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.
Highlights
Purkinje neurons (PNs) are the sole output neurons of the cerebellar cortex[1]
We show that our protocol allows for the generation of an expandable PN progenitor population that can be matured both in vitro and in vivo, paving the way for future studies that focus on the integration of ES cell derived PNs into the intact adult mouse cerebellum
We chose to isolate cerebellar progenitors from embryoid bodies (EBs), because (i) the use of a cerebellar progenitor population allows for the generation of an intermediate and stable cell state[30] and (ii) the number of PNs that can be generated directly from ES cell cultures is limited[19,20,21,22]
Summary
Purkinje neurons (PNs) are the sole output neurons of the cerebellar cortex[1]. Degeneration of PNs causes severe motor coordination deficits, referred to as ataxia[2, 3]. Until now standardization of differentiation protocols of neural progenitor cells (NPCs) has not led to consistent and robust generation of cerebellar neurons from transgenic mouse models and/or human patients with cerebellar disorders To date, it has remained unclear what is the best strategy to consistently mature PNs derived from pluripotent stem cells at high numbers in vitro, allowing subsequent functional implantation and integration in adult animals in vivo. We generated PN progenitors from mouse ES cells implementing a cerebellar differentiation protocol of embryoid bodies (EBs) using insulin, FGF2 and cyclopamine so as to fine-regulate the balance between cerebellar neurons and glia cells, while evaluating the expression of cell-specific factors like Engrailed 2 (En2) and Neph[324] We subsequently promoted their proliferation and expansion by using an NPC medium containing FGF2, B27 and N2 on a substrate of the neuroprotectant laminin[25]. We show that our protocol allows for the generation of an expandable PN progenitor population that can be matured both in vitro and in vivo, paving the way for future studies that focus on the integration of ES cell derived PNs into the intact adult mouse cerebellum
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