Generation of hiPSC-Derived Functional Dopaminergic Neurons in Alginate-Based 3D Culture.

Valentina Gilmozzi,Marcelo D Rosato-Siri,Simona Casarosa,Alessandra Zanon,Giovanna Gentile,Peter P Pramstaller,Luciano Conti,Christian X Weichenberger,Irene Pichler,Alexandros A Lavdas,Emanuela Kerschbamer,Diana A Riekschnitz,Michael Von Troyer,Andrew A Hicks

doi:10.3389/fcell.2021.708389

Abstract

Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of patient-specific dopaminergic (DA) neurons, overcoming the hurdle of restricted accessibility to disease-affected tissue for mechanistic studies on Parkinson’s disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Neurons differentiated in a three dimensional (3D) in vitro culture system might better mimic the in vivo cellular environment for basic mechanistic studies and represent better predictors of drug responses in vivo. In this work we established a new in vitro cell culture system based on the microencapsulation of hiPSCs in small alginate/fibronectin beads and their differentiation to DA neurons. Optimization of hydrogel matrix concentrations and composition allowed a high viability of embedded hiPSCs. Neural differentiation competence and efficiency of DA neuronal generation were increased in the 3D cultures compared to a conventional 2D culture methodology. Additionally, electrophysiological parameters and metabolic switching profile confirmed increased functionality and an anticipated metabolic resetting of neurons grown in alginate scaffolds with respect to their 2D counterpart neurons. We also report long-term maintenance of neuronal cultures and preservation of the mature functional properties. Furthermore, our findings indicate that our 3D model system can recapitulate mitochondrial superoxide production as an important mitochondrial phenotype observed in neurons derived from PD patients, and that this phenotype might be detectable earlier during neuronal differentiation. Taken together, these results indicate that our alginate-based 3D culture system offers an advantageous strategy for the reliable and rapid derivation of mature and functional DA neurons from hiPSCs.

Highlights

In biomedical research, key features of human pluripotent stem cells, including human embryonic stem cells and human induced pluripotent stem cells, are their selfrenewal capability and the competence to generate unlimited numbers of specialized cell types
It has been previously reported that Rho-associated protein kinase inhibitor (RI) is beneficial for viability and cell aggregation of human pluripotent stem cells (hPSCs) (Ohgushi et al, 2010; Horiguchi et al, 2014), and it was shown to be essential for sustaining viability of human embryonic stem cells (hESCs) in Alg microcapsules (Chayosumrit et al, 2010; Kim et al, 2013)
Most in vitro neurodegenerative disease modeling studies so far are based on conventional 2D cell culture systems, which show a variable reproducibility with relatively low degrees of functional maturation, whereas 3D cell culture models are thought to better mimic cell growth encountered in vivo and support the expression of tissue-specific genes and proteins (Geckil et al, 2010)

Summary

Introduction

Key features of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are their selfrenewal capability and the competence to generate unlimited numbers of specialized cell types These intrinsic properties enable their use as a unique tool for in vitro modeling of a large number. PD is the second most frequent neurodegenerative disorder affecting 2–3% of the population over 65 years (Poewe et al, 2017) It is clinically characterized by resting tremor, rigidity, bradykinesia, and postural instability (Hoehn and Yahr, 1967). While about 90% of PD cases are classified as idiopathic, in the past two decades, inherited mutations in more than 20 genes have been linked to rare, familial forms of PD and parkinsonism (Blauwendraat et al, 2020) These genetic findings have led to significant advancement of the understanding of the molecular pathways contributing to the loss of DA neurons. Disease modeling efforts have uncovered that midbrain DA neurons generated from PD patients-derived hiPSCs exhibit mitochondrial dysfunction and α-synuclein aggregation as major cellular disease phenotypes (Devi et al, 2008; Byers et al, 2011; Cooper et al, 2012; Imaizumi et al, 2012; Ryan et al, 2013; Flierl et al, 2014; Shaltouki et al, 2015; Chung et al, 2016; Kouroupi et al, 2017)

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