Generation of a human induced pluripotent stem cell line (FSMi001-A) from fibroblasts of a patient carrying heterozygous mutation in the REEP1 gene

Abstract

Hereditary spastic paraplegias (HSPs) a group of rare, clinically, and genetically heterogeneous disorders characterized by progressive degeneration of the corticospinal tract. Among these HSPs, SPG31 is due to autosomal dominant mutations in the receptor expression-enhancing protein 1 (REEP1) gene. Over 80 genes have been associated with HSPs, and the list is constantly growing as research progresses. This study is aimed to create a patient-derived human induced pluripotent stem cell (hiPSC) line with a specific nonsense mutation to better characterize the etiopathogenesis of the disease.

1. Introduction

Resource Table

Unique stem cell line identifier	FSMi001-A
Alternative name(s) of stem cell line	FSM-SPG31-2
Institution	Department of Neurobiology and Molecular Medicine, IRCCS Fondazione Stella Maris, Calambrone (PI), Italy
Contact information of distributor	Matteo Baggiani – matteo.baggiani@fsm.unipi.it
	Devid Damiani – devid.damiani@fsm.unipi.it
Type of cell line	hiPSC
Origin	Human
Additional origin info required for human ESC or iPSC	Age: 47 years Sex: Female Ethnicity: Caucasian
Cell Source	Skin fibroblasts
Clonality	Clonal
Method of reprogramming	Episomal vectors encoding OCT4/sh-p53, SOX2/KLF4, EBNA-1, and L-MYC/LIN28
Genetic Modification	NO
Type of Genetic Modification	None
Evidence of the reprogramming transgene loss (including genomic copy if applicable)	RT/qPCRs for plasmid-specific transcripts show no expression for reprogramming genes
Associated disease	SPG31 (Spastic Paraplegia 31)
Gene/locus	REEP1 (c.337C > T; p. Arg113*)
Date archived/stock date	03 July 2013
Cell line repository/bank	https://hspcreg.eu/cell-line/FSMi001-A
Ethical approval	Written informed consent was obtained from patient

2. Resource utility

Generation of hiPSCs by means of somatic cell reprogramming from patients harboring different genetic variants offers an invaluable tool to understand disease mechanisms and explore novel treatment options for rescuing axonal defects and diverse cellular processes, especially in neurological diseases as SPG31 in which etiopathogenesis has been shown to be strictly dependent on the identity of mutations.

3. Resource details

Hereditary spastic paraplegias (HSPs) are inherited neurological disorders characterized by progressive degeneration of the corticospinal tract, leading to weakness and spasticity in the lower limb. Genetic alterations in more than 80 genes have been associated with HSPs (Elsayed et al., 2021). Among them, Spastic paraplegia type 31 (SPG31; MIM #610250) is an autosomal dominant form due to mutations in the REEP1 gene and characterized by lower limb spasticity, gait abnormalities, and muscle weakness. Most SPG31 patients are affected by a “pure” HSP form with onset between the second and the fourth decade of life. However, a not negligible percentage of the patients also display a “complex form”, presenting additional symptoms as sensory-motor peripheral neuropathy, bladder disturbances, white matter lesions, cerebellar ataxia, and dementia (Goizet et al., 2011, Toft et al., 2019). At the molecular level, several studies has involved the gene product in the regulation of lipid droplet metabolism and ER-mitochondrial membrane interface, with alteration of mitochondrial morphology, localization (Table 1), and functionality in case of heterozygous REEP1 mutations (Lavie et al., 2016, Naef et al., 2023).

Table 1.

Characterization and validation.

