (Lonza), DMEM, 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). After 7 days, the freshly transduced cells were transferred on feeders (mytomycin-inactivated CF1 mouse

embryonic (E12.5) fibroblasts), (Charles River) on 0.1 % Gelatin (Sigma Aldrich) coated dishes in KODMEM/F-12 with 20 % KO serum, 1 % NEAA, 0.1 mM β-mercaptoethanol, 1 mM glutamax, 1 % penicillin/streptomycin (Gibco) and 5 ng/mL bFGF (Miltenyi Biotec). Several iPSC clones were passaged and expanded.

### 3.2. Pluripotent stem cell cultures

IPSC were first maintained on a feeder layer and they then adapted on 0.1 % Geltrex (Gibco)) coated dishes and maintained in mTeSR Plus medium (Stemcell Technologies). Colonies were mechanically passaged every 4–7 days into clumps. 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).

### 3.3. PCR analysis

Total RNA was extracted from fibroblasts and iPSCs (P20) with the RNAeasy micro kit (Qiagen). cDNA were obtained using the SupercriptIII kit (Invitrogen). Disappearance of the Sendai virus vectors and expression of mesodermal markers was assessed by PCR using specific primers (Table 2). PCR products were analysed on 2 % agarose gel and the picture was acquired using Ebox Vilber System. For the expression of the endogenous genes of pluripotency, quantitative PCR analysis was performed using the Light Cycler LC480 device (Roche Diagnostics) using SYBRGreen I Master Kit (Roche Diagnostics). The relative quantification was calculated using the 2-ΔΔCt method with non-transduced fibroblasts as controls and GAPDH as housekeeping gene (Primers are listed in Table 2) using H9 human embryonic stem cell mRNA as reference and positive control.

### 3.4. In vitro directed differentiation

IPS cells (p24) were dissociated as single cells (Gentle Dissociation Reagent, Stemcell Technologies) and differentiated into each germ layers (mesoderm, endoderm, ectoderm) in parallel with StemMACS Trilineage Differentiation Kit (Miltenyi). After 7 days, cells were fixed for immunocychemistery.

### 3.5. Immunofluorescence

iPSC colonies (p22) or EB were fixed on coverslips in 4 % PFA during 15 min and washed 3 times with DPBS (Gibco). Cells were permeabilized

