# Generation of induced pluripotent stem cells (iPSCs) from a microvillus inclusion disease patient with a homozygous missense mutation in UNC45A

Celine Banal, Eddy Quelennec, Cécile Talbotec, Anis Khiat, Fabienne Charbit-Henrion, Nadine Cerf-Bensussan, Nathalie Lefort, Corinne Lebreton

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## To cite this version:

Celine Banal, Eddy Quelennec, Cécile Talbotec, Anis Khiat, Fabienne Charbit-Henrion, et al. Generation of induced pluripotent stem cells (iPSCs) from a microvillus inclusion disease patient with a homozygous missense mutation in UNC45A. Stem Cell Research, 2023, 68, 10.1016/j.scr.2023.103057. hal-04088625

HAL Id: hal-04088625

https://u-paris.hal.science/hal-04088625

Submitted on 4 May 2023

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Lab Resource: Single Cell Line

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# Generation of induced pluripotent stem cells (iPSCs) from a microvillus inclusion disease patient with a homozygous missense mutation in UNC45A

Celine Banal<sup>a</sup>, Eddy Quelennec<sup>a</sup>, Cécile Talbotec<sup>b</sup>, Anis Khiat<sup>c</sup>, Fabienne Charbit-Henrion<sup>b,d</sup>, Nadine Cerf-Bensussan<sup>c</sup>, Nathalie Lefort<sup>a,*</sup>, Corinne Lebreton<sup>c,*</sup>

## ABSTRACT

Mutations in UNC45A, a co-chaperone for myosins, were recently found causative of a syndrome combining cholestasis, diarrhea, loss of hearing and bone fragility. We generated induced pluripotent stem cells (iPSCs) from a patient with a homozygous missense mutation in UNC45A. Cells from this patient, which were reprogrammed using integration-free Sendaï virus, have normal karyotype, express pluripotency markers and are able to differentiate into the three germ cell layers.

## 1. Resource Table

Unique stem cell line identifier
Alternative name(s) of stem cell line
Institution

Contact information of distributor

Type of cell line
Origin
Additional origin info required
for human ESC or iPSC

Cell Source
Clonality
Method of reprogramming

Genetic Modification
Type of Genetic Modification
Evidence of the reprogramming
transgene loss (including genomic
copy if applicable)
Associated disease

IMAGINi020-A
IMAGINE020c24
Université Paris Cité, Imagine Institute,
INSERM UMR U1163
Corinne Lebreton (corinne.
lebreton@inserm.fr)Nadine Cerf-
Bensussan
(nadine.cerf-bensussan@inserm.fr)
Nathalie Lefort
(nathalie.lefort@inserm.fr)

iPSC
human
Age: 4
Sex: female
Ethnicity if known: caucasian

Cryopreserved peripheral blood
mononuclear cells (PBMCs)
clonal
Non-integrating SeV-mediated delivery of
OCT4, SOX2, c-MYC and KLF4
Yes
Inherited recessive mutation.
RT-/q-PCR scorecard assay

microvillus inclusion disease

(continued on next column)

(continued)

<table><tr><td>Gene/locus</td><td>UNC45, Chr15(GRCh37):g.91485689 T &gt; C; NM_018671.4: c.710 T &gt; C</td></tr><tr><td>Date archived/stock date</td><td>May 2018</td></tr><tr><td>Cell line repository/bank</td><td>https://hpscreg.eu/cell-line/IMA_GiNi020-A</td></tr><tr><td>Ethical approval</td><td>hiPSC culture and storage protocols were performed with approval from French Research Ministry (DC 2015-2595, 09/05/2016)</td></tr></table>

## 2. Resource utility

UNC45A mutated-iPSCs can be used to differentiate into intestinal organoids in order to characterize and compare their phenotype and function to cells and organoids without the mutation. They will be useful to explore the cellular mechanisms leading to microvillus inclusion diseases.

## 3. Resource details

Monogenic intestinal disorders form an expanding spectrum of rare but severe diseases revealed by chronic diarrhea starting early in life. Next generation sequencing (NGS) performed in a 3 year-old girl with a syndrome combining cholestasis, diarrhea, loss of hearing and bone fragility revealed a homozygous missense mutation in UNC45A(Chr15

<footnote><sup>a</sup> Université Paris Cité, Imagine Institute, INSERM U1163, iPS Core Facility, 75015 Paris, France</footnote>

<footnote><sup>b</sup> Department of Pediatric Gastroenterology, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, F-75015 Paris, France</footnote>

<footnote><sup>c</sup> Université Paris Cité, Imagine Institute, INSERM U1163 and Laboratory of Intestinal Immunity, 75015 Paris, France</footnote>

