Molecular phenotyping of midbrain dopaminergic neurons derived from human induced pluripotent stem cells

The objective of this study is to investigate the generation, maturation, and function of midbrain dopaminergic (mDA) neurons derived from human induced- pluripotent stem cells (iPSCs), alongside examining the mechanisms underlying their degeneration in Parkinson's disease (PD). This research aims to advance cell replacement therapies, disease modeling, and the identification of potential therapeutic targets.

To achieve this, we first developed a novel mDA neuron differentiation protocol using human embryonic stem cells (hESCs) that accurately recapitulates key aspects of in vivo human ventral midbrain development (Paper I). This approach employs a combination of dual WNT activation, CHIR boost and LN511 coating to improve midbrain patterning. In addition, neurogenesis and differentiation were further enhanced by activating liver X receptors and inhibiting fibroblast growth factor (FGF) signaling.

We next leveraged the mDA neuron differentiation protocol as a blueprint for iterative refinements, aiming to adapt and optimize it for various human induced stem pluripotent cell (hiPSC) lines to ensure broad applicability. These efforts led to a streamlined differentiation protocol, which was validated across multiple iPSC lines (Paper II). Additionally, a time-course multiomic analysis of mDA neuron differentiation identified discrepancies in gene expression and regulation patterns compared to human midbrain development. This information was utilized to correct the initial protocol, optimizing regional specificity, cell type composition, molecular signatures, and maturation of mDA neurons, ultimately leading to enhanced recovery in PD animal models (Paper III).

On the other hand, we explored phenotypic changes associated with PD, focusing on mutations in the GBA gene, which encodes the glucosylceramidase enzyme, by comparing GBA E326K and N370S mutated iPSCs to their isogenic controls. Through secretome profiling of cerebrospinal fluid (CSF) and iPSC- derived mDA neurons from PD patients, we determined mDA neuron-attributed alterations in CSF protein composition and found that FKBP4, a member of immunophilin protein family, has a potential linkage to pathological progression and psychiatric symptoms in GBA mutation-associated PD (Paper IV). Using both 2D monocultures and a microfluidic brain-on-a-chip model, we revealed that GBA E326K mutated mDA neurons exhibited accelerated differentiation and altered axonal morphology. These findings are consistent with molecular phenotypes identified through the single-nuclei RNA sequencing, which showed that mutated midbrain progenitors displayed expedited neurogenesis, while their descendant neurons progressed towards neurodegenerative phenotypes (Paper V).

In summary, this study advances our understanding of mDA neuron biology and PD pathogenesis, providing a strong foundation for further research in regenerative medicine and therapeutic development.

List of scientific papers

I. Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation. Kaneyasu Nishimura, Shanzheng Yang, Ka Wai Lee, Emilía Sif Ásgrímsdóttir, Kasra Nikouei, Wojciech Paslawski, Sabine Gnodde, Guochang Lyu, Lijuan Hu, Carmen Saltó, Per Svenningsson, Jens Hjerling-Leffler, Sten Linnarsson, Ernest Arenas. Stem Cell Reports. 2023. https://doi.org/10.1016/j.stemcr.2022.10.016

II. A reference human induced pluripotent stem cell line for large-scale collaborative studies. Caroline B Pantazis, Andrian Yang, Erika Lara, Justin A McDonough, Cornelis Blauwendraat, Lirong Peng, Hideyuki Oguro, Jitendra Kanaujiya, Jizhong Zou, David Sebesta, Gretchen Pratt, Erin Cross, Jeffrey Blockwick, Philip Buxton, Lauren Kinner-Bibeau, Constance Medura, Christopher Tompkins, Stephen Hughes, Marianita Santiana, Faraz Faghri, Mike A Nalls, Daniel Vitale, Shannon Ballard, Yue A Qi, Daniel M Ramos, Kailyn M Anderson, Julia Stadler, Priyanka Narayan, Jason Papademetriou, Luke Reilly, Matthew P Nelson, Sanya Aggarwal, Leah U Rosen, Peter Kirwan, Venkat Pisupati, Steven L Coon, Sonja W Scholz, Theresa Priebe, Miriam Öttl, Jian Dong, Marieke Meijer, Lara J M Janssen, Vanessa S Lourenco, Rik van der Kant, Dennis Crusius, Dominik Paquet, Ana-Caroline Raulin, Guojun Bu, Aaron Held, Brian J Wainger, Rebecca M C Gabriele, Jackie M Casey, Selina Wray, Dad AbuBonsrah, Clare L Parish, Melinda S Beccari, Don W Cleveland, Emmy Li, Indigo V L Rose, Martin Kampmann, Carles Calatayud Aristoy, Patrik Verstreken, Laurin Heinrich, Max Y Chen, Birgitt Schüle, Dan Dou, Erika L F Holzbaur, Maria Clara Zanellati, Richa Basundra, Mohanish Deshmukh, Sarah Cohen, Richa Khanna, Malavika Raman, Zachary S Nevin, Madeline Matia, Jonas Van Lent, Vincent Timmerman, Bruce R Conklin, Katherine Johnson Chase, Ke Zhang, Salome Funes, Daryl A Bosco, Lena Erlebach, Marc Welzer, Deborah Kronenberg-Versteeg, Guochang Lyu, Ernest Arenas, Elena Coccia, Lily Sarrafha, Tim Ahfeldt, John C Marioni, William C Skarnes, Mark R Cookson, Michael E Ward, Florian T Merkle. Cell Stem Cell. 2022. https://doi.org/10.1016/j.stem.2022.11.004

III. Single-cell multiomic sequencing informs strategies to enrich the differentiation of human iPS cells in SOX6+ midbrain dopaminergic neurons. Guochang Lyu, Anqi Xiong, Rika Kojima, Judith Kreutzmann, Wojciech Paslawski, Chiara Tremolanti, Carmen Salto, Per Uhlén, Per Svenningsson, Ernest Arenas. [Manuscript]

IV. Secretome Analyses Identify FKBP4 as a GBA1-Associated Protein in CSF and iPS Cells from Parkinson's Disease Patients with GBA1 Mutations. Rika Kojima, Wojciech Paslawski, Guochang Lyu, Ernest Arenas, Xiaoqun Zhang, Per Svenningsson. International Journal of Molecular Sciences. 2024. https://doi.org/10.3390/ijms25010683

V. A comprehensive phenotypical characterization of midbrain dopaminergic neurons derived from GBA E326K Parkinsonian iPSCs. Guochang Lyu, Rika Kojima, Anqi Xiong, Judith Kreutzmann, Wojciech Paslawski, Carmen Salto, Inês Sousa Pereira, Clelia Introna, Maria José Lopez Martinez, Josep Samitier Martí, Josep M. Canals Coll, Per Uhlén, Per Svenningsson, Ernest Arenas. [Manuscript]