Dr. Shapiro: In our previous discussion, Birgitt, you had mentioned that the cell lines made available as part of the collaboration between ATCC and MJFF were derived from a donor with an SNCA genomic triplication. Can you tell us a bit more about how the isogenic cell lines in the collection were developed? What is the advantage to having 3 clones of each alpha-synuclein knockout all derived from the same individual?
Dr. Schüle: In 2009, we successfully obtained skin fibroblasts from the aforementioned patient and subsequently reprogrammed them into induced pluripotent stem cells (iPSCs). The patient’s comment during the skin biopsy was “I am taking one for the team!” These iPSCs were then differentiated into neurons. Notably, the neurons derived from the iPSCs exhibited noteworthy alterations in oxidative stress, mitochondrial health, and the process of neuronal differentiation when compared to control cultures consisting of cells from unaffected siblings (references available).
To further investigate the impact of SNCA gene dosage on these observed effects, we devised an isogenic model through CRISPR/Cas9 gene editing, which is now available at ATCC. This approach involved the introduction of frameshift mutations into exon 2 of the SNCA coding region within human iPSCs originating from a patient belonging to the Iowa kindred. We successfully generated and characterized multiple clones, each harboring distinct frameshift mutations within the SNCA gene. This innovative isogenic SNCA gene dosage model now enables us to comprehensively explore the physiological consequences as well as the detrimental outcomes associated with varying levels of alpha-synuclein expression.
In adherence to principles of rigor and reproducibility, we deliberately created multiple clones for each SNCA gene dosage variant, each bearing distinct frameshift mutations. This strategic approach not only ensures consistency and reliability within our experimental design but also affords greater flexibility when structuring and conducting experiments.
Dr. Shapiro: How do these cell lines impact Parkinson’s disease research, and how can researchers use these cell models in their research?
Dr. Schüle: The accumulation and aggregation of alpha-synuclein protein constitutes a pivotal event in the pathophysiology of Parkinson's disease, resulting in the impairment of neuronal function and contributing to the demise of dopaminergic neuronal cells. Given its central role in Parkinson's, alpha-synuclein has emerged as a prime target for the development of novel therapies with the goal of modifying the course of the disease. These findings have generated a consensus that reducing the content of alpha-synuclein or eliminating toxic alpha-synuclein species within cells could offer a promising strategy for decelerating, reversing, or potentially even preventing Parkinson's disease and other alpha-synuclein-related disorders. Nonetheless, the optimal extent of downregulation of a-syn that confers benefits and neuroprotection remains to be definitively ascertained.
These isogenic human iPSC lines in which alpha-synuclein is ‘titrated’ from four functional gene copies to zero functional gene copies will allow researchers to test the varying protein levels of alpha-synuclein using cellular biology, 'omics' analyses, the establishment of screening assays, and the facilitation of drug development. This versatile system enables the exploration of various facets, from molecular pathways to potential therapeutic interventions.
Dr. Shapiro: What type of characterization data did you generate on these cell lines prior to deposit at ATCC?
Dr. Schüle: Before we deposited the cell lines with ATCC, we performed extensive characterization according to the guidelines by Stem Cell Research, such as morphology, karyotype, pluripotency, genotyping, random plasmid integration events, quantitative RT-PCR for alpha-synuclein gene expression, multilineage differentiation potential, and mycoplasma testing.
More specifically, we used optical genome mapping as a high-density karyotype, which can detect structural variants down to 500 bps. We also developed a specific genotyping assay—a targeted allele-specific PCR (fragment analysis with fluorescently labeled primers and capillary electrophoresis)—that can distinguish the various frameshift mutations in these clones. All results and links for raw data files are published in Zafar et al. 2022.9
Researchers are also encouraged to visit the ATCC website to learn more about the cell lines and the patient with the SNCA genomic triplication.
Stay tuned for the third interview in this series where Dr. Shapiro will meet with Ms. Pamela Wood, a QC Supervisor at ATCC, to discuss how the SNCA knockout isogenic cell lines were authenticated and characterized at ATCC.
Did you know?
The SNCA knockout iPSCs available from ATCC harbor a triplication, duplication, two copies, single copy, or complete knockout of the α-synuclein (SNCA) gene.