<footnote><sup>d</sup> Department of Genomic Medicine for Rare Diseases, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, F-75015 Paris, France</footnote>

<footnote>* Corresponding authors.
E-mail addresses: nathalie.lefort@inserm.fr (N. Lefort), corinne.lebreton@inserm.fr (C. Lebreton).</footnote>
A

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B

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D

<table><thead><tr><th>samples</th><th>IMAGINi020-A</th></tr></thead><tbody><tr><td>PBMC020</td><td>99.30%</td></tr></tbody></table>

E

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G

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H

<figure><img src="image_9.png" /><figcaption>Fig. 1. Cellular and molecular characterization of IMAGINi020-A cell line.</figcaption></figure>

C

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F

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<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>Phase contrast bright field microscopy</td><td>Normal morphology</td><td>Fig. 1 panel A</td></tr><tr><td>Phenotype</td><td>Qualitative analysis by Immunocytochemistry</td><td>Expression of the pluripotency markers: OCT4 and SOX2</td><td>Fig. 1 panel F</td></tr><tr><td></td><td>Quantitative analysis by TaqMan assay</td><td>POU5F1, SOX2 and NANOG</td><td>Fig. 1 panel E</td></tr><tr><td>Genotype</td><td>SNP array (&gt;240000 markers)</td><td>Normal karyotypeArr (1-22, X)x2</td><td>Fig. 1 panel C</td></tr><tr><td>Identity</td><td>SNP array (&gt;240000 markers)</td><td>SNP array to compare iPSC line and the starting PBMC. Matching genotype confirms the origins of iPSCs</td><td>Fig. 1 panel D</td></tr><tr><td></td><td>STR analysis</td><td>Matched to parental PBMC at 8/8 STR loci</td><td>'Available with the authors'</td></tr><tr><td>Mutation analysis (IF APPLICABLE)</td><td>Sequencing Southern Blot OR WGS</td><td>Homozygous c.710 T &gt; C N/A</td><td>Fig. 1 panel B N/A</td></tr><tr><td>Microbiology and virology</td><td>Mycoplasma</td><td>Mycoplasma testing by luminescence. Negative</td><td>Supplementary Fig. 1</td></tr><tr><td>Differentiation potential</td><td>Direct differentiation into three germ layers</td><td>TaqMan® hPSC Scorecard™ analysis; negative score for self-renewal gene expression and positive score for trilineage gene expression positive immunostaining for β-III-TUBULIN, α-smooth muscle actin and α-fetoprotein</td><td>Fig. 1 panel G and H</td></tr><tr><td>Donor screening (OPTIONAL)</td><td>HIV 1 + 2 Hepatitis B, Hepatitis C</td><td>Negative</td><td>Not shown</td></tr><tr><td>Genotype additional info (OPTIONAL)</td><td>Blood group genotyping HLA tissue typing</td><td>N/A N/A</td><td>N/A N/A</td></tr></tbody></table>

(GRCh37):g.91485689 T > C; NM_018671.4: c.710 T > C) (Duclaux-Loras et al 2022). Given the difficulties to obtain intestinal biopsies from this patient, we choose to derive iPSCs from somatic cells. Peripheral blood mononuclear cells (PBMCs) were obtained from the patient harboring the UNC45A mutation and utilized to generate iPSCs using the non-integrating cytotune™-iPS20 Sendai virus reprogramming system (Fusaki et al., 2009). After two to three weeks, colonies with a morphology consistent with an iPSC identity became visible under phase contrast microscopy and were manually picked (Fig. 1A). Sanger sequencing of the mutation-variant at passage 11 confirmed the presence of the homozygous missense mutation in UNC45A (Chr15 (GRCh37):g.91485689 T > C; NM_018671.4: c.710 T > C) (Fig. 1B). Absence of mycoplasma contamination was ascertained before PBMC reprogramming and on iPSCs at passage 17, using the MycoAlert™ kit (Supp Fig. 1). Genomic integrity of iPSCs was assessed at passage 12 by SNP array analysis and showed a normal female karyotype (Arr(1-22, X) x2) (Fig. 1C). This test also confirmed the matching genotype of patient's PBMCs and of the iPSCs line (Fig. 1D). Furthermore, short tandem repeat (STR) analysis of 8 different loci demonstrated the iPSCs matched to the starting PBMC (STR analysis, Table 1). Following confirmation of genomic integrity, up-regulation of pluripotent stem cell markers was assessed by RT-qPCR analyses to demonstrate expression of POU5F1, SOX2 and NANOG mRNA (Fig. 1E) and by immunocytochemistry to show OCT4 and SOX2 protein expression (Fig. 1F). IPSCs' pluripotency was next estimated by evaluating their ability to spontaneously differentiate into cells of the three germ layers. Scorecard assays revealed that iPSC line thereafter called IMAGINi020-A iPSCs did not show any lineage bias and expressed mRNA levels comparable to the reference standard (Fig. 1G). Immunocytochemistry analyses confirmed that cells also expressed ectodermal (β-III-tubulin), mesodermal (α-smooth muscle actin, α-SMA) and endodermal proteins (α-fetoprotein, AFP) (Fig. 1H).