Classification	Test	Result	Data
Morphology	Photography Bright field	Normal morphology of human iPS cells	Fig. 1 A
Phenotype	Qualitative analysis by Immunocytochemistry	hiPS cells express stemness markers: Oct4, Nanog, Tra 1–60, SSEA-4.	Fig. 1 B
	Qualitative analysis by RT-qPCR	Expression of stemness markers compared to ACS-1019 (CTRL) reference cell line/parental fibroblasts	Fig. 1 C
Genotype	hPSC Genetic Analysis Kit; CGH array (60 kb)	No abnormalities detected. 46, XX. No abnormalities detected	Fig. 1 D; Suppl. Fig. 1 B
Identity	Microsatellite PCR (mPCR) OR	Not performed	N/A
	STR analysis	16 markers tested- matched	submitted in archive with journal
Mutation analysis	Sequencing	heterozygous, type of mutation. REEP1 (c.337C > T; p.Arg113Ter)	Fig. 1 E
	Southern Blot OR WGS	N/A	N/A
Microbiology and virology	Mycoplasma detection by PCR	Negative	Fig. 1 F
Differentiation potential	Directed differentiation	Expression of layer-specific markers: Ectoderm (Nestin, β3- tubulin), Mesoderm (BRACHYURY) and Endoderm (SOX17).	Fig. 1 G
Donor screening (OPTIONAL)	HIV 1 + 2 Hepatitis B, Hepatitis C	N/A	N/A
Genotype additional info (OPTIONAL)	Blood group genotyping	N/A	N/A
	HLA tissue typing	N/A	N/A

The purpose of this study was to generate a hiPSC line derived from a patient carrying a heterozygous nonsense mutation in exon 5 of REEP1, namely c.337C > T/(p. Arg113Ter). Our future goal is to use this line for in vitro differentiation in cortical tissue, in order to get a better characterization of etiopathogenesis of the disease, as previously done in other models of HSP (Damiani et al., 2024). A recent work has generated heterozygous and homozygous cell lines harboring a c.329-391del p.Lys112Serfs*54 variant in REEP1 by means of CRISPR-Cas9 technology (Korneck et al., 2024). Despite being very useful resources, this approach could not sufficient to fully describe the heterogeneity of the complex genetic background found in SPG31 patients. In particular, previous research identified numerous mutations hitting this gene. Insertions, deletions, and nucleotide changes were shown to result in frameshift, premature termination of translation, exon skipping and even changes in the 3′ UTR region, affecting the binding site of a known microRNAs, such as miR-140 and miR-691. In most cases, haploinsufficiency due to absence of protein produced by a single mutant allele seems to be the leading mechanism of the disease, as most terminating mutations are predicted to lead to nonsense-mediated decay. In other cases, variants of full length or truncated REEP1 protein could be produced, leading to alteration of subcellular targeting or even possible toxic gain-of-function. Most importantly, heterogeneity of genetic background in SPG31 patients directly reflects diversity in their symptomatology (Beetz et al., 2012, Beetz et al., 2008, Falk et al., 2014, Goizet et al., 2011, Hewamadduma et al., 2009, Maroofian et al., 2019, Toft et al., 2019, Züchner et al., 2006). In our case, the mutation was shown to result in haploinsufficiency due to nonsense-mediated decay of the transcript (Naef et al., 2023). Having received written informed consent, fibroblasts were derived and amplified from a skin-punch biopsy taken from a 47-year-old Italian woman. Cells were successively reprogrammed via episomal transfection of 3 plasmids encoding the classical Yamanaka factors. Pluripotent cell clones, emerging about 2 weeks after transfection, were picked, propagated as single cell lines, and passed at least 7 times before full immunocytochemical, molecular and genomic characterization. Among them, one clone was selected for further amplification and cryopreservation.

4. Materials and methods

4.1. Reprogramming of skin fibroblasts

A small sample of skin tissue was harvested from the patient via punch biopsy. Human dermal fibroblasts (HDFs) derived from the sample were cultured and amplified in HDF medium (DMEM High Glucose, 10 % Fetal Bovine Serum, L-Glutamine 2 mM, 100 U/ml PenStrep; Euroclone, Milan, Italy).

