Sameehan Mahajani

Homogenous generation of dopaminergic neurons from multiple hipsc lines by transient expression of transcription factors

• Authors Details :  
• Sameehan Mahajani,  
• Anupam Raina,  
• Claudia Fokken,  
• Sebastian Kuegler,  
• Mathias Baehr

Journal title : Cell Death & Disease

Publisher : Springer Science and Business Media LLC

Online ISSN : 2041-4889

Journal volume : 10

Journal issue : 12

856 Views Research reports

A major hallmark of Parkinson's disease is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The pathophysiological mechanisms causing this relatively selective neurodegeneration are poorly understood, and thus experimental systems allowing to study dopaminergic neuron dysfunction are needed. Induced pluripotent stem cells (iPSCs) differentiated toward a dopaminergic neuronal phenotype offer a valuable source to generate human dopaminergic neurons. However, currently available protocols result in a highly variable yield of dopaminergic neurons depending on the source of hiPSCs. We have now developed a protocol based on HBA promoter-driven transient expression of transcription factors by means of adeno-associated viral (AAV) vectors, that allowed to generate very consistent numbers of dopaminergic neurons from four different human iPSC lines. We also demonstrate that AAV vectors expressing reporter genes from a neuron-specific hSyn1 promoter can serve as surrogate markers for maturation of hiPSC-derived dopaminergic neurons. Dopaminergic neurons differentiated by transcription factor expression showed aggravated neurodegeneration through α-synuclein overexpression, but were not sensitive to γ-synuclein overexpression, suggesting that these neurons are well suited to study neurodegeneration in the context of Parkinson’s disease.

Article DOI & Crossmark Data

DOI : https://doi.org/10.1038/s41419-019-2133-9

Article Subject Details

• Neurobiology
• Neurology
• Neuroscience
• Stem cell

Article Keywords Details

• Dopaminergic neurons
• IPSCs
• Neurodifferentiation
• Parkinson's Disease
• Patterning

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Article References

• (1). Polymeropoulos, M. H. et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047 (1997).
• (2). Spillantini, M. G. et al. Alpha-synuclein in Lewy bodies. Nature 388, 839–840 (1997).
• (3). Goedert, M., Spillantini, M. G., Del Tredici, K. & Braak, H. 100 years of Lewy pathology. Nat. Rev. Neurol. 9, 13–24 (2013).
• (4). Surmeier, D. J., Obeso, J. A. & Halliday, G. M. Selective neuronal vulnerability in Parkinson disease. Nat. Rev. Neurosci. 18, 101–113 (2017).
• (5). Halliday, G. M. et al. Neuropathology of immunohistochemically identified brainstem neurons in Parkinson’s disease. Ann. Neurol. 27, 373–385 (1990).
• (6). Taschenberger, G. et al. Aggregation of alphaSynuclein promotes progressive in vivo neurotoxicity in adult rat dopaminergic neurons. Acta Neuropathol. 123, 671–683 (2012).
• (7). Taschenberger, G. et al. beta-synuclein aggregates and induces neurodegeneration in dopaminergic neurons. Ann. Neurol. 74, 109–118 (2013).
• (8). Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).
• (9). Friling, S. et al. Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells. Proc. Natl Acad. Sci. USA 106, 7613–7618 (2009).
• (10). Kirkeby, A. et al. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep. 1, 703–714 (2012).
• (11). Xi, J. et al. Specification of midbrain dopamine neurons from primate pluripotent stem cells. Stem Cells 30, 1655–1663 (2012).
• (12). Doi, D. et al. Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation. Stem Cell Rep. 2, 337–350 (2014).
• (13). Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480, 547–551 (2011).
• (14). Awad, O. et al. Altered differentiation potential of Gaucher’s disease iPSC neuronal progenitors due to Wnt/beta-catenin downregulation. Stem Cell Rep. 9, 1853–1867 (2017).
• (15). Beevers, J. E. et al. MAPT genetic variation and neuronal maturity alter isoform expression affecting axonal transport in iPSC-derived dopamine neurons. Stem Cell Rep. 9, 587–599 (2017).
• (16). Sheng, Y. et al. Using iPSC-derived human DA neurons from opioid-dependent subjects to study dopamine dynamics. Brain Behav. 6, e00491 (2016).
• (17). Sundberg, M. et al. Improved cell therapy protocols for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons. Stem Cells 31, 1548–1562 (2013).
• (18). Ishikawa, T. et al. Genetic and pharmacological correction of aberrant dopamine synthesis using patient iPSCs with BH4 metabolism disorders. Hum. Mol. Genet. 25, 5188–5197 (2016).
• (19). Romero-Moya, D. et al. Genetic rescue of mitochondrial and skeletal muscle impairment in an induced pluripotent stem cells model of coenzyme Q10 deficiency. Stem Cells 35, 1687–1703 (2017).
• (20). Arenas, E., Denham, M. & Villaescusa, J. C. How to make a midbrain dopaminergic neuron. Development 142, 1918–1936 (2015).
• (21). Vazin, T. et al. Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: a model system to study neurotoxicity in Alzheimer’s disease. Neurobiol. Dis. 62, 62–72 (2014).
• (22). Theka, I. et al. Rapid generation of functional dopaminergic neurons from human induced pluripotent stem cells through a single-step procedure using cell lineage transcription factors. Stem Cells Transl. Med. 2, 473–479 (2013).
• (23). Chung, S. et al. Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway. Cell Stem Cell 5, 646–658 (2009).
• (24). Lin, W. et al. Foxa1 and Foxa2 function both upstream of and cooperatively with Lmx1a and Lmx1b in a feedforward loop promoting mesodiencephalic dopaminergic neuron development. Dev. Biol. 333, 386–396 (2009).
• (25). Hsu, C. C. et al. Targeted methylation of CMV and E1A viral promoters. Biochem. Biophys. Res. Commun. 402, 228–234 (2010).
• (26). Thiel, G., Lietz, M. & Cramer, M. Biological activity and modular structure of RE-1-silencing transcription factor (REST), a repressor of neuronal genes. J. Biol. Chem. 273, 26891–26899 (1998).
• (27). Ballas, N., Grunseich, C., Lu, D. D., Speh, J. C. & Mandel, G. REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis. Cell 121, 645–657 (2005).
• (28). Tiburcy, M. et al. Defined engineered human myocardium with advanced maturation for applications in heart failure modeling and repair. Circulation 135, 1832–1847 (2017).
• (29). Kugler, S., Hahnewald, R., Garrido, M. & Reiss, J. Long-term rescue of a lethal inherited disease by adeno-associated virus-mediated gene transfer in a mouse model of molybdenum-cofactor deficiency. Am. J. Hum. Genet. 80, 291–297 (2007).
• (30). Mahajani, S. et al. Lamin B1 levels modulate differentiation into neurons during embryonic corticogenesis. Sci. Rep. 7, 4897 (2017).
• (31). Zahur, M., Tolo, J., Bahr, M. & Kugler, S. Long-term assessment of AAV-mediated zinc finger nuclease expression in the mouse brain. Front. Mol. Neurosci. 10, 142 (2017).
• (32). Giacomini, C., Mahajani, S., Ruffilli, R., Marotta, R. & Gasparini, L. Lamin B1 protein is required for dendrite development in primary mouse cortical neurons. Mol. Biol. Cell 27, 35–47 (2016).