(759g) High-Efficiency Generation of Human Neurons for Modeling Neurodegenerative Diseases By Microfluidic Technology

Neurodegenerative diseases including Alzheimer’s and Parkinson’s have become a global threat to human health. Neurodegenerative diseases involve the progressive loss of neural cell function. Although extensive research efforts aim to develop new disease-modifying therapeutics, there is no significant progress.

The biggest limitation in neurodegenerative disease research is the lack of in vitro human models recapitulating the pathological hallmarks: current protocols for in vitro generation of human neurons are still often lengthy, expensive, complex and yield heterogenous populations.

We developed an innovative microfluidic technology for high-efficiency and cost-effective generation of in vitro human neurons, suitable for modelling of patient-specific biology in heathy and diseased conditions.

Taking advantage of highly efficient fibroblast reprogramming protocols we recently developed in microfluidics^1,2, we integrated a subsequent neural differentiation via NGN2 viral induction³ all inside a single microfluidic platform.

Starting from fibroblasts of patients with neurodegenerative diseases, we will first conduct partial reprogramming, immediately followed by forward neuronal programming.

With as little as 3 days of transgenic expression of reprogramming transcription factors OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28 (OSKMNL) based on mRNAs, the transient and epigenetically unstable intermediary cells formed will be efficiently neuronal differentiated by inducible expression (doxycycline-inducible systems) of a single transcription factor Neurogenin-2 (Ngn2) with a marked cost reduction.

In particular, the high quality and purity of the resulting hiPSCs allowed direct differentiation into functional neurons-like cells without additional expansion and passages.

We were able to generated patient-specific neurons in less than 15 days with cost of a few hundred euros.

The model has been characterized by immunostaining of the main mature neural markers (bIIITub, Ngn2, Map2, Fox3, HuD, NeuN).

Thanks to the intrinsic advantages of the microfluidic technology (i.e., cost-effectiveness and high-throughput), the production costs to make our in vitro model available and suitable for large- scale studies is highly reduced, ensuring at the same time a robust generation of human neurons from patient-specific cells.

This methodology has been successfully applied to Familial Alzheimer’s Disease cellular models with specific mutations in Presenilin 1 (PSEN1) gene.

Our technological approach has the potential to revolutionize the study of neurodegenerative disease. By accelerating, downscaling, and increasing homogeneity, microfluidics can speed up drug discovery and improve our mechanistic understanding, even for studies of large cohort of patients, which are otherwise not economically sustainable using conventional technologies.

References

1. Gagliano, O. et al. Microfluidic reprogramming to pluripotency of human somatic cells. Nat. Protoc. 14, (2019).
2. Giulitti, S. et al. Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics. Nat. Cell Biol. 21, 275–286 (2018).
3. Zhang, Y. et al. Rapid Single-Step Induction of Functional Neurons from Human Pluripotent Stem Cells. NIH Public Access. 78, 785–798 (2013).