Generation of two human induced pluripotent stem cell lines (iPSCs) with mutations of the α-synuclein (SNCA) gene associated with Parkinson’s disease; the A53T mutation (LCSBi003) and a triplication of the SNCA gene (LCSBi007)

Abstract

Mutations in the SNCA (α-synuclein, PARK1) gene significantly contribute to Parkinson’s disease (PD) and SNCA inclusions are strongly associated with PD. Fibroblasts from a 51-year-old female patient with disease onset at 39 years, carrying the A53T SNCA mutation (LCSBi003, ND40996), and fibroblasts with a triplication of the SNCA gene obtained from a 55-year-old female patient with disease onset at 52 years (LCSBi007, ND27760), were reprogrammed into human induced pluripotent stem cells (iPSCs) using Sendai virus. The presence of other genetic variants was determined using array comparative genomic hybridization. Presence of SNCA triplication was confirmed by FISH analysis.

1. Resource table

Unique stem cell lines identifier	LCSBi003-A
	LCSBi003-C
	LCSBi007-A
	LCSBi007-B
Alternative names of stem cell lines	ND40996-SNCA-A53T-clone 1 (LCSBi003-A)
	ND40996-SNCA-A53T-clone 10 (LCSBi003-C)
	ND27760-SNCA/ PARK1 triplication clone 2 (LCSBi007-A)
	ND27760-SNCA/PARK1 triplication clone 3 (LCSBi007-B)
Institution	The Gladstone Institutes, CA, USA
Contact information of distributor	Gabriela Novak gabriela.novak@alumni.utoronto.ca
	Alexander Skupin alexander.skupin@uni.lu
Type of cell lines	iPSC
Origin	Human
Cell Source	Fibroblasts
Clonality	Clonal
Method of reprogramming	CytoTune-iPS Sendai Reprogramming kit
	(ThermoFisher Scientific).
	transgenes/vectors used:
	CytoTuneTM 2.0 KOS
	CytoTuneTM 2.0 hc-Myc
	CytoTuneTM 2.0 hKlf4
	Clearance was confirmed using Scorecard, which detects the presence of Sendai virus, passage number is listed in Fig. 1
Multiline rationale	Two different mutations in the same gene, both leading to PD
Gene modification	YES
Type of modification	Familial, spontaneous mutation
Associated disease	Parkinson’s disease
Gene/locus	SNCA (PARK1) A53T 4q22.1
	SNCA (PARK1) triplication, 4q22.1
Method of modification	N/A
Name of transgene or resistance	N/A
Inducible/constitutive system	N/A
Date archived/stock date	2016
Cell line repository/bank	IBBL, Luxembourg
Ethical approval	Samples were collected in accordance with the US Government guidelines and are subject to MTA issued by Coriell Institute for Medical Research NINDS Cell Repository.
	The iPSC reprogramming protocol was approved by the Committee on Human Research at the University of California San Francisco. For description of Coriell collection protocol see Supplement section C3.

Fig. 1.

2. Resource utility

Parkinson’s disease (PD) is characterized by the loss of midbrain dopaminergic neurons. Their loss is a major obstacle to the study of the disease. Neurons differentiated from iPSCs carrying PD-associated mutations provide a window into the underlying molecular mechanisms and constitute an invaluable resource for the study of this disease.

3. Resource details

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder, characterized by the loss of midbrain dopaminergic neurons (mDA). There is no treatment that can slow the progression of the disease. PD presents with heterogeneous pathology due to its complex underlying mechanisms, which are only partly understood. About 90% of mDA neurons die by the end point of the disease, making the study of human mDA neurons affected by PD difficult. With the development of somatic cell reprogramming into iPSCs, followed by the differentiation of iPSCs into mDA neurons, an essential resource for the study of PD became available. Furthermore, because iPSCs can be generated from skin cells of PD patients known to carry a PD-associated mutation, the mechanism of disease development due to that mutation can be studied (Novak et al., 2022).

Duplications or triplications of α-synuclein (SNCA/PARK1) are known to significantly increase α-synuclein levels and lead to characteristic inclusions found in PD (Petrucci et al., 2016; Waxman and Giasson, 2009). The severity of the disease correlates with the number of SNCA copies. The presence of four copies, as in the case of a heterozygous triplication in the ND27760 cell line, leads to a severe early onset disease in the fourth to sixth decade of life, a decade earlier than other PD mutations, with a rapid progression (Petrucci et al., 2016).