<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>Morphology</td><td>Photography Bright field</td><td>Normal</td><td>Fig. 1A</td></tr><tr><td rowspan="2">Phenotype</td><td>Qualitative analysis (Immunocytochemistry)</td><td>Positive for OCT4, SOX2, SSEA4</td><td>Fig. 1D</td></tr><tr><td>Quantitative analysis (RT-qPCR)</td><td>Expression of pluripotency markers: OCT4, SOX2, NANOG</td><td>Fig. 1C</td></tr><tr><td>Genotype</td><td>Karyotype and resolution</td><td>46XY, Resolution: 400</td><td>Fig. 1F</td></tr><tr><td rowspan="2">Identity</td><td>Recurrent genomic abnormalities</td><td>No recurrent genetic abnormalities detected</td><td rowspan="2">NA</td></tr><tr><td>Microsatellite PCR (mPCR) OR STR analysis</td><td>Not performed</td></tr><tr><td></td><td></td><td>Matched to parental STR</td><td>Available with authors</td></tr><tr><td>Mutation analysis (IF APPLICABLE)</td><td>Sequencing</td><td>NA</td><td>NA</td></tr><tr><td rowspan="2">Microbiology and virology</td><td>Southern Blot OR WGS</td><td>NA</td><td>NA</td></tr><tr><td>Mycoplasma</td><td>Mycoplasma testing by luminescence. Negative</td><td>Supplementary data</td></tr><tr><td>Differentiation potential</td><td>In vitro directed differentiation</td><td>Expression of PAX6, βIII-tubulin, SOX17 and FOXA2 by immunocytochemistry. Expression of Brachyury and αSMA by RT-PCR.</td><td>Fig. 1E</td></tr><tr><td>List of recommended germ layer markers</td><td>mRNA (RT-PCR) and protein (IF) levels, at least 2 markers need to be shown per germ layer</td><td>Ectoderm: PAX6, TUBB3/TUJ1<br>Endoderm: SOX17, FOXA2,<br>Mesoderm: BRACHYURY, αSMA</td><td>Fig. 1E</td></tr><tr><td>Donor screening (OPTIONAL)</td><td>HIV 1 + 2 Hepatitis B, Hepatitis C</td><td>NA</td><td>NA</td></tr><tr><td rowspan="2">Genotype additional info (OPTIONAL)</td><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>
<figure><img src="image_3.png" /><figcaption>Fig. 1.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 1.</figcaption></figure>
<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: AB_628051</td></tr><tr><td>Rabbit anti-SOX2</td><td>1:100</td><td>Bethyl<br>Cat#301-739A</td><td>RRID: AB_1211354</td></tr><tr><td rowspan="3">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: 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_2,313,773</td></tr><tr><td>Rabbit anti-PAX6</td><td>1:20</td><td>Thermo Fisher<br>Scientific Cat# PA5-25970</td><td>RRID: AB_2543470</td></tr><tr><td rowspan="5">Secondary antibodies</td><td>Rabbit anti-HNF3beta/FOXA2</td><td>1:75</td><td>Millipore Cat# 07-633</td><td>RRID: 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: AB_2195646</td></tr><tr><td>Donkey Anti-Mouse IgG, Alexa Fluor 555-conjugated</td><td>1:1000</td><td>Molecular<br>Probes Cat# A21424</td><td>RRID: AB_141780</td></tr><tr><td>Goat anti-Rabbit IgG, Alexa Fluor 488-conjugated</td><td>1:1000</td><td>Molecular<br>Probes Cat# A-11008</td><td>RRID: AB_143165</td></tr><tr><td>Donkey anti-Rabbit IgG, Alexa Fluor 488-conjugated</td><td>1:1000</td><td>Molecular<br>Probes Cat# A21206</td><td>RRID: AB_2535792</td></tr><tr><td rowspan="3">Donkey anti-Goat IgG, Alexa Fluor 555-conjugated</td><td>Donkey anti-Goat IgG, Alexa Fluor 488-conjugated</td><td>1:1000</td><td>Thermo Fisher<br>Scientific Cat# A21432</td><td>RRID: AB_2535853</td></tr><tr><td>Donkey anti-Mouse IgG, Alexa Fluor 488-conjugated Primers</td><td>1:1000</td><td>Molecular<br>Probes Cat# A21202</td><td>RRID: AB_141607</td></tr><tr><td>Target</td><td>Size of band</td><td colspan="3">Forward/Reverse primer (5'-3')</td></tr><tr><td rowspan="3">Transgenes from SeV vector (PCR)</td><td>SeV</td><td>181 bp</td><td colspan="3">Forward: GGA TCA CTA GGT<br>GAT ATC GAG C<br>Reverse: ACC AGA CAA GAG<br>TTT AAG AGA TAT GTA TC</td></tr><tr><td>c-Myc</td><td>532 bp</td><td colspan="3">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>GAPDH</td><td>300 bp</td><td colspan="3">Forward: CTG GCG TCT TCA<br>CCA CCA TGG<br>Reverse: CAT CAC GCC ACA GTT<br>TCC CGG</td></tr><tr><td rowspan="3">Mesodermal markers (PCR)</td><td>BRACHYURY/<br>TBXT</td><td>218/<br>392/<br>395 bp</td><td colspan="3">Forward: ACC CAG TTC ATA<br>GCG GTG AC<br>Reverse: CAA TTG TCA TGG<br>GAT TGC AG</td></tr><tr><td>αSMA</td><td>222 bp</td><td colspan="3">Forward: TTC AAT GTC CCA<br>GCC ATG TA<br>Reverse: GAA GGA ATA GCC<br>ACG CTC AG</td></tr><tr><td>OCT4</td><td>150 bp</td><td colspan="3">Forward: TGT ACT CCT CGG<br>TCC CTT TC<br>Reverse: TCC AGG TTT TCT TTC<br>CCT AGC</td></tr><tr><td rowspan="3">Pluripotency Markers (qPCR)</td><td>SOX2</td><td>144 bp</td><td colspan="3">Forward: ATG CAC CGC TAC<br>GAC GTG A<br>Reverse: CTT TTG CAC CCC TCC<br>CAT TTC</td></tr><tr><td>NANOG</td><td>354 bp</td><td colspan="3">Forward: TGC CTC ACA CGG<br>AGA CTG TC</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="3">House-Keeping Genes (qPCR)</td><td rowspan="3">GAPDH</td><td rowspan="3">NA</td><td>Reverse: TGC TAT TCT TCG GCC AGT TG</td></tr><tr><td>Forward: CGC TCT CTG CTC CTC CTG TT</td></tr><tr><td>Reverse: CCA TGG TGT CTG AGC GAT GT</td></tr></tbody></table>

