# Generation of human induced pluripotent stem cell lines (iPSC) from adipose-derived mesenchymal stromal cells from two patients with systemic sclerosis

Mélanie Vélier, Romain Appay, Robin Arcani, Romain Desprat, Stéphanie Simoncini, Anouck Coulange Zavarro, D. Noel, Lenha Bendaass, Christian Jorgensen, Quentin Gomes de Pinho, et al.

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Mélanie Vélier, Romain Appay, Robin Arcani, Romain Desprat, Stéphanie Simoncini, et al. Generation of human induced pluripotent stem cell lines (iPSC) from adipose-derived mesenchymal stromal cells from two patients with systemic sclerosis. Stem Cell Research, 2025, 82, pp.103624. 10.1016/j.scr.2024.103624. hal-04951562

HAL Id: hal-04951562

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Submitted on 17 Feb 2025

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# Generation of human induced pluripotent stem cell lines (iPSC) from adipose-derived mesenchymal stromal cells from two patients with systemic sclerosis

Mélanie Velier<sup>a,b,*</sup>, Romain Appay<sup>c,d</sup>, Robin Arcani<sup>a,e</sup>, Romain Desprat<sup>f</sup>,
Stéphanie Simoncini<sup>a</sup>, Anouck Zavarro<sup>a</sup>, Danièle Noel<sup>g</sup>, Lenha Bendaass<sup>a</sup>,
Christian Jorgensen<sup>f,g,i</sup>, Quentin Gomes De Pinho<sup>a,h</sup>, Audrey Benyamine<sup>a,h</sup>, Brigitte Granel<sup>a,h</sup>,
Aurélie Daumas<sup>a,e</sup>, Francoise Dignat George<sup>a</sup>, Florence Sabatier<sup>a,b</sup>, Jérémy Magalon<sup>a,b</sup>

## ABSTRACT

Systemic sclerosis (SSc) is a rare and complex connective tissue disease associated with high morbidity and mortality. SSc is characterized by ischemic vasculopathy, cutaneous and visceral fibrosis and a dysimmune state (Denton and Khanna, 2017; Volkmann et al., 2023; Barnes and Mayes, 2012). We have derived induced pluripotent stem cell (iPSC) lines from two SSc patients aged 38 and 67 years with severe vascular phenotype. These iPSC lines expressed pluripotent markers, exhibited normal and stable genome, and differentiated into trilineage embryonic layers in teratoma formation assays. These SSc-specific iPSC lines can be differentiated into endothelial cells, providing a valuable model to elucidate vascular dysfunction and develop personalized therapeutic approaches.

## 1. Resource table

<table><thead><tr><th>Alternative name(s) of stem cell lines</th><th>REGi011-A,REGi012-A</th></tr></thead><tbody><tr><td>Institution</td><td>Safe IPS facility</td></tr><tr><td>Contact information of distributor</td><td>Mélanie VELIER melanie.velier@ap-hm.fr</td></tr><tr><td>Type of cell lines</td><td>iPSC</td></tr><tr><td>Origin</td><td>human</td></tr><tr><td>Additional origin info required<br>for human ESC or iPSC</td><td>Age: 38 and 67<br>Sex: Female</td></tr><tr><td>Cell Source</td><td>Adipose-derived mesenchymal<br>stromal/stem cells</td></tr><tr><td>Clonality</td><td>Pool</td></tr><tr><td>Method of reprogramming</td><td>Sendai</td></tr><tr><td>Genetic Modification</td><td>YES</td></tr><tr><td>Type of Genetic Modification</td><td>Spontaneous mutation</td></tr><tr><td>Evidence of the reprogramming transgene loss<br>(including genomic copy if applicable)</td><td>RNA seq, Phosphatase alkaline,<br>Cytometry</td></tr></tbody></table>

(continued on next column)

(continued)