For reprogramming reaction, we followed a modified procedure based on protocols described in literature (Okita et al., 2013). HDFs were transfected with a mix of episomes for integration-free expression of human “Yamanaka factors” (L-MYC, LIN28, OCT3/4, SOX2, and KLF4). In particular, pCXLE-hOCT3/4-shp53-F + mCherry-2A-puro (gift from Ann Zovein, Addgene plasmid # 74947; Addgene, Watertown, MA), pCXLE-hUL (Addgene plasmid #27080), pCXWB-EBNA1 (Addgene, #37624), and pCXLE-hSK (Addgene, #27078, all gifts from Shinya Yamanaka) were nucleofected in a cell suspension via Amaxa Nucleofector 2b (Lonza, Tampa, FL), following manufacturer instructions. Transfected cells were plated on dishes coated with Geltrex (Thermo Fisher Scientific, Waltham, MA) and cultured with E6 containing 100 ng/ml FGF2 (named E7 medium), 0.5 mM sodium butyrate, 1 µM hydrocortisone. Successful transfection was checked after 2 days by positive expression of mCherry (Schmitt et al., 2017). To further increase the percentage of transfected cells, 0.5 µg/ml puromycin was added to the medium (PAC is encoded from Plasmid #74947). After 10 days, confluent cells were dissociated into single cell suspension with Trypsin (Euroclone), plated on Geltrex coated dishes at 5.000 cells/cm², and cultured with E7 medium adding 0.5 mM sodium butyrate only for 2 days. hiPSC “islands” emerged after 14 days after transfection. At that time, medium was switched to Stemflex medium (Thermo Fisher Scientific) to grant iPSC survival and growth. hiPSC clones with uniform flat and round shaped morphology were picked in sterility conditions and propagated as single cell lines. To decrease the odds of integration of reprogramming sequences, clones were carefully checked to be completely free of mCherry-positive cells (Suppl Fig. 1A).

4.2. Culture of hiPSCs

hiPSCs were cultured at 37 °C and 5 % CO2 on Geltrex coated 6-well-plates in Stemflex medium, refreshing medium every other day. Cells were passed every 5–7 days with ReLeSR™ Passaging Reagent (Stem Cell Technologies, Vancouver, Canada), following manufacturer instruction (Fig. 1A).

Fig. 1.

4.3. Expression of stemness markers

Total RNA from hiPSCs and parental HDFs was extracted with All-prep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized with PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan). Relative expression of markers for the undifferentiated state OCT4, SOX2, and L-MYC was determined via RT-qPCR on a Mic qPCR Cycler (Bio Molecular Systems, Upper Coomera, Australia), normalizing values to expression of the established ACS-1019 hiPSC line (ATCC, VA), named CTRL, via 2^−ΔΔCT method (Fig. 1 C). Human GAPDH was used as housekeeping gene. Moreover, to exclude the presence of episomes, the relative expression of eOCT4, eSOX2, eLIN28, and eKLF4 was measured via RT-qPCR on a Mic qPCR Cycler on tHDFs (transfected HDFs), CTRL, and FSMi001-A cell lines (Fig. 1 H). Primers used for qPCR reactions are listed in Table 2.

Table 2.

Reagents details.