Fibroblasts heterozygous for the SNCA triplication obtained from a 55-year-old female patient with disease onset at 52 years (LCSBi007, Coriell Institute ND27760) and fibroblasts from a 51-year-old female patient heterozygous for the A53T SNCA mutation with disease onset at 39 years (LCSBi003, Coriell Institute ND40996) were reprogrammed using the Sendai virus reprogramming method (performed by Yale Stem Cell Core), which does not introduce changes into the genome (Table 1). Their iPSC status was determined by staining for the pluripotency markers Oct4 & Tra-1–60 (Fig. 1, panel a) and expression of OCT4, SOX and NANOG was confirmed by qPCR (Table 3). The iPSC marker expression in these cell lines was compared to iPSCs which have undergone neurodifferentiation into mDA neurons and to a published iPSC cell line ND40066–8 (Fig. 1, panel b; Table 2 and 3). The expression of iPSC markers was further compared to a library of well characterized cell lines is shown in Supplement sections A5 and B5 via Scorecard analysis. Pluripotency and trilineage differentiation potential, as well as absence of Sendai virus, were also determined using Scorecard (Fig. 1, panel d; Table 2). Due to shortage of required media, ND27760–2 EBs were not generated successfully, but this cell line was differentiated into neurons in a separate experiment, using a protocol published in (Novak et al., 2022). The presence of the A53T mutation was confirmed by sequencing (Fig. 1, panel c; Table 2). The presence of SNCA triplication was confirmed using FISH analysis in fibroblasts and both iPSC clones (Supplement section A1). Normal karyotype was confirmed in fibroblasts of both cell lines (Supplement) and in iPSCs (Supplement section A1 and B1). The genotypic variation was determined by aCGH analysis (Supplement section A1 and B1). It should be noted that comparative genomic hybridization (CGH) array does not detect translocations or inversions, alterations in chromosome structure, mosaicism or polyploidy, hence we used FISH analysis to detect a-synuclein triplication (Supplement section A1).

Table 1.

Summary of lines.

iPSC line names	Abbreviation in figures	Gender	Age	Ethnicity	Genotype of locus	Disease
ND40996-SNCA-A53T-clone 1 (LCSBi003-A)	ND40996-1	Female	51	Caucasian	A53T	Parkinson’s disease
ND40996-SNCA-A53T-clone 7 (LCSBi003-B)	ND40996-7
ND40996-SNCA-A53T-clone 10 (LCSBi003-C)	ND40996-10
ND27760-SNCA3x clone 2 (LCSBi007-A)	ND27760-2	Female	55	Caucasian	SNCA triplication	Parkinson’s disease
ND27760-SNCA3x clone 3 (LCSBi007-B)	ND27760-3

Table 3.

Reagents details.

Antibodies
	Antibody	Dilution	Company Cat # and RRID
Pluripotency Markers	Mouse anti-Human POU5F1 (Oct3/4) Mouse anti-Human Tra-1-60	1:500 1:500	Santa Cruz Biotechnology sc-5279 RRID AB_628051 Merck Millipore MAB4360 RRID AB_2119183
Secondary antibodies	Donkey anti-Mouse IgG, Alexa Fluor Plus 555	1:1000	ThermoFisher A32773 RRID AB_2762848
Nuclear stain	DAPI	300 nM	Thermofisher D1306
Primers	Target	Forward/Revers e primer (5′–3′)
Sequencing primers	SNCA A53T	5′-GCAGAAGCAGCAGGAAAGAC-3′ 5′-TTCTGGGCTACTGCTGTCAC-3′
qPCR primers	OCT4	5′-TCGAGAACCGAGTGAGAGG-3′ 5′-GAACCACACTCGGACCACA-3′
qPCR primers	SOX2	5′-GCCGAGTGGAAACTTTTGTCG-3′ 5′-GCAGCGTGTACTTATCCTTCTT-3′
qPCR primers	NANOG	5′-TTCCCTCCTCCATGGATCTG-3′ 5′-TGTTTCTTGACTGGGACCTTGTC-3′
qPCR primers	B2M Beta-2-microglobulin	5′-GAGTATGCCTGCCGTGTG-3′ 5′-AATCCAAATGCGGCATCT-3′
Trilineage markers
Trilineage markers (qPCR)	Ectoderm, Mesoderm, Endoderm	Scorecard – Thermofisher – A15870

Table 2.