<table><thead><tr><th>Alternative name(s) of stem cell lines</th><th>REGi011-A,REGi012-A</th></tr></thead><tbody><tr><td>Associated disease</td><td>Systemic sclerosis</td></tr><tr><td>Gene/locus</td><td>N/A</td></tr><tr><td>Date archived/stock date</td><td>31/07/2024</td></tr><tr><td>Cell line repository/bank</td><td>hPSCReg (https://hpscreg.eu/user/dashboard)</td></tr><tr><td>Ethical approval</td><td>CPP Sud Mediterranee V (ref 14.051)<br>N° 2014-003023-22.</td></tr></tbody></table>

## 2. Resource utility

Vascular alterations represent an early and critical event in SSc. However, direct access to endothelial cells from SSc patients remains challenging (Denton and Khanna, 2017; Fioretto et al., 2023). The generation of iPSC from SSc patients offers an alternative to overcome

<footnote><sup>a</sup> Aix-Marseille Univ, C2VN, INSERM 1263, INRAE1260, Marseille, France</footnote>

<footnote><sup>b</sup> Culture and Cell Therapy Laboratory, INSERM CIC BT 1409, Hôpital de la Conception, Assistance Publique Hôpitaux de Marseille (AP-HM), Marseille, France</footnote>

<footnote><sup>c</sup> Department of Pathological Anatomy and Neuropathology, Hôpital de La Timone, Public Assistance Publique Hôpitaux de Marseille (AP-HM), Marseille, France</footnote>

<footnote><sup>d</sup> Aix-Marseille Univ, CNRS, Institute of Neurophysiopathology, Marseille, France</footnote>

<footnote><sup>e</sup> Department of Internal Medicine and Therapeutics Department, Hôpital de La Timone, Assistance Publique Hôpitaux de Marseille (AP-HM), Marseille, France</footnote>

<footnote><sup>f</sup> Plateforme SAFE iPS, CHU Montpellier, IRMB, France</footnote>

<footnote><sup>g</sup> IRMB, University of Montpellier, INSERM, Montpellier, France</footnote>

<footnote><sup>h</sup> Internal Medicine Department, Hôpital Nord, Assistance Publique Hôpitaux de Marseille (AP-HM), Marseille, France</footnote>

<footnote><sup>i</sup> Clinical Immunology and Osteoarticular Disease Therapeutic Unit, Department of Rheumatology, CHU Montpellier, Montpellier, France</footnote>

<footnote>* Corresponding author.</footnote>
this limitation, providing a valuable and renewable model for investi-
gating the mechanisms driving microvascular dysfunction in SSc.

3. Resource details

The experimental approach for the development of a cellular model
of systemic sclerosis with induced pluripotent stem cells (iPSC) is out-
lined in Fig. 1A. Adipose-derived mesenchymal stromal cells (ASC) were
reprogrammed using the Sendai virus gene delivery system to express

four key reprogramming factors—OCT4, SOX2, SSEA4 and NANO-
G—without genomic integration. The pluripotency of both cell lines was
demonstrated by subcutaneous injection and the subsequent formation
of teratoma. Histologic analysis of these teratoma, stained with May-
Grünwald Giemsa, confirmed the presence of all three germ layers
(Fig. 1B). In addition, the pluripotency of iPSC lines M018 and M027
was confirmed by enzyme staining for alkaline phosphatase (Fig. 1C),
and corroborated by flow cytometry analysis of the undifferentiated
human iPSC markers, which revealed the expression of OCT4, SSEA4,

<figure><img src="image_4.png" /><figcaption>Fig. 1.</figcaption></figure>
SOX2, and NANOG (Fig. 1C). The results of the transcriptome analysis of the two iPSC lines are shown in Fig. 1D: a heatmap shows the supervised clustering of genes involved in pluripotency (left panel), and RNA sequencing data of the iPSC used in the ScoreCard pluripotency assessment and Sequin platform (https://sequin.ncats.io/app/), which confirmed the pluripotent nature of the cells (right panel). Finally, the genomic stability of iPSC M018 and M027 was analyzed by CGH array (Fig. 1E), and no chromosomal abnormalities were detected. A summary of the characterization and validation of iPSC lines M018 and M027 can be found in Table 1.