with 0.5 % Triton X-100 (Sigma Aldrich), 3x10min for nuclear staining or 3 min in 0.3 % Triton X-100 for the other immunostainings. Non-specific binding sites were blocked with 0.2 % Bovine Serum Albumin (MP BIOMEDICALS) in DPBS (1 h, RT). Primary and secondary antibodies were diluted (Table 2) in 0.2 % BSA-DPBS solution, and applied overnight at 4 °C and 1 h, RT respectively. Nuclei were labelled with DAPI (Life technologies) in DPBS. Cells were washed twice in DPBS and water. Coverslips were mounted with Fluorsave (Millipore). Samples were imaged with AxioObserver Zeiss.

### 3.6. Karyotype and recurrent genomic instability

Genomic stability tests and Karyotypes were performed by StemGenomics at passage 33 (CT1) and passage 35 (CT4), (https://www stemsgenomics.com/).

### 3.7. Short tandem repeat analysis

Microsatellite analyses were performed on DNA extracted from parental fibroblasts and iPSCs, using the following short tandem repeats: D1S439, D9S1784, D14S986, D19S913, D5S818, D13S317, D7S820, D16S539, vWA, Th01, TPOX, CSF1PO) + amelogenin.

### 3.8. Mycoplasma testing

Absence of mycoplasma contamination was assessed on cell culture supernatants (p25) using the MycoAlert® Mycoplasma Detection assay (Lonza).

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Crozet reports financial support was provided by Fondation pour la Recherche Médicale. CLUA-PROVOST reports financial support was provided by Alzheimer Foundation.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2023.103250.

## 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, 1-8.

* Auboyer, L., Monzo, C., Wallon, D., Rovelet-Lecrux, A., Gabelle, A., Gazagne, I., Cacheux, V., Lehmann, S., Crozet, C., 2019a. Generation of induced pluripotent stem cells (iPSCs) IRMBi001-A) from an Alzheimer's disease patient carrying a G217D mutation in the PSEN1 gene. Stem Cell Res 34, 101381.

* Auboyer, L., Monzo, C., Wallon, D., Rovelet-Lecrux, A., Gabelle, A., Gazagne, I., Cacheux, V., Lehmann, S., Crozet, C., 2019b. Generation of induced pluripotent stem cells (iPSCs) IRMBi002-A) from an Alzheimer's disease patient carrying a D694N mutation in the APP gene. Stem Cell Res 37, 101438.
<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: AB_628051</td></tr><tr><td>Rabbit anti-SOX2</td><td>1:100</td><td>Bethyl<br>Cat#301-739A</td><td>RRID: AB_1211354</td></tr><tr><td rowspan="5">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: 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_2,313,773</td></tr><tr><td>Rabbit anti-PAX6</td><td>1:20</td><td>Thermo Fisher<br>Scientific Cat# PA5-25970</td><td>RRID: AB_2543470</td></tr><tr><td>Rabbit anti-HNF3beta/FOXA2</td><td>1:75</td><td>Millipore Cat# 07-633</td><td>RRID: 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: AB_2195646</td></tr><tr><td rowspan="10">Secondary antibodies</td><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 Primers</td><td>1:1000</td><td>Molecular<br>Probes Cat# A21424</td><td>RRID: AB_141780</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 Primers</td><td>1:1000</td><td>Molecular<br>Probes Cat# A-11008</td><td>RRID: AB_143165</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 Primers</td><td>1:1000</td><td>Molecular<br>Probes Cat# A21206</td><td>RRID: AB_2535792</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 Primers</td><td>1:1000</td><td>Thermo Fisher<br>Scientific Cat# A21432</td><td>RRID: AB_2535853</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 Primers</td><td>1:1000</td><td>Molecular<br>Probes Cat# A21202</td><td>RRID: AB_141607</td></tr><tr><td>Target</td><td>Size of band</td><td colspan="3">Forward/Reverse primer (5'-3')</td></tr><tr><td rowspan="3">Transgenes from SeV vector (PCR)</td><td>SeV</td><td>181 bp</td><td colspan="3">Forward: GGA TCA CTA GGT<br>GAT ATC GAG C<br>Reverse: ACC AGA CAA GAG<br>TTT AAG AGA TAT GTA TC</td></tr><tr><td>c-Myc</td><td>532 bp</td><td colspan="3">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>GAPDH</td><td>300 bp</td><td colspan="3">Forward: CTG GCG TCT TCA<br>CCA CCA TGG<br>Reverse: CAT CAC GCC ACA GTT<br>TCC CGG</td></tr><tr><td rowspan="3">Mesodermal markers (PCR)</td><td>BRACHYURY/<br>TBXT</td><td>218/<br>392/<br>395 bp</td><td colspan="3">Forward: ACC CAG TTC ATA<br>GCG GTG AC<br>Reverse: CAA TTG TCA TGG<br>GAT TGC AG</td></tr><tr><td>αSMA</td><td>222 bp</td><td colspan="3">Forward: TTC AAT GTC CCA<br>GCC ATG TA<br>Reverse: GAA GGA ATA GCC<br>ACG CTC AG</td></tr><tr><td>OCT4</td><td>150 bp</td><td colspan="3">Forward: TGT ACT CCT CGG<br>TCC CTT TC<br>Reverse: TCC AGG TTT TCT TTC<br>CCT AGC</td></tr><tr><td rowspan="3">Pluripotency Markers (qPCR)</td><td>SOX2</td><td>144 bp</td><td colspan="3">Forward: ATG CAC CGC TAC<br>GAC GTG A<br>Reverse: CTT TTG CAC CCC TCC<br>CAT TTC</td></tr><tr><td>NANOG</td><td>354 bp</td><td colspan="3">Forward: TGC CTC ACA CGG<br>AGA CTG TC</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 AGT TG</td></tr><tr><td>Forward: CGC TCT CTG CTC CTC CTG TT<br>Reverse: CCA TGG TGT CTG AGC GAT GT</td></tr></tbody></table>

