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

Abstract

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.

Dopamine promotes the neurodegenerative potential of β‐synuclein

“…This line was generated from dermal skin fibroblasts, by Sendai virus‐mediated transcription factor expression. Patterning into dopaminergic neurons or glutamatergic neurons was performed essentially as described (Kriks et al., 2011) (Vazin et al., 2014) with the modifications specified in (Mahajani, Raina, Fokken, Kügler, & Bähr, 2019), as shown in Figure 4a and b. Cells were seeded at 150.000 cells/well in 24‐well plates.…”

Mind the translational gap: using iPS cell models to bridge from genetic discoveries to perturbed pathways and therapeutic targets

“…Zhang et al [154] NGN2 expression and WNT/dual-SMAD inhibition Nehme et al [47] GABAergic inhibitory neurons Transient expression of TFs (Ascl1 and Dlx2) Yang et al [155] Dopaminergic neurons Transient expression of TFs (rLmx1a, rNurr1 or rPitx3) Mahajani et al [156] Sensory peripheral neurons Small molecule-mediated direct differentiation, followed by human epidermal keratinocytes-conditioned medium Guimareãs et al [157] Interneurons Small molecule-mediated direct differentiation Maroof et al [158] transcriptional patterning, to generate cortical excitatory glutamatergic neurons [47]. After three weeks of maturation, transcriptomic analysis confirmed homogenous maturation of cultured iPSCs into upper layer cortical projection neurons.…”

The application of in vitro‐derived human neurons in neurodegenerative disease modeling

“…An ongoing challenge in neurodegenerative disease modeling is the ability to understand selective vulnerability, where for a particular disorder a specific neuronal subtype is more affected than others. Refinements in neuronal differentiation protocols have made significant advances in producing distinct neuronal subtypes such as CA3 hippocampal neurons (Sarkar et al, 2018), cortical interneurons (Nestor et al, 2015), medium spiny neurons (MSNs; Stanslowsky et al, 2016), dopaminergic neurons (Mahajani, Raina, Fokken, Kugler, & Bahr, 2019; Theka et al, 2013; Zhang, Xia, & Reijo Pera, 2014), motor neurons (Hester et al, 2011; Shimojo et al, 2015), and Purkinje neurons (Watson, Wong, Vowles, Cowley, & Becker, 2018). Furthermore, studies that transplant hiPSC‐derived neurons into slice cultures show that the transplanted cells take on the specific properties of the region where they were transplanted, such as CA1 or the dentate gyrus (Hiragi et al, 2017).…”