<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>Visual record of the lines is normal</td><td>Supplementary Fig. 1</td></tr><tr><td>Phenotype</td><td>Immunocytochemistry,<br>Quantitative analysis<br>Flow cytometry</td><td>Assess staining/expression of undifferentiated human iPSC markers: Oct3/4, Nanog, Sox2, Tra 1–60, SSEA-4, SSEA-1, Tra-1–80.<br>Assess % of positive cells or transcripts for antigen & cell surface markers e.g. Oct3/4: 97 %Tra 1–60: 98 %SSEA-4: 99 %SSEA-1: 6 %<br>E.g. 46XX,</td><td>Data not shown<br>Fig. 1 panel C</td></tr><tr><td>Genotype</td><td>CGH array</td><td>Resolution 450–500<br>DNA Profiling e. g. Performed/not performed<br>12 STR ATCC</td><td>Fig. 1 panel E</td></tr><tr><td>Identity</td><td>STR analysis</td><td>Mycoplasma testing by luminescence.<br>Negative<br>Describe expression of genes in embryoid bodies: e.g. muscle actin, β-tubulin and α-feto protein.<br>OR proof of three germ layers formation</td><td>Supplementary Table1<br>e.g. submitted in archive with journal<br>Supplementary Table2</td></tr><tr><td>Microbiology and virology<br>Differentiation potential</td><td>Mycoplasma<br>Teratoma formation</td><td>Mycoplasma testing by luminescence.<br>Negative<br>Describe expression of genes in embryoid bodies: e.g. muscle actin, β-tubulin and α-feto protein.<br>OR proof of three germ layers formation</td><td>Fig. 1 panel B</td></tr><tr><td>List of<br>recommended<br>germ layer<br>markers</td><td>Expression of these markers has to be demonstrated at mRNA (RT PCR) or protein (IF) levels, at least 2 markers need to be shown per germ layer</td><td></td><td>not shown but available with author</td></tr><tr><td>Donor screening</td><td>HIV 1 + 2 Hepatitis B, Hepatitis C</td><td>Negative</td><td>not shown but available with author</td></tr></tbody></table>

# 4. Materials and methods

## 4.1. Reprogramming ASC to iPSC

ASC were isolated from two SSc patients using surgical residues obtained from routine lipotransfer procedures with informed consent provided for the scientific use of these samples. The collected lipoaspirate was washed and enzymatically digested at 37 °C for 45 min. The resulting cell suspension was then plated into a T75 flask to isolate ASC (adherent cells). The ASC were then reprogrammed using the CytoTune ®-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, cat#A34546) containing Klf4, Oct3/4, Sox2, and c-Myc vectors, according to the manufacturer's protocol. The morphology of the IPS is presented in Supplementary Fig. 1.

## 4.2. Alkaline phosphatase activity

The cells were stained with Vector® Red AP substrate (Vector Laboratories kit #SK-5100) following the manufacturer's instructions. The culture media was removed before incubation of the cells with the substrate working solution for 20–30 min in the dark. The images are presented in Fig. 1C.

## 4.3. iPSC culture and genomic DNA extraction

The iPSC were cultured on hPSC-qualified Matrigel substrate as extracellular matrix (Fisher Scientific, cat#354277, lot number: 5194015), with a recommended dilution factor of 1:83. Cells were maintained in Essential 8™ culture media (Thermo Fisher Scientific, cat#A15169-01) following the manufacturer's instructions. Cultures were incubated at 37 °C under conditions of 5 % O₂ and 5 % CO₂. For cell dissociation, Versene solution (Thermo Fisher Scientific; cat#15040066) was used.

