Combining NGN2 programming and dopaminergic patterning for a rapid and efficient generation of hiPSC-derived midbrain neurons

Abstract

The use of human derived induced pluripotent stem cells (hiPSCs) differentiated to dopaminergic (DA) neurons offers a valuable experimental model to decorticate the cellular and molecular mechanisms of Parkinson’s disease (PD) pathogenesis. However, the existing approaches present with several limitations, notably the lengthy time course of the protocols and the high variability in the yield of DA neurons. Here we report on the development of an improved approach that combines neurogenin-2 programming with the use of commercially available midbrain differentiation kits for a rapid, efficient, and reproducible directed differentiation of hiPSCs to mature and functional induced DA (iDA) neurons, with minimum contamination by other brain cell types. Gene expression analysis, associated with functional characterization examining neurotransmitter release and electrical recordings, support the functional identity of the iDA neurons to A9 midbrain neurons. iDA neurons showed selective vulnerability when exposed to 6-hydroxydopamine, thus providing a viable in vitro approach for modeling PD and for the screening of small molecules with neuroprotective proprieties.

A dual hit of α-synuclein internalization and immune challenge leads to the formation and maintenance of Lewy body-like inclusions in human dopaminergic neurons

“…AIW002-02 iPSCs expressing doxycycline-inducible NGN2 were generated as described previously 107 . Briefly, the parental AIW002-02 iPSC line was split with ReLesR (STEMCELL Technologies, 100-0484) and seeded at a density of 135,000 cells per well in a 6-well plate in mTesR1 media plus Y27632 one day prior to simultaneous transduction with separate lentiviruses encoding Ngn2 and rtTA.…”

Transcription factor-mediated generation of dopaminergic neurons from human iPSCs – a comparison of methods

“…The following protocol was adopted from Sheta et al [9] to differentiate the iPSCs into iDAs. With an established protocol to generate iPSC-derived cortical neurons (i 3 Ns) [18,22], our i 3 N protocol was combined with the iDA protocol from Sheta et al to modify the media compositions and generate purer populations of iDAs.…”

“…The versatility of iPSCs has been harnessed in other fields of research, such as cardiac and renal diseases [3,4]. Many groups have embarked on differentiating skin fibroblast-derived iPSCs into dopaminergic neurons, from both healthy and Parkinson's disease affected individuals, using different approaches such as small molecules, transcriptions factors, and microRNAs [1,[5][6][7][8][9][10][11][12][13]. The original protocol published more than a decade ago by Kriks et al [6] used small molecules to generate human dopaminergic neurons from iPSCs, however, the protocol took more than two months to achieve mature human dopaminergic neurons.…”

“…Other studies [14][15][16] have used modified protocols with small molecules and have obtained purer populations of dopaminergic neurons, however, the time to develop the mature neurons was still more than a month. Since 2013, several researchers have developed protocols for transcription factor-mediated differentiation, using various transcription factors [7][8][9][10][11], and have successfully generated iPSC-derived human dopaminergic neurons in a relatively short amount of time (less than one month). Theka et al [5] was one of the first to generate iPSCderived dopaminergic neurons through transcription factor-mediated differentiation, omitting the neural progenitor cell stage.…”

Protective effects of polyphenol-rich extracts against neurotoxicity elicited by paraquat or rotenone in cellular models of Parkinson’s disease

“…Polyphenols are naturally occurring molecules in plants and fruits that have been studied extensively for their potential therapeutic benefit in a number of diseases, including mild cognitive impairment 16,17 and various neurodegenerative disorders [18][19][20] . Our group and others have shown that polyphenols can alleviate neurotoxicity in preclinical models of PD, including flavonoids (a subclass of polyphenols) such as green tea polyphenols [21][22][23] , anthocyanins (ANC) [24][25][26][27] , and isoflavones [28][29][30] , as well as the phenolic acid derivative curcumin 31 and stilbenes such as resveratrol and oxyresveratrol 24,32 . Polyphenols have been shown to protect against oxidative stress in a variety of disease models by scavenging free radicals and up-regulating endogenous cytoprotective responses 33,34 .…”