	Antibodies used for immunocytochemistry
	Antibody	Dilution	Company Cat #
Stemness Markers	Rabbit anti-OCT4 Rabbit anti-Nanog (D73G4) Mouse anti-TRA-1–60 Mouse anti-SSEA4 (clone MC813)	1:500 1:200 1:500 1:500	Abcam Cat# ab19857 Cell Signaling Technology Cat# 4903 Cell Signaling Technology Cat # 4746 Cell Signaling Technology Cat #4755
Differentiation Markers	Mouse anti-Nestin Mouse anti-Beta-3 Tubulin Rabbit anti-Brachyury (D2Z3J) Rabbit anti-SOX17 (D1T8M)	1:500 1:1000 1:500 1:500	Abcam Cat# ab18102 Abcam Cat# ab7751 Cell Signaling Cat# 81694 Cell Signaling Cat# 81778
Secondary antibodies	Goat Anti-Mouse Alexa Fluor 488 Goat Anti-Rabbit Alexa Fluor 555	1:500 1:500	Thermo Fisher Scientific Cat# a11029 Thermo Fisher Scientific Cat# a21429
	Primers
	Target	Size of band	Forward/Reverse primer (5′-3′)
Episomal Plasmids (qPCR)	eOCT4 eLIN28 eSOX2 eKLF4	70–150 bp 70–150 bp 70–150 bp 70– 150 bp	Fwd: CAT TCA AAC TGA GGT AAG GG Rev: TAG CGT AAA AGG AGC AAC ATA G Fwd: AGC CAT ATG GTA GCC TCA TGT CCG C Rev: TAG CGT AAA AGG AGC AAC ATA G Fwd: TTC ACA TGT CCC AGC ACT ACC AGA Rev: TTT GTT TGA CAG GAG CGA CAA T Fwd: CCA CCT CGC CTT ACA CAT GAA GA Rev: TAG CGT AAA AGG AGC AAC ATA G
Stemness Markers (qPCR)	OCT4 L-MYC SOX2	70–150 bp 70–150 bp 70–150 bp	Fwd: CCC CAG GGC CCC ATT TTG GTA CC Rev: ACC TCA GTT TGA ATG CAT GGG AGA GC Fwd: GCG AAC CCA AGA CCC AGG CCT GCT CC Rev: CAG GGG GTC TGC TCG CAC CGT GAT G Fwd: TTC ACA TGT CCC AGC ACT ACC AGA Rev: TCA CAT GTG TGA GAG GGG CAG TGT GC
House-Keeping Genes (qPCR)	GAPDH	70–150 bp	Fwd: GGA AGG ACT CAT GAC CAC AGT Rev: GGA TGA TGT TCT GGA GAG CCC
Genotyping	chr2:86,251,853–86,252,186 (spanning REEP1 Exon 5)	334 bp	Fwd: CCC AGC AAG AAC AAG GAT TG Rev: CCA CTG ATT GGT CCT TAG CC

For immunofluorescence staining the hiPSCs were fixed with 4 % paraformaldehyde for 12 min at RT, permeabilized in PBS with 0.5 % Triton X-100 for 10 min at RT, blocked in PBS with 5 % Normal Goat Serum, 0.3 % Triton X-100 for 1 h at RT, and incubated with primary antibodies (Table 2) in PBS with 3 % Normal Goat Serum, 0.2 % Triton X-100 at 4 °C overnight. On the next day, the stained cells were washed 3 times with PBS and incubated in the same antibody solution with the corresponding secondary antibody (Table 2) for 1 h at RT. Nuclei were counterstained with DAPI (1 µg/ml; Merck, Darmstadt, Germany). All images were acquired with a Zeiss LSM 900 confocal microscope (Zeiss, Oberkochen, Germany) and processed with Fiji software.

4.4. Trilineage differentiation

With the purpose to have a formal demonstration of effective pluripotency of the generated cell line, we performed a series of differentiation protocols aimed to obtain the three different embryonic germ layers, namely ectoderm, endoderm, and mesoderm. To this end, hiPSCs were cultured in trilineage differentiation medium (DMEM High Glucose, Glutamax 2 mM, 100 U/ml PenStrep, MEM-NEAA 1x, β-mercaptoethanol 0.1 %; Thermo Fisher Scientific) to get spontaneous differentiation in neural progenitors (positive for neural stem cell marker Nestin, Fig. 1 G1), or neuronal marker β3-tubulin (Fig. 1 G2), as previously reported (Maria Turco et al., 2022). Modifying Lam et al., 2014 guidelines, to acquire an endodermal identity, 5 µM CHIR99021 (Stem Cell Technologies) for 1 day and then 100 ng/ml Activin A (Stem Cell Technologies,) for 3 days were added, generating cells positive to endodermal marker SOX17 (Fig. 1 G3). Instead, to induce mesodermal differentiation, 5 µM CHIR99021 (Stem Cell Technologies) was added for 2 days, obtaining cells positive to the mesodermal marker Brachyury (Fig. 1 G4). Medium was changed every day.