## 4.4. Flow cytometry analysis

For flow cytometry analysis, a single-cell suspension (at P12) for each cell line was fixed in a 4 % paraformaldehyde solution in phosphate-buffered saline (PBS). Following fixation, the cells were permeabilized and labelled according to the established protocols of the BD Stemflow Human and Mouse Pluripotent Stem Cell Analysis Kit (catalog no. 560477) and the BD Stemflow Human Pluripotent Stem Cell Transcription Factor Analysis Kit (catalog no. 560589). The staining of specific undifferentiated human iPSC markers was performed using dedicated antibodies. In the flow cytometry results, the white peaks correspond to IgG staining, while the black peaks represent specific markers, confirming the pluripotent status of the cells. Cell analysis was conducted using a Becton Dickinson CANTO II instrument, and the acquired data were processed using FlowJo software. The results are presented in Fig. 1C and the antibodies used are listed in Table 2.

## 4.5. RNA sequencing and supervised hierarchical clustering

Total RNA extraction was performed on 2 iPSC lines using the RNeasy kit (#74104, Qiagen) with on-column DNase digestion with the RNase-Free DNase Set (#79254, Qiagen) following the original manufacturer's protocol. RNA integrity was assessed using the RNA Screen Tape (Agilent 4150 TapeStation system). Bulk mRNA sequencing in paired-end mode (150 bp) was performed on BGISEQ sequencing platform by BGI Genomics. Filtered clean reads were aligned to the genome version reference sequence: homo_sapiens_9606.NCBI. GCF_000001405.40_GRCh38.p14.v2201, by HISAT, and then mapped to the reference genes using Bowtie2. Gene expression quantification was performed using RSEM software (v1.3.1) and heatmaps illustrating gene expression clustering across different samples were generated using pheatmap (v1.0.8). DESeq2 (v1.4.5) was used for differential gene
<table><caption>**Table 2**
Reagents details Characterization and validation.</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>Pluripotency Markers Flow cytometry</td><td>Pluripotency Markers Flow cytometry</td><td>1:200</td><td>Cell Signaling Technology Cat# 4903</td><td>RRID: AB_10559205</td></tr><tr><td>Pluripotency Markers Flow cytometry</td><td>Oct-4A Rabbit mAb (Clone C30A3) IgG</td><td>1:200</td><td>Cell Signaling Technology Cat# 2840</td><td>RRID: AB_2167691</td></tr><tr><td>Pluripotency Markers Flow cytometry</td><td>Sox2 XP® Rabbit mAb (Clone D6D9) IgG</td><td>1:200</td><td>Cell Signaling Technology Cat# 3579</td><td>RRID: AB_2195767</td></tr><tr><td>Pluripotency Markers Flow cytometry</td><td>SSEA4 Mouse mAb (Clone MC813) IgG3</td><td>1:200</td><td>Cell Signaling Technology Cat# 4755</td><td>RRID: AB_1264259</td></tr><tr><td>Pluripotency Markers Flow cytometry</td><td>TRA-1-60(S) Mouse mAb (Clone TRA-1-60(S)) IgM</td><td>1:200</td><td>Cell Signaling Technology Cat# 4746</td><td>RRID: AB_2119059</td></tr></tbody></table>

<table><thead><tr><th rowspan="2"></th><th colspan="2">Primers</th><th rowspan="2">Forward/Reverse primer (5'-3')</th></tr><tr><th>Target</th><th>Size of band</th></tr></thead><tbody><tr><td>Sendai Virus detection</td><td>Sendai virus detection</td><td>181 bp</td><td>F: GGATCACTAGGTGATATCGAGC<br>R: ACCAGACAAGAGTTTAAGAGATATGTATC</td></tr><tr><td>Sendai Virus detection</td><td>c-Myc detection</td><td>532 bp</td><td>F: TAACTGACTAGCAGGCTTGTCG<br>R: TCCACATACAGTCCTGGATGATGATG</td></tr><tr><td>Sendai Virus detection</td><td>Cluster KOS detection</td><td>528 bp</td><td>F: ATGCACCGCTACGACGTGAGCGC<br>R: ACCTTGACAATCCTGATGTGG</td></tr></tbody></table>

detection. A revised list of pluripotency genes was employed to generate the heatmap for supervised hierarchical clustering (Fig. 1D).

