An expandable embryonic stem cell-derived Purkinje neuron progenitor population that exhibits in vivo maturation in the adult mouse cerebellum

Abstract

The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not been possible to efficiently generate Purkinje neuron (PN) progenitors from human or mouse pluripotent stem cells, let alone to develop a methodology for in vivo transplantation in the adult cerebellum. Here, we present a protocol to obtain an expandable population of cerebellar neuron progenitors from mouse embryonic stem cells. Our protocol is characterized by applying factors that promote proliferation of cerebellar progenitors. Cerebellar progenitors isolated in culture from cell aggregates contained a stable subpopulation of PN progenitors that could be expanded for up to 6 passages. When transplanted into the adult cerebellum of either wild-type mice or a strain lacking Purkinje cells (L7cre-ERCC1 knockout), GFP-labeled progenitors differentiated in vivo to establish a population of calbindin-positive cells in the molecular layer with dendritic trees typical of mature PNs. We conclude that this protocol may be useful for the generation and maturation of PNs, highlighting the potential for development of a regenerative medicine approach to the treatment of cerebellar neurodegenerative diseases.

ADNP promotes neural differentiation by modulating Wnt/β-catenin signaling

“…We hypothesized that loss of Adnp may cause developmental and functional defects of embryonic cerebellum. To test this hypothesis, we took use of ESC-based in vitro cerebellar neuron differentiation as a model system, and adapted the published SFEB protocols that have been shown to efficiently induce ESCs into GABA receptor alpha 6 + granule cells and L7 + Purkinje neurons via Math1 and Ncam expressing precursors 24–27 (Fig. 8a).…”

“…The Math1 or Zic1 positive precursors could be further induced into GABA receptor alpha 6 positive granule cells and L7 positive Purkinje neurons on day 20 of ESC in vitro differentiation 24, 27 . The expression of Purkinje cell marker L7 was detected by immunofluorescence staining of day 20 neuronal cell cultures.…”

“…ESCs were differentiated into cerebellar neuron cells using an approach adapted from the previously described protocols 24–27 . The differentiation medium (SFEM) was prepared as follows: DMEM supplemented with 5% KSR, 0.1 mM nonessential amino acids (Gibco, 11140050), 1 mM sodium pyruvate, 2 mM L-Glutamine and 0.1 mM 2-mercaptoethanol.…”

Synthetic Polymers Provide a Robust Substrate for Functional Neuron Culture

“…Hence, we set out to discover new synthetic substrates that support neuron cell culture looking at both primary mouse neurons and mouse embryonic stem cell–derived neural progenitor cells (NPCs) . Primary neurons are an archetypal source for cell replacement therapy in human neurodegenerative diseases, while the electrical responses of NPCs are well characterized in a range of neurobiological studies, and as such are highly relevant for the evaluation of new biomaterials suitable for functional neuron culture.…”

Upregulation of Neural Cell Adhesion Molecule 1 and Excessive Migration of Purkinje Cells in Cerebellar Cortex

“…Several studies have shown that BGCs play a crucial role in regulating the movement of granule cell progenitors from the external germinal zone (EGZ) to the granular layer. BGCs have been suggested to share similar interactions with other neurons such as PCs and promote their migration (Higuera et al, 2017). Therefore, the PCs-BGCs intercellular crosstalk at P2-P7, is a likely prerequisite for a coupled migration and for the ability to form a definitive PC layer (Rahimi-Balaei et al, 2018).…”