Meet the scientists
Birgitt Schüle, MD
Associate Professor, Department of Pathology, Stanford University School of Medicine
Birgitt Schüle, MD, is an Associate Professor in the Department of Pathology at Stanford University School of Medicine. Her research focuses on medical genetics and stem cell modeling to uncover disease mechanisms and pathways involved in neurodegeneration in Parkinson's disease and related disorders. She is dedicated to developing novel therapeutic strategies that contribute to the advancement of precision medicine.
Dr. Schüle received her medical training from the Georg-August University Göttingen and Medical University Lübeck, Germany, during between 1993 and 2001. In 2001, she obtained her doctoral degree in medicine (Dr. med.) from the Georg-August University Göttingen in neurophysiology. Following, from 2001 to 2002, she completed her neurology internship at the Medical University of Lübeck, where she was mentored by Prof. Christine Klein in neurogenetics of Parkinson’s disease and dystonia syndromes. Continuing, Dr. Schüle pursued a postdoctoral fellowship in human genetics at Stanford University School of Medicine from 2003 to 2005, under the guidance of Prof. Uta Francke.
Between 2005 and 2019, Dr. Schüle built and led critical clinical research programs and establishing biospecimen and cell line repositories in the fields of neurogenetics, translational stem cell research, and brain donation at the Parkinson's Institute and Clinical Center.
Dr. Schüle serves as Associate Core Leader, Neuropathology, within the Stanford Alzheimer Research Center (ADRC). Within this role, she has made significant contributions to the ADRC, particularly in genetic characterization, biobanking, and the establishment of a human induced pluripotent stem cell and post-mortem leptomeninges tissue bank. These resources are made accessible to the scientific community through repositories at the National Institutes of Health (NIH), thereby promoting collaborative research.
Dr. Schüle's expertise in the field of neurodegeneration have been instrumental in advancing medical knowledge. She is highly regarded within the scientific community for her significant contributions and plays an essential role in the pursuit of effective treatments and precision medicine approaches for neurodegenerative conditions.
Brian Shapiro, PhD
Marketing Segment Manager, Oncology, ATCC
Brian A Shapiro, PhD, works to communicate the scientific breakthroughs of ATCC’s product development laboratories to the biomedical research community. Brian is the Executive Producer of ATCC's Podcast, Behind the Biology. Previously, he worked at Virginia Commonwealth University, where he investigated the role of pre-mRNA splicing in the multi-drug resistance of lung cancer. Dr. Shapiro attended the Medical College of Georgia, where his research focused on adrenal physiology as well as diseases of the epidermis.
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MoreReferences
- Singleton A.B., et al. Alpha-synuclein locus triplication causes Parkinson’s disease. Science 302, 841 (2003).
- Zafar, F. et al. Genetic fine-mapping of the Iowan SNCA gene triplication in a patient with Parkinson's disease. NPJ Parkinsons Dis 4, 18 (2018).
- Mittal, S. et al. beta2-Adrenoreceptor is a regulator of the alpha-synuclein gene driving risk of Parkinson's disease. Science 357, 891-898 (2017).
- Oliveira, L.M. et al. Elevated alpha-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells. Cell Death Dis 6, e1994 (2015).
- Flierl, A. et al. Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication. PLoS One 9, e112413 (2014).
- Chung, C.Y. et al. Identification and rescue of alpha-synuclein toxicity in Parkinson patient-derived neurons. Science 342, 983-7 (2013).
- Mak, S.K., Tewari, D., Tetrud, J.W., Langston, J.W. & Schüle, B. Mitochondrial Dysfunction in Skin Fibroblasts from a Parkinson’s Disease Patient with an alpha-Synuclein Triplication. Journal of Parkinson's disease 1, 175-183 (2011).
- Byers, B. et al. SNCA triplication Parkinson's patient's iPSC-derived DA neurons accumulate alpha-synuclein and are susceptible to oxidative stress. PLoS One 6, e26159 (2011).
- Zafar, F., Nallur Srinivasaraghavan, V., Yang Chen, M., Alejandra Morato Torres, C. & Schüle, B. Isogenic human SNCA gene dosage induced pluripotent stem cells to model Parkinson's disease. Stem Cell Res 60, 102733 (2022).