## 4.6. CGH array

Genomic DNA extracted from the iPSC line at passage 10 and was analyzed by ChromoStem facility of Montpellier using the chromosomal microarray was performed using SurePrint G3 Human CGH microarray Kit, 8 × 60 K with overall median probe 41 Kb genome-wide median probe spacing. Experiments were conducted according manufacturer's protocol. Commercial reference DNA (male and female) provided by Agilent Technologies were used for the analysis. Results are presented in Fig. 1E.

## 4.7. Sendai clearance

To confirm the absence of the Sendai virus, RNA was extracted from cells using the RNeasy kit (QIAGEN) according to the manufacturer's instructions. After 10 passages, of iPSC, extracted RNA was used as a matrix for RT-qPCR with 5x One-Step RT-PCR Mastermix (BioLabs). RT-PCR was conducted using primers provided with the Cytotune kit to detect the SeV backbone and OSKM genes under the following conditions: denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and elongation at 72 °C for 30 s for a total of 30–35 cycles. Results are presented in Supplementary Table 3.

## 4.8. Teratoma induction and histological analysis

The evaluation of differentiation potential was accomplished through in vivo teratoma formation. Clusters containing approximately 3 × 10⁶ iPSC cells were introduced into anesthetized NOD SCID gamma

(NOD.CgPrkdcscidIl2rg tm1Wjl/SzJ) mice. Following a latency period of 4–8 weeks, a 100 % efficiency in teratoma formation was observed. These teratoma were subsequently fixed, embedded in paraffin blocks, subjected to Hematoxylin-Eosin-Saffron staining, and subsequently analysed by a pathologist. This analysis aimed to identify structures corresponding to the three primary embryonic germ layers (Fig. 1B).

## 4.9. Short tandem repeat analysis (STR)

Cell lines were authenticated using analysis performed on 17 STR. Genomic DNA from each iPSC was sent to ATCC (ATCC Manassas, Virginia 20110 USA).

## 4.10. Mycoplasma detection

To detect mycoplasma, the MycoAlert® Detection Kit (Lonza) was used according to manufacturer's instructions. Cells culture was done without any antibiotic affecting myoplasm growth. Results are presented in Supplementary Table 2.

### CRediT authorship contribution statement

**Mélanie Velier:** Writing – review & editing, Writing – original draft, Methodology, Conceptualization. **Romain Appay:** Methodology, Data curation. **Robin Arcani:** Conceptualization. **Romain Desprat:** Methodology, Data curation. **Stéphanie Simoncini:** Conceptualization. **Anouck Zavarro:** Conceptualization. **Danièle Noel:** Writing – review & editing. **Lenha Bendaass:** Conceptualization. **Christian Jorgensen:** Supervision. **Quentin Gomes De Pinho:** Conceptualization. **Audrey Benyamine:** Conceptualization. **Brigitte Granel:** Writing – review & editing, Conceptualization. **Aurélie Daumas:** Conceptualization. **Françoise Dignat George:** Supervision. **Florence Sabatier:** Validation, Supervision, Conceptualization. **Jérémy Magalon:** Validation, Supervision, Conceptualization.

### 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.

### Acknowledgments

This work was supported by the Fondation pour la Recherche Médicale (grant #REP202210016160).

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2024.103624.

### Data availability

Data will be made available on request.

### References

* Barnes, J., Mayes, M.D., 2012. Epidemiology of systemic sclerosis: incidence, prevalence, survival, risk factors, malignancy, and environmental triggers. Curr. Opin. Rheumatol. 24 (2), 165–170.

* Denton, C.P., Khanna, D., 2017. Systemic sclerosis. Lancet 390 (10103), 1685–1699.

* Fioretto, B.S., Rosa, I., Matucci-Cerinic, M., Romano, E., Manetti, M., 2023. Current trends in vascular biomarkers for systemic sclerosis: a narrative Review. IJMS 24 (4), 4097.

* Volkmann, E.R., Andréasson, K., Smith, V., 2023. Systemic sclerosis. Lancet 401 (10373), 304–318.