Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Abstract

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain "organoid" protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

Bioelectric Potential in Next-Generation Organoids: Electrical Stimulation to Enhance 3D Structures of the Central Nervous System

“…These are important factors regulating brain development, differentiation, survival and maturation ( Allen et al, 2013 ; Popova et al, 2017 ). Accordingly, BDNF and GDNF have been implemented in vitro to guide differentiation of brain organoids, including region-specific forebrain ( Birey et al, 2017 ; Qian et al, 2018 ), midbrain ( Kim et al, 2019 ), cerebellar ( Holmes and Heine, 2017 ), telencephalic ( Mariani et al, 2015 , 1), as well as cerebral organoids ( Quadrato et al, 2017 ; Watanabe et al, 2017 ). Specifically, addition of GDNF and BDNF is shown to enhance the maturation of neuronal networks and improve electrophysiological function in forebrain organoids ( Qian et al, 2018 ).…”

SCN2A channelopathies in the autism spectrum of neuropsychiatric disorders: a role for pluripotent stem cells?

“…As described above, these cells express Na V 1.2 throughout development and maintain high expression of this channel throughout life, rather than replacing it with Na V 1.6 (as occurs in the cerebral cortex). There are a small number of published protocols for generating cerebellar tissue types from iPSCs [103][104][105], and given the increasing interest in using iPSCs to model human neurodevelopment, more differentiation protocols are likely to follow. These will open the door for scientists to interrogate the unusual Na V 1.2 expression pattern observed in the cerebellum and investigate its contribution to SCN2A syndromes.…”

Layer-By-Layer: The Case for 3D Bioprinting Neurons to Create Patient-Specific Epilepsy Models

“…Such spreading creates particular stresses in the cell body that are particular to the 2D situation. In particular, anchorage dependant cells such as neurons require adhesive interactions with neighbouring cells and ECM to recapitulate normative cell morphology in vitro [52,53]. (e) In the body, cell adhesion sites surround the cell, while in 2D these adhesion sides are restricted to the x-y plane [54,55,56].…”

“…In 3D bioprinting cell laden hydrogels are typically deposited in liquid form in the desired shape prior to being crosslinked into a gel within minutes [172]. Prior to cell proliferation the bioink serves as a rudimentary ECM [52,53,155,170,173]. Extrusion printing and inkjet printing are commonly employed methods of 3D bioprinting.…”