Generation of eight human induced pluripotent stem cell lines from Parkinson's disease patients carrying familial mutations

We generated eight induced pluripotent stem cell (iPSC) lines from Parkinson's disease (PD) patients with di ﬀ erent familial mutations using non-integrating episomal plasmids. All iPSC lines have a normal karyotype, express pluripotent genes including POU5F1, NANOG, and show alkaline phosphatase activity, as well as the ability to di ﬀ erentiate into all three germ layers. These PD iPSC lines can be used for disease modeling to identify PD mechanisms and for the development or strati ﬁ cation of new drugs.


Resource utility
A bank of Parkinson's disease (PD) iPSC lines from a broad range of familial PD patients can be used to study early disease mechanisms and those involved in its progression, which may be relevant for sporadic cases, and provide a platform for the development or stratification of new drugs.

Resource details
Parkinson disease is the second most common neurodegenerative disorder, which affects a broad segment of the aging population in our society.The majority of PD cases are sporadic; however, more than 10% of cases are hereditary (Marti et al., 2003).Hereditary cases, where a high penetrant pathogenic variant has been identified, provide the opportunity to investigate PD related mechanism that may also be relevant for sporadic cases.Induced pluripotent stem cells (iPSCs) offer new opportunities to use these patient cells and generate specific cell type to model PD in vitro in a human context (Soldner et al., 2011).Establishing a bank of PD iPSC lines from a broad range of familial PD patients will enable the analysis of patient-specific neurons from various familial PD genetic backgrounds, which can potentially uncover disease relevant mechanisms and help accelerate the development of new drugs.
The fibroblasts and lymphoblasts were reprogrammed by transfection with POU5F1, SOX2, KLF4, MYCL and LIN28 using non-integrating episomal vectors.After 3-4 weeks, we observed cell morphological changes.Subsequently, iPSCs clones were picked and cultured on feeders for expansion and further characterization.Chromosomal analysis from all iPSCs showed normal karyotypes 46, XX or 46, XY (Supplementary Fig. 1A) and the familial mutations in PD-iPSCs were confirmed (Supplementary Fig. 2).All iPSCs were alkaline phosphatase positive (Fig. 1A) and expressed the pluripotent markers, POU5F1, and NANOG (Fig. 1B, C).Quantitative assessment of pluripotency was determined by counting the percentage of POU5F1 + /DAPI and NANOG + /DAPI cells from three different colonies for each cell line (Supplementary Figure 1B and Supplementary Table 1).
All the iPSC lines reported in this paper were confirmed to be free from random integration of the reprogramming plasmids, which were analyzed by qPCR (Supplementary Fig. 1C).All iPSC lines successfully formed embryoid bodies and at day 14 cultures contained cell types representative of the three germ layers, indicated by positive staining for SOX17/FOXA2 (endoderm), TBX6 (mesoderm), and TUBB3 (ectoderm) (Fig. 1D-F).In addition, the absence of mycoplasma for all the lines was confirmed by PCR (Supplementary Figure 1D).Cell line identities were confirmed to match the original donors by a genetic profile of a set of STR loci on each cell line (Table 2).

Pluripotency markers and embryoid body formation assay
iPSCs analyzed by immunocytochemistry were first fixed in 4% PFA at 4 °C for 10 min and washed briefly in PBS and blocked for 1 h at room temperature (RT) with 5% donkey serum in PBT (PBS with 0.25% triton-X) solution.Primary antibodies diluted in blocking solution were applied at 4 °C overnight followed by washes in PBT, after which the corresponding secondary antibodies were applied for 1 h at RT (antibodies shown in Table 3).Nuclei were counterstained with 4,6diamidino-2-phenylindole (DAPI; 1 μg/ml, Sigma).Positive cells for POU5F1 and NANOG staining was counting from three different colonies and data expressed as a percentage of POU5F1 + /DAPI and NANOG + /DAPI.

Genomic analysis
Karyotype analysis was performed on Q-banded metaphase spreads that were prepared according to standard protocol at a clinical accredited laboratory.Ten metaphases were counted and two analysed according to clinical standards.Briefly, growth medium was renewed and colcimide was added to the cultures at 0.1 µg/ml and incubated at 37 °C for 60-120 min depending on the donor.The PD iPSC cells were harvested by trypsinization (0.025% W/V in Hanks buffered saline) at 37 °C.The trypsinization was stopped by adding serum-containing medium.Cells were collected by centrifugation and then incubated in hypotonic KCl 0.56% at 37 °C for 30 min in a water bath.Cells were collected by centrifugation and resuspended in fixation buffer (1 part glacial acetic acid and 3 parts methanol).The cells were spun down by centrifugation and resuspended in fixative.This step was repeated once.The resuspended cells were added dropwise to slide glasses, dried, stained by quinacrine and mounted for fluorescence microscopy.
Genomic DNA were collected and purified using GeneJet Genomic DNA purification kit (Cat #K0721, ThermoFisher Scientific).Familial mutations for each of the PD iPSC lines and their parental cell lines were validated by either standard PCR or qPCR.The standard PCR amplification was done with Thermo Scientific™ Arktik™ Thermal Cycler with the following program: initial denaturation at 94 °C for 30 s; 35 cycles of (94 °C for 30 s, 60 °C for 30 s, 68 °C for 30 s); final extension at 68 °C for 5 min and hold at 15 °C.PCR products were extracted and cleaned with DNA Clean and concentrator kit (Cat#D4005, Zymo Research) and then samples were prepared and sent to Eurofins Genomics for Sanger sequencing using primers in Table 3. QPCR were done with 7500 Fast Real-Time PCR system (Applied Biosystems) using Taqman Universal Master Mix II no UNG (Cat#444040, ThermoFisher Scientific) to confirm the mutations of SNCA duplication of the DANi-009C and the original parental lymphoblasts.iPSC clones were tested for random integration of episomal plasmids by qPCR with a Go-Taq®qPCR System kit (Cat#A6001, Promega) for EBNA/OriP sequences using primers in Table 3 and those positive for plasmid integration were excluded.Cell line identity was performed by the Department of Molecular Medicine (MOMA) at Aarhus University Hospital with the GenePrint® 10 system.

Fig. 1 .
Fig. 1.Characterization of eight human iPSC lines generated from Parkinson's disease patients carrying familial mutations.

Table 1
Summary of lines.

Table 2
Characterization and validation.