Generation of TWO G51D SNCA missense mutation iPSC lines (CRICKi011-A, CRICKi012-A) from two individuals at risk of Parkinson ’ s disease

A B S T R A C T Mutations or multiplications of the SNCA (Synuclein Alpha) gene cause rare autosomal dominant Parkinson ’ s disease (PD). The SNCA G51D missense mutation is associated with a synucleinopathy that shares PD and multiple system atrophy (MSA) characteristics. We generated induced pluripotent stem cell (iPSC) lines from two individuals with SNCA G51D missense mutations at risk of PD. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. The resulting human iPSCs displayed normal morphology, expressed markers associated with pluripotency, and differentiated into the three germ layers. The iPSC lines could facilitate disease-modelling and therapy development studies for synucleinopathies.


Resource Table:
Unique stem cell line identifier

Resource utility
We report two patient-derived iPSC lines for use as a disease-specific cellular model to further understand how SNCA G51D missense mutation influences pathology. The iPSC lines could facilitate diseasemodelling and therapy development studies for synucleinopathies (see Table 1).

Resource details
Multiple missense mutations of the SNCA gene, which encodes the protein alpha-synuclein, have been identified in patients with autosomal dominant forms of Parkinson's disease (PD). The SNCA missense mutation G51D presents as a synucleinopathy which incorporates a mixture of PD and MSA pathological hallmarks, with both neuronal and oligodendroglial alpha-synuclein positive inclusions (Kiely et al., 2013(Kiely et al., , 2015. Here, we report the generation and characterisation of two iPSC lines derived from two individuals from the same family with G51D SNCA mutation. Patient dermal fibroblasts were obtained from the individuals at UCL Queen Square Institute of Neurology and reprogrammed using the nonintegrating mRNA-based protocol (StemRNA™ 3rd Gen Reprogramming Kit, REPROCELL) that combines non-modified RNA (NM-RNA) and microRNA technology. It contains six reprogramming factors, Oct4, Sox2, Klf4, cMyc, Nanog, and Lin 28, together with three immune evasion factors E3, K3 and B18. Cells were reprogrammed in a feederfree system according to the manufacturer's instructions. Colonies with a typical pluripotent stem cell morphology were individually and manually selected to establish clonal feeder-free iPSC lines.
Cells showed typical iPSC morphology after a few passages (Fig. 1A). Dideoxynucleotide sequencing (Sanger Sequencing) confirmed the presence of the pathogenic c.G152A in Exon 3 of the SNCA gene (Fig. 1B). Copy number variation analysis by chromosomal microarray confirmed the sex of the individuals (Fig. 1C). Stem cell identity of the CRICKi011-A and CRICKi012-A was confirmed by the expression of pluripotency markers OCT4 and SSEA4 on Flow Cytometry analyses (Fig. 1D).
In vitro differentiation (direct and spontaneous) confirmed the cell line's ability to differentiate into all three germ layers ( Fig. 1E and F). The identical genetic identity of the donor of each iPSC was confirmed by short tandem repeat (STR) profiling. These results prove we have successfully produced iPSC lines from two patients with G51D SNCA mutation.

iPSC cell generation and expansion
We thawed the dermal fibroblasts at passage 4 for CRICKi011-A and passage 5 for CRICKi012-A and seeded at a density of 5 × 10 4 /well in 2 wells/ each of a 6-well plate coated with iMatrix (Stemgent). They were plated in Fibroblast Expansion Medium (DMEM(Gibco)/ Glutamax (Gibco)/10% Hyclone FBS (Thermo Scientific)) and cultured for 24 h in 37 • C, 5% CO 2 and 21% O 2. Then, on the first day of reprogramming, Day 0, the medium was switched to NutriStem medium (Stemgent), and cells were transferred to a hypoxic incubator at 37 • C, 5% CO 2 and 5% O 2 .
Cells were reprogrammed 1-day post-seeding using the StemRNA™ 3rd Gen Reprogramming Kit (Stemgent) according to the manufacturer's instructions. Briefly, the NM-RNA cocktail was added to RNAi-MAX transfection reagent (Gibco) and transfected into the cells for four consecutive days with medium change 6 h post-transfection.
iPSC-like colonies started to show 9 to 12 days post the first day of transfections. We manually picked those with iPSC-like morphology and transferred them to Matrigel-coated 6-well plates with mTeSR1 medium (StemCell Technologies) containing 10 µM Y-27362. The medium was changed after 24 h. Colonies were expanded by splitting at a 1:3 to 1:6 ratio every 4-6 days and maintained in a hypoxic incubator at 37 • C, 5% CO2 and 5% O2.