with 0.5 % Triton X-100 (Sigma Aldrich), 3x10min for nuclear staining or 3 min in 0.3 % Triton X-100 for the other immunostainings. Non-specific binding sites were blocked with 0.2 % Bovine Serum Albumin (MP BIOMEDICALS) in DPBS (1 h, RT). Primary and secondary antibodies were diluted (Table 2) in 0.2 % BSA-DPBS solution, and applied overnight at 4 °C and 1 h, RT respectively. Nuclei were labelled with DAPI (Life technologies) in DPBS. Cells were washed twice in DPBS and water. Coverslips were mounted with Fluorsave (Millipore). Samples were imaged with AxioObserver Zeiss.

### 3.6. Karyotype and recurrent genomic instability

Genomic stability tests and Karyotypes were performed by StemGenomics at passage 33 (CT1) and passage 35 (CT4), (https://www stemsgenomics.com/).

### 3.7. Short tandem repeat analysis

Microsatellite analyses were performed on DNA extracted from parental fibroblasts and iPSCs, using the following short tandem repeats: D1S439, D9S1784, D14S986, D19S913, D5S818, D13S317, D7S820, D16S539, vWA, Th01, TPOX, CSF1PO) + amelogenin.

### 3.8. Mycoplasma testing

Absence of mycoplasma contamination was assessed on cell culture supernatants (p25) using the MycoAlert® Mycoplasma Detection assay (Lonza).

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Crozet reports financial support was provided by Fondation pour la Recherche Médicale. CLUA-PROVOST reports financial support was provided by Alzheimer Foundation.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2023.103250.

## 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, 1-8.

* Auboyer, L., Monzo, C., Wallon, D., Rovelet-Lecrux, A., Gabelle, A., Gazagne, I., Cacheux, V., Lehmann, S., Crozet, C., 2019a. Generation of induced pluripotent stem cells (iPSCs) IRMBi001-A) from an Alzheimer's disease patient carrying a G217D mutation in the PSEN1 gene. Stem Cell Res 34, 101381.

* Auboyer, L., Monzo, C., Wallon, D., Rovelet-Lecrux, A., Gabelle, A., Gazagne, I., Cacheux, V., Lehmann, S., Crozet, C., 2019b. Generation of induced pluripotent stem cells (iPSCs) IRMBi002-A) from an Alzheimer's disease patient carrying a D694N mutation in the APP gene. Stem Cell Res 37, 101438.