Characterization and validation.

Classification	Test	Result	Data
Morphology	Photography	Photography	Normal Fig. 1 panel a Supplement section C2
Phenotype	Qualitative analysis:	Confirmed by staining for pluripotency markers: Oct4 & Tra-1–60	Positive Fig. 1 panel a Supplement sections A4 and B4
	Quantitative analysis:	Determined by expression of iPSC-specific transcripts via qPCR (OCT4, NANOG, SOX2), by Scorecard and by FACS (staining for SSEA4 with more than 90% cells positive).	Fig. 1 panel b Fig. 1 panel d Supplement section C4 Supplement – FACS
Genotype	Karyotype	ND40996: 46, XX normal human female karyotype ND27760: 46, XX, normal human female karyotype. Also confirmed in iPSCs.	Supplement sections A1 and B1
Identity	Array comparative genomic hybridization for ND40996 STR analysis ND40996 and ND27760	aCGH Probes: Pass SNP Probes: Pass attached	Supplement section B1 Submitted in archive with journal
Mutation analysis	Sequencing FISH analysis	Heterozygous A53T mutation in ND40996 Heterozygous for α-synuclein triplication in ND27760	Fig. 1 panel c Supplement section B5 Supplement section A1
Microbiology and virology	Mycoplasma	Mycoplasma testing by luminescence: Negative	Supplement sections A2, B2 (at reprogramming) and C1 (recent)
Differentiation potential	Scorecard	Embryotic bodies show ability to differentiate into all three lineages	Fig. 1 panel d
Donor screening (OPTIONAL)	HIV 1 + 2 Hepatitis B, Hepatitis C	All samples negative for HIV 1, Hepatitis B, & Hepatitis C	Supplement sections A2 and B2
Genotype additional info (OPTIONAL)	Blood group genotyping	N/A
	HLA tissue typing	N/A

All cell lines were screened and found negative for HIV 1, Hepatitis B, Hepatitis C and mycoplasma (Supplement sections A2 and B2). FACS analysis was performed (Supplement sections A3 and B3).

Both cell lines (ND27760 and ND40996) were successfully differentiated into astrocytes in a study which showed that SNCA mutant astrocytes had increased mitochondrial fragmentation and decreased mitochondrial connectivity compared to controls, and reduced mitochondrial bioenergetic function (Barbuti et al., 2020).

4. Materials and methods

Fibroblasts cell lines ND27760 and ND40996 were obtained from the Coriell Institute and were cultured as described in recently published methods section pertaining to cells processed in parallel (Novak et al., 2021). Live adherent fibroblasts in culture media were sent to be karyotyped (Cell Line Genetics, Madison, WI, USA) (Supplement sections A1 and B1) and confirmed to have a normal karyotype. The reprogramming of fibroblasts into pluripotent stem cells was done using Sendai virus at the Yale Human Embryonic Stem Cell Core, New Haven CT, USA and early passage iPSCs were then passaged and characterised. The ND40996 iPSC clones were analysed using Array Comparative Genomic Hybridization (aCGH, high resolution karyotype analysis for the detection of unbalanced structural and numerical chromosomal alterations) and confirmed to be normal (Supplement section B1). The resulting iPSC cell lines were maintained as described for cells processed in parallel (Novak et al., 2021). Their iPSC status was confirmed by immunocytochemistry, as described for cells processed at the same time (Novak et al., 2021) (Fig. 1 panel a), for individual channels see Supplement sections A4 and B4. We used primary antibodies for POU5F1 (also known as Oct3/4) and Tra-1–60 and a secondary antibody (Donkey anti-Mouse IgG, Alexa Fluor Plus 555), these are listed in Table 3. The A63T mutation in ND40996 was confirmed in the iPSC cell lines as described in Novak et al. 2021(Novak et al., 2021), using primers listed in Table 3. Their iPSC status and their trilineage potential was further confirmed by a TaqMan iPSC Scorecard Assay (ThermoFisher Scientific, Fig. 1 panel d and Supplement section C4). The expression via qPCR was compared to iPSCs differentiated into neurons and to an established iPSC cell line published previously (Fig. 1 panel b) using a method described for cells processed in parallel (Novak et al., 2021). To confirm the presence of SNCA triplication, FISH analysis was performed (Cell Line Genetics, Madison, WI, USA) (Supplement section A1).