4.5. Mutation analysis

Genomic DNA from hiPSCs and their parental HDFs was extracted with All-prep DNA/RNA Mini Kit (Qiagen). The exon 5 of REEP1 was amplified by PCR (PCR primers are listed in Table 2). The identity of mutation was confirmed via Sanger sequencing (Fig. 1 E).

4.6. STR analysis

The identity of hiPSCs with parental HDFs was confirmed via gDNA typing with PowerPlex 16HS multiplex STR system (Promega, Madison, WI), including all 13 CODIS STR markers, Amelogenin for gender determination, Penta D and Penta E loci. The PCR products labeled with fluorescent dyes were detected with ABI-3500 Genetic Analyzer and data were analyzed with GeneMapper 5 (Thermo Fisher Scientific).

4.7. Genomic analysis via CGH array

High-resolution whole-genome array-based Comparative Genomic Hybridization (aCGH) analysis was conducted on genomic DNA extracted from patient derived hiPSCs, using the SurePrint G3 Human CGH Microarray 8 × 60 k (Agilent Technologies, Santa Clara, CA), a dual-color array containing 60-mer high-quality probes with 41 Kb genome-wide median probe spacing. Copy Number Variants (CNVs) were analyzed and mapped using the Human Genome Assembly GRCh37/hg19. Slides were scanned using an Agilent G2600D Microarray Scanner (Agilent Technologies) and processed using Feature Extraction software (v12.1.0.3). Agilent CytoGenomics software (v5.3.0.14) was used to analyze the results with default settings. Imbalances with at least three consecutive probes with abnormal log2 ratios for deletions and at least four consecutive probes for duplications were included into results. The Database of Genome Variants (https://dgv.tcag.ca/dgv/app/home), DECIPHER (https://www.deciphergenomics.org), UCSC genome browser (https://genome.ucsc.edu), Database of Human CNVs (https://gvarianti.homelinux.net/gvariantib37/index.php), SFARI (Simon’s Foundation Autism Research Initiative), and Gene Database (https://gene.sfari.org) databases were used in the interpretation of the results (Fig. 1D). Coincident results were obtained when the same experiment was performed on fibroblast patient’s genomic DNA.

Moreover, genomic instability was tested on SPG31 hiPSCs at P7 and was ruled out using hPSC Genetic Analysis Kit (Stem Cell Technologies) on 7500 Fast Real Time Pcr System (Thermo Fisher Scientific), capable of detecting the most common karyotypic abnormalities reported in human ES and iPS cells by comparing the copy number of loci in 9 different chromosomes via qPCR (Suppl Fig. 1B).

4.8. Mycoplasma testing

Absence of mycoplasma was assessed by PCR via Mycoplasma PCR Detection Kit (ABM, Richmond, Canada), according to the manufacturer instruction (Fig. 1 F).

Funding

This research was partly supported by RF-2019-12370112 (to AT), SG-2021-12375552 (to SM), and 2023 RC Linea 3 from Italian Ministry of Health, and Fondazione Telethon Grant GJC21131 (to FMS and DD). MB’s position is supported by the Fondazione Telethon grant GJC21131.

CRediT authorship contribution statement

Matteo Baggiani: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Filippo Maria Santorelli: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Funding acquisition, Data curation, Conceptualization. Serena Mero: Writing – review & editing, Visualization, Supervision, Investigation, Data curation. Flavia Privitera: Writing – review & editing, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Devid Damiani: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Alessandra Tessa: Writing – review & editing, Writing – original draft, Visualization, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, 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.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Fig. 1.