Abstract
Human induced pluripotent stem cells (hiPSCs) are invaluable to study developmental processes and disease mechanisms particularly in the brain. hiPSCs can be differentiated into mature and functional dopaminergic (DA) neurons. Having robust protocols for the generation of differentiated DA neurons from pluripotent cells is a prerequisite for the use of hiPSCs to study disease mechanisms, for drug discovery, and eventually for cell replacement therapy. Here, we describe a protocol for generating and expanding large numbers of homogeneous midbrain floor plate progenitors (mFPPs) that retain efficient DA neurogenic potential over multiple passages and can be cryobanked. We demonstrate that expanded mFPPs have increased DA neuron potential and differentiate more efficiently and rapidly than progenitors generated by standard protocols. In addition, this novel method results in increased numbers of DA neurons that in vitro show characteristic electrophysiological properties of nigrostriatal DA neurons, produce high levels of dopamine, and integrate into host mice when grafted in vivo. Thus, we describe a robust method for producing human mesencephalic DA neurons from hiPSCs.
Highlights
Human pluripotent stem cells, including embryonic stem cells and Human induced pluripotent stem cells (hiPSCs), can be expanded in vitro and retain their capacity to differentiate into any cell type of the three germ layers[1, 2]
We show that expanded midbrain floor plate progenitors (mFPPs) can be frozen and thawed and that they generate mature DA neurons in vitro with higher efficiency than those generated by standard protocols
This protocol allows for expansion and banking of expanded mFPP for large-scale generation of mature and functional DA neurons in vitro and in vivo and circumvents some of the variability often seen with protocols that require differentiation from pluripotent cells without the possibility of progenitor expansion
Summary
Human pluripotent stem cells, including embryonic stem cells and hiPSCs, can be expanded in vitro and retain their capacity to differentiate into any cell type of the three germ layers[1, 2] They represent an unlimited source of cells for several applications including drug screening and cell replacement therapy for treatment of neurological disorders. Numerous protocols have been developed to generate human DA neurons in vitro from pluripotent cells[8,9,10,11,12,13,14] These methods rely on the directed differentiation of pluripotent cells using small molecules and growth factors either through an embryoid body or neurosphere step or in adherent culture[8,9,10,11,12,13,14,15,16,17,18]. The generation of expandable mFPPs on a large scale makes this protocol advantageous for understanding cellular and molecular mechanisms of early human DA neuron development, generating large numbers of mFPPs or DA neurons for drug screening, and transplantation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