# 4. Materials and methods

## 4.1. Ethics statements

The generation, use and storage of human iPSCs were performed with approval from French Research Ministry (DC 2015-2595, 09/05/

2016). Formal informed consent was obtained from both parents.

## 4.2. Cell culture

Cryopreserved PBMCs were reprogrammed as previously described (Banal and Lefort, 2022). Briefly, PBMC were cultured for 4 days in StemPro™-34 SLM medium (ThermoFisher Scientific) with SCF, FLT-3 (100 ng/ml), IL-3, and IL-6 (20 ng/ml) (PeproTech) cytokines. Then, $2.5 \times 10^5$ cells were transduced using CytoTune-iPS 2.0 Sendai Reprogramming Kit according to manufacturer's instructions (ThermoFisher Scientific). Three days later, cells were transferred on vitronectin-coated culture dishes (StemCell technologies). At day 7, the medium was gradually replaced by ReproTeSR™ medium (StemCell technologies). After two weeks, colonies appeared and were manually picked and expanded on vitronectin-coated culture dishes with daily renewal of mTeSR™1 medium (StemCell technologies). For pluripotency assays, cells were cut into small clumps and cultured in suspension in low-attachment dishes for 8 days. Half of the resulting embryoid bodies (EBs) were collected for transcriptional analyses. The remaining EBs were plated onto gelatin-coated dishes for additional 7 days for immunocytochemical analyses as previously described (Quelennec et al., 2020).

Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂, 20% O₂.

## 4.3. Genomic analyses

DNA was extracted at passage 12 with the PureLink™ Genomic DNA Mini Kit (Invitrogen). Total RNA was extracted from iPSC colonies at passage 13 and from EBs derived from these cells using RNeasy Mini Kit (Qiagen). RT-PCR analyses: cDNA was synthesized using high capacity cDNA RT kit (ThermoFisher Scientific) from 1 µg of total RNA. The expression of pluripotency markers was analyzed using TaqMan® probes (probes ID listed in Table 1) and TaqMan® Gene Expression Master Mix (Applied Biosystems). Cycling conditions were as follows: 95 °C/5 min, (95 °C/10 s, 60 °C/15 s) × 45 cycles, followed by melt curve analysis. All quantitative gene expression data were normalized to the expression levels of 18S and compared to the control IMAGINi005-A
<table><caption>**Table 2**
Reagents details.</caption><thead><tr><th colspan="3">Antibodies used for immunocytochemistry</th></tr></thead><tbody><tr><td>Pluripotency Markers</td><td>Antibody<br>Monoclonal Anti-OCT3/4 (Mouse)</td><td>Dilution<br>1:100</td></tr><tr><td>Pluripotency Markers</td><td>Monoclonal Anti-Sox2 (Rabbit)</td><td>1:1000</td></tr><tr><td>Differentiation markers</td><td>Monoclonal anti-Tubulin β 3 (Mouse)</td><td>1:1000</td></tr><tr><td>Differentiation markers</td><td>Monoclonal Anti-Actin, α-Smooth Muscle - FITC (Mouse)</td><td>1:300</td></tr><tr><td>Differentiation markers</td><td>Monoclonal Anti-alpha-Foetoprotein (Mouse)</td><td>1:60</td></tr><tr><td>Secondary antibodies</td><td>goat anti-rabbit alexa fluor plus 488</td><td>1:1000</td></tr><tr><td>Secondary antibodies</td><td>goat anti-mouse alexa fluor plus 555</td><td>1:1000</td></tr><tr><td>Primers</td><td></td><td></td></tr><tr><td>Targeted mutation analysis</td><td>Target<br>UNC45A (202 bp)</td><td>Forward/Reverse primer (5'-3')<br>Forward 5'-TGCCATCCATTCTGTGTTGT-3'Reverse 5'-CCCAGGATGGAGACTACTCG-3'</td></tr><tr><td>Pluripotency Markers (qPCR)</td><td>NANOG (109 bp)</td><td>Hs02387400_g1 (TaqMan® probe ID)</td></tr><tr><td>Pluripotency Markers (qPCR)</td><td>POU5F1 (77 bp)</td><td>Hs04260367_gH (TaqMan® probe ID)</td></tr><tr><td>Pluripotency Markers (qPCR)</td><td>SOX2 (91 bp)</td><td>Hs01053049_s1 (TaqMan® probe ID)</td></tr><tr><td>House-Keeping Gene (qPCR)</td><td>18S (187 bp)</td><td>Hs99999901_s1 (TaqMan® probe ID)</td></tr></tbody></table><table><tbody><tr><td>Company Cat # and RRID</td><td>RRID</td></tr><tr><td>Santa Cruz Biotechnology</td><td>AB_628051</td></tr><tr><td>Cat# sc-5279</td><td></td></tr><tr><td>Millipore Cat# AB5603</td><td>AB_2286686</td></tr><tr><td>BioLegend Cat# 801,202</td><td>AB_10063408</td></tr><tr><td>Sigma-Aldrich Cat# F3777</td><td>AB_476977</td></tr><tr><td>R and D Systems Cat# MAB1369</td><td>AB_2258005</td></tr><tr><td>Thermo Fisher Scientific Cat# A32723</td><td>AB_2633275</td></tr><tr><td>Thermo Fisher Scientific Cat# A32732</td><td>AB_2633281</td></tr></tbody></table>