Pluripotency markers
We evaluated the pluripotency status of CRICKi011-A and CRICKi012-A line at passages 9 and 10, respectively, by Flow Cytometry using the BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit (BD) as per the manufacturer's instruction. Briefly, we detached the cells using Accutase (Sigma-Aldrich) and passed through a 70 μM cell strainer to eliminate cell clumps. The cells were then washed with DPBS (without Ca 2 Mg 2 ) (Thermo Fisher Scientific) and resuspended at 1 × 10 6 cells/ml before adding the Live/ Dead staining (Thermo Fisher) for 30 min at room temperature. After another cell wash step with DPBS, cells were fixed in 4% paraformaldehyde (BD Stemflow Analyses kit component) for 20 min. For the permeabilization step, we used the 1X Perm/ Wash buffer (BD Stemflow Analyses kit component) for 10 min, followed by a wash in the same buffer. We incubated the cells with the antibodies (Table 2) for 30 min. We used DIVA software to analyse the cells and FlowJo to analyse the data.

Spontaneous differentiation into three germ layers
We tested the differentiation capacity of the CRICKi011-A and CRICKi012-A lines at passages 11 and 12, respectively, by spontaneous differentiation and embryoid body (EB) formation. Using the Aggre-Well™ 800 Microwell Plates, we seeded 5 × 10 6 /well in APEL 2 medium (STEMCELL Technologies) and cultured the EBs for 14 days in a hypoxic incubator at 37 • C, 5% CO2 and 5% O2.
The expression of the lineage-specific markers was assessed by TaqMan hPSC Scorecard Assay (Thermo Scientific). Briefly, we isolated RNA using the QIAgen RNeasy mini kit and measured concentration with a Nanodrop. We used the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher) for cDNA preparation. We analysed the data using the hPSC Scorecard™ -Analysis group (Thermo Fisher).

Direct differentiation into three germ layers
For direct differentiation, we used the STEMdiff™ Trilineage Differentiation Kit (STEMCELL Technologies) as per the manufacturer's instructions. We evaluated the differentiation potential of CRICKi011-A and CRICKi012-A lines at passages 11 and 12, respectively, by  Fig. 1. L.G. Devito et al. immunostaining for lineage-specific markers on Day 5 (Mesoderm and Endoderm) and Day 7 (Ectoderm) as described (Devito et al., 2021). Differentiated cells were washed twice in DPBS (Ca 2 Mg 2 ) (Thermo Fisher Scientific) before fixation with 3.7% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature, then incubated with primary antibodies (Table 2) overnight at 4 • C. The following day, cells were washed twice with DPBS and incubated with secondary antibodies (Table 2) for 30 min at room temperature.

Dideoxynucleotide sequencing
We performed PCR amplification (primers listed in Table 2) using the Q5 High-Fidelity 2X Master-Mix (BioLabs) on both cell lines at passage 7. The genomic DNA was extracted using the QIAamp DNA micro Kit (Qiagen). Then we purified the PCR product using the Monarch PCR and DNA cleanup Kit (BioLabs). Finally, we sent the samples for Sanger sequencing to Source Biosciences (UK) and analysed the data using SnapGene software.

Chromosomal microarray
Using the genomic DNA of each iPSC line, Thermo Scientific (USA) performed the KaryoStat assay (Thermo Scientific, USA), an array comparative genomic hybridization (CGH).

Short tandem repeat (STR) profiling
The Cell Services, a Science Technology Platform (STP) within the Francis Crick Institute, performed the STR profiling on DNAs from the parental sample and iPSC line using the Powerplex 16 HS System (Promega). Since reprogramming started, all lines were sent regularly for STR profiling (every 3 passages).

Mycoplasma detection test
The Cell Services (STP) confirmed the absence of mycoplasma contamination using PCR amplification using the Universal Mycoplasma Detection Kit (ATCC 30-1012 K) for PCR amplification. Cells were regularly sent for Mycoplasma testing (every 3 passages) since reprogramming started.

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.