iPSC line (Quellennec et al., 2020). The TaqMan® hPSC Scorecard™ assay evaluated the trilineage differentiation potential of the cells and also revealed absence of SeV in iPSC. **Sanger sequences** of the mutation in UNC45 gene (Chr15(GRCh37):g.91485689 T > C; NM_018671.4: c.710 T > C) were analyzed using the automated 3500xl DNA Analyzer (Applied-Biosystems). **Karyotype analysis:** For molecular karyotype, Infinium Core-24 v1.2 Kit (Illumina) containing 300,000 SNPs was used. Data were analyzed with BeadStudio/GenomeStudio software (Illumina). All gene positions were based upon Human Genome Build 37 (HG19).

## 4.4. Short tandem repeat (STR) analysis

STR analysis was performed on 8 microsatellite markers (D3S3031, D3S4554, D4S3351, D15S1510, D17S1307, D19S393, D21S2196, D21S1446).

## 4.5. Immunofluorescence staining

Cells were fixed at room temperature with 4% paraformaldehyde for 15 min, washed in PBS-1 %BSA blocking solution for 1 h and incubated overnight at 4 °C with primary antibodies. After 3 washes in PBS, cells were incubated with a secondary antibody 2 h at room temperature. Antibodies were diluted in PBS added with 1 %BSA and 0.1% triton solution (Table 2). Nuclei were stained with a DAPI solution. Immunofluorescence staining was visualized with the Celena S™ Digital Imaging System (Logos Biosystems).

## 4.6. Mycoplasma detection

Mycoplasma contamination was checked using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza) according to manufacturer's instructions.

## 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 institutional grants from INSERM, by European grant ERC-2013-AdG-339407-IMMUNOBIOTA, by ANR-20-CE17-0020-01, by the Fondation Princesse Grace, and by the Fondation Maladies Rares. Imagine Institute is supported by Investissement d'Avenir grant ANR-10-IAHU-01. We thank the Imagine Institute's clinical research department for its help. We thank the patient and her parents for accepting to participate in research on UNC45A deficiency.

We thank Nadine Gigarel and Julie Steffann from the « laboratoire de génétique, service de médecine génomique des maladies rares Hôpital Necker-Enfants Malades » who performed STR assays.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2023.103057.

## References

* Banal, C., Lefort, N., 2022. Generation and characterization of IMAGINi013-A, an induced pluripotent stem cell line generated from a healthy donor. Stem Cell Res. 61, 102755.

* Duclaux-Loras, R., Lebreton, C., Berthelet, J., Charbit-Henrion, F., Nicolle, O., Revenu de Courtils, C., Waich, S., Valovka, T., Khiat, A., Rabant, M., Racine, C., Guerrera, I.C., Baptista, J., Mahe, M., Hess, M., Durel, B., Lefort, N., Banal, C., Parisot, M., Talbotec, C., Lacaille, F., Ecochard-Dugelay, E., Demir, A.M., Vogel, G., Faivre, L., Rodrigues, A., Fowler, D., Janecke, A., Müller, T., Huber, L., Rodrigues-Lima, F., Ruemmele, F., Uhlig, H., Del Bene, F., Michaux, G., Cerf-Bensussan, N., Parlato, M., 2022. Deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking. J. Clin. Invest. 132 (10), e154997.

* Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M., 2009. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad., Ser. B 85 (8), 348-362.

* Quellennec, E., Banal, C., Hamlin, M., Clémantine, D., Michael, M., Lefort, N., 2020. Generation of two induced pluripotent stem cell lines IMAGINi004-A and IMAGINi005-A from healthy donors. Stem Cell Res. 